A gene encoding neurotrophin-3, gene products and process for preparing thereof

The present invention is about brain neurotrophic factor (BDNF / NGF) neurotrophin-3 (NT-3), a novel neurotrophic factor family of factor / nerve growth genes. The gene encoding NT-3 was cloned and sequenced; recombinant NT-3 was expressed in mammalian cells. NT-3 was found to have diverse biological activity, different from BDN and NGF. The present invention provides derivatives of a nucleic acid sequence encoding NT-3, a purified NT-3 protein; as well as antibodies to and derivatives of NT-3 protein. The products of the gene encoding the NT-3 protein can be used for diagnosis, treatment of neurological disorders, including peripheral neuropathy, Alzheimer's disease and Parkinson's disease.

with peptide fragments; or

2. The origins of the invention

2.1. Role of neurotrophic factors in the nervous system

The development and activity of the nervous system are dependent on proteins known as neurotrophic factors. The normal development of the central and peripheral nervous systems is accompanied by large-scale neuronal cell death, which is obviously a crucial process for regulating the number of neurons that protect the target area (Berg, DK, 1982, Neurons Development, 297-331; Couvant et al. .; 1984, 225, 1258-65). During developmental examination of peripheral target tissues, during lysis and transplantation, neuronal cells have been found to compete with neurons for their supporting factors (neurotrophic factors), which are synthesized in their defense domain. These observations have led to the identification of nerve growth factor (NGF), which is best characterized (Levi-Montalcini and Angeletti P., U. 1988, Physiol. Rev. 48, 53469; Thanen H., and Barole J., A. 1980, Physiol Rev.

60, 1284-335). Finding out the role and mechanism of action of NGF was greatly facilitated by the fact that it was. a source rich in this protein has been found - mice swollen forearm glands. This enabled the protein to be purified and cloned (Ullrich et al., 1983, Nature 303, 821-5,

Scott et al., 1983, Nature 302, 538-40), as well as obtaining neutralizing antibodies to this protein. NGF supports only a limited number of neurons, and other neurotrophic factors have been postulated to exist (Varon S. and Adler R. 1981, Adv. Cellular

Neurobiol., 2, 115-63, Barde et al., 1987, Prog. Brain

Res., 71, 185-9, Shide WD and Johnson EM, 1989, Ann. Neurol., 16, 489-506).

It is now clear that such factors exist, but their low concentration makes it difficult to characterize these factors at the molecular level. However, purification of a small amount of cerebral neurotrophic neurotrophic partial factor (BDNF) and ciliary (CNTF) has recently been successful in sequencing (Leibrock et al. , 1989, Nature, 341, 149-52; Stockli et al., 1989, Nature, 342, 21-28 and Lin et al., Science, 246, 1023-25). Although specific neuronal populations exhibit specificity, BDNF and NGF (but not CNTF) have sufficient structural homology, which allows them to be treated as members of a single gene family (Leibrock et al., 1989, Nature, 341, 146-52).

2.2. Other neurotrophic factors

Over the last decade, there have been many reports of neurotrophic activity in extracts of various tissues and in culture fluid of various cells. In almost all cases, purification and characterization of these proteins was complicated by the low concentration of these proteins from picogram to nonagram per gram of tissue. In addition, peripheral neurons have undergone adequate biological assays and replicate specific tests on central nervous system neurons have failed. Individual types of peripheral neurons have been detected in discrete, easily distinguishable ganglia, at which time central nervous system (CNS) neurons are highly heterogeneous. Specific markers, such as antibodies against the cell surface or cytostructural components or specific histological staining, are required for identification, and the selection of these markers is limited. Therefore, it is very difficult to characterize neurotrophic factors because of (1) their very low concentration, in contrast to NGF, (2) the difficulty of conducting experiments with them, and (3) their amounts too small to produce antibodies against them.

2.2.1 Comparison of growth factor secreted from brain and nerve growth factor.

Neurotrophic activity that maintains the viability of dorsal basal ganglion neurons in chicken embryos was identified in conditioned media cultured in rat glioma C-6 cells (Barde et al., 1978, Nature, 274, 818). This activity was not neutralized with antibodies to mouse NGF, suggesting another neurotrophic factor in this medium. Similar activities have subsequently been described that were not neutralized with antibodies to NGF in astrogenic cultures of normal adult rat brain cells (Lindsay, 1979, Nature, 282, 80-82; Lindsay et al., 1982, Brain Res., 243, 329-343). and brain extracts of developing and adult rats (Barde et al., 1980,

77, 1199-1203),, and in the chicken spinal cord (Lindsay and Peters, 1984, Neuroscience, 12, 45-51). In either case, however, no activity factor (s) were purified or identified and it remains unclear whether the observed

Proc. Natl. Acad. developing and

Sci. In the USA, mature activities are the same factor, or different factors.

Using a pig source as the primary source, Barde et al. (1982, EMBO, 1, 549-553) reported a factor, now called the brain (secreted from the brain) neurotrophic factor (BDNF), which apparently supported the viability of E10 / E11 dorsal basal ganglion neurons in chicken embryos. Neurotrophic activity was found to be a highly alkaline protein (isoelectric point pI> 10.1) with a molecular weight of 12.3 kD as determined by SDS PAGE electrophoresis. The purification factor was 1.4x10⁶, but the yield is very low at about 1 µg BDNF per 1.5 kg of pig brain. In addition, the final purification step was electrophoresis, so BDNF activity is not completely restored due to · residual SDS (Sodium Dodecylsulfate) (Barde and Thanen, 1985, Hormons and Cell Regulation Vol. 9, Damont et al., Eds. Elservier Science Publishers, 385- 390). It is noted that the BDNF protein is close to the NGF monomer in terms of isotope and molecular weight. But BDNF exhibits properties that are different from known NGF properties in that (a) in vitro antibodies to NGF did not alter BDNF activity, (b) BDNF effects were complementary to NGF, and (c) unlike NGF, BDNF had no effect on the E12 sympathetic chick embryo. for neurons. Additionally, in brain extracts, it was found that neuronal activity from this source appears to affect sensory neurons in later stages of development than NGF. Using dissociated culture of chicken embryonic neurons cultured on polycationic substrate polylysine or polyiornitine, BDNF was found to maintain more than 30% viability of dorsal basal ganglion neurons of E10 / E11 (i.e., 10 or 11 days developmental embryo), but which has a mild effect on the viability of the same neurons from E6 (Barde et al., 1980, Natl. Acad. USA, 77, 1199-1203, cf.

above). under similar conditions, NGF maintains 30-40% viability of E6 DRG neurons. Interestingly, it was later found that when cultured on a substrate coated with an extracellular matrix glycoprotein, laminin like NGF, BDNF maintained viability in 50% of DRG neurons from E6-E12-old chicken embryos (Lindsay et al., 1985, Develop Biol. , 319-328). Subsequent studies have shown that the effects of NGF and BDNF are complementary when present in high concentrations.

In previous studies on the neuron specificity of Levi-Montalcini (1966, The Harvey lectures 60, 217-259), NGF was thought to be not a ubiquitous neuronal factor, even for sensory neurons, because NGF was found not to affect certain sensory ganglion neurons in the chick. scalp nerve, especially nodular ganglion. Subsequently, in vivo studies (Johnson et al., 1980, Science 210, 916-918, Pearson et al., 1983, Development Biol. 96, 32-36) showed that removal during embryogenesis did not alter neuronal viability in most rats. of the sensory ganglia of the head, while such an operation significantly reduced the number of neurons in the sensory ganglia of the neural crest. For more detailed in vitro studies (Lindsay and Rohrer, 1985, Develop., Biol., 112, 30-48, Davies and Lindsay, 1985, Develop. Biol., Lll., 62-72, Lindsay et al., 1985, J. Cell. Sci. Suppl., 3, 115-129), it was clearly found that NGF maintains the viability of most neurons taken from the neural crest but has no significant effect on the viability of the scalp sensory neurons.

The first demonstration of the difference in BDNF neuronal specificity from NGF was an in vitro assay in which purified NGF maintained 40-50% of the sensory neurons dissociated from nodular ganglion derived from the chicken patch embryo E6, E9 or E12 (Lindsay et al., 1985 J. Cell Sci. Supp., 3, 115-129). NGF had no appreciable effect on these neurons either alone or in combination with BDNF. Later, in explant exploration cultures, BDNF was shown to preserve viability and regrowth of neurites from other sensory ganglia taken from the placenta, including the incisor, lateral body, and lateral trigeminal ganglia (Davies et al., 1986, J. Neuro. Sci., 6, 1897-1904), none of which was sensitive to NGF. In all of the above assays, neutralizing antibodies to NGF did not alter BDNF activity. In addition to these effects on cultured neurons from the peripheral ganglia, BDNF has been shown to stimulate viability and neuronal differentiation of cells taken from the quail neuron ridge (Kalcheim and Gendrean, 1988, Develop. Brain Res., 41, 79-86).

Prior to the present invention, the inability to obtain sufficient amounts of BDNF for immunization hindered the production of anti-BDNF antibodies required to compare them with the anti-NGF antibodies in their neuronal populations and BDNF / NGF cross-neutralization experiments could not be performed. Two recent studies on BDNF (Kalcheim et al., 1987, EMBO, 6, 2871-2873,

Barde, 1988, Nature, 331, 261-262) have shown, however, that BDNF has a physiological role in the development of the peripheral nervous system (PNS). If a mechanical septum was inserted in ovo (egg) between the E3 / E4 DRG (3 or 4 embryonic day dorsal basal ganglia) and their target in the central nervous system, then most DRG neurons were killed (Kalcheim and de Donarin, 1986, Develop Biol. 116, 451-466). It has been suggested that this neuronal death may be associated with the loss of neurotrophic factor from the CNS (neural tube). It was later observed that BDNF when attached to laminin

Hofer and that, however, an avian neural tube was observed with a padded sialastic avoidable cell (1987, EMBO, 6, 2871-2873).

membranes, may be fatal (Kalcheim - et al.,

Injections of BDNF into eggs of developing quail have been shown to reduce natural cell death in nodular ganglia, an effect that does not occur with NGF (Hofer and Barde, 1988, Nature 331, 261262). In addition to these effects on peripheral sensory neurons, both from the neural crest as well as from the neural plexus, BDNF has been shown to maintain viability of developing CNS neurons. Johnson et al. (1986, J. Neurosci., 6, 3031-3938) have reported that BDNF maintains the viability of cultured retinal ganglion cells taken from E17 rat embryos. This is a follow-up to previous studies which found that conditioned media and brain extracts from retinal ganglion cells in the target area apparently support the viability of these neurons (Mc Caffery et al., 1982, Ex. Brain Res., 48, 37-386, Sarthy et al., 1983, J. Neurosci, 3, 25322544, Turner et al., 1983, Dev. Brain Res.).

In addition to its effects on the viability of developing neurons in culture, BDNF has been shown to affect cultured mature neurons in the peripheral and central nervous system. BDNF, like NGF, has been shown to stimulate axonal regeneration in cultured DRG neurons taken from an adult rat (Lindsay, 1988, J. Neurosci, 8, 2394-2405) while mature sensory neurons do not require neurotrophic factors to be preserved in vitro for longer periods of 3 or 4 weeks. In addition, in culturing adult rat retinal cells, BDNF has been shown to maintain not only viability but also elongate axons taken from retinal ganglion cells (Thanos et al., 1989, Eur. J. Neurosci, 1, 19-26).

A comparison of the biological effects of NGF and BDNF is presented in Table 1.

Table 1.

Biological effects of NGF and BDNF *

Viability * * BDNF NGF

<td>Peripheral I.</td><td>nerve system E6 chicken DRG</td><td></td><td> + +</td>

<td></td><td>E10 chicken DRG</td><td> +</td><td> + +</td>

<td></td><td>E12 Chicken Symp. (Barde</td><td> -</td><td> + +</td>

<td>II</td><td>et al., 1980, cf. above) E6-E12 chicken DRG</td><td> + +</td><td> + +</td>

<td></td><td>E6-E12 chicken, spine</td><td> + +</td><td> -</td>

<td></td><td>E12-sympathetic, chick</td><td> -</td><td> + +</td>

<td></td><td>E12-ciliary, chick</td><td> -</td><td> -</td>

<td>III</td><td>E3-E14, chicken: neck</td><td> + /++</td><td> + +</td>

<td></td><td>DM-tee</td><td> + /++</td><td> ++</td>

<td></td><td>beige</td><td> + / + +</td><td> -</td>

<td></td><td>of the road</td><td> + / + +</td><td> -</td>

<td></td><td>VL-tee</td><td> ++</td><td> -</td>

<td></td><td>vestibular</td><td> -</td><td> -</td>

<td></td><td>mesencephalic</td><td> ++</td><td> -</td>

<td>(Davies et al (Barde et al.,</td><td>., 1986, cf. above) , 1987, Prog. Brain Res., 71,</td><td> 185-189)</td><td></td>

<td colspan="2">Central nerve system I E17 rat retina</td><td> + +</td><td></td>

ganglion cells (Johnson et al., 1986, J.

Neurosci., 6, 3031-3038) * - Chronological order by date of publication;

effects tested in vitro ** - no viability - (-), moderate viability (-), good viability - (++).

2.2.2. Neural targets of brain neurotrophic factor

Sensory neurons in the peripheral nerve ganglia originate, as it has been found, from two transient embryonic structures distinct from one another, namely, the neural crest and the neuronal patches. The neural crest is the origin of the neurons, the autonomic ganglion satellite cells, and the sensory ganglia of the spinal nerve, ie DRGs. The contribution of the neural crest and neuronal plaques to the formation of the cranial nerve sensory ganglia has been investigated using the chimeric (quail / chick) transplant system developed by Le Donarin (1973, Develop. Biol. 20, 217-22, Noolen, 1978, Develop. Biol. 67). , 313-329, Narayanan and Narayanan, 1980, Anat. Rec., 196, 71-82, Ayer-LeZiev and Le Denarin, 1982, develop Biol., 94, 291-310, D'Amico-Martel and Soden, 1983, Am. J. Ann., 166, 445-468). As noted by Lindsay et al. (1985, J. Cell. Sci. Supp., 3, 115-129), it is now believed that the smallest birds, the distal ganglion neurons of the VII, IX, and X cranial nerves (knee, incisor, nodosum, respectively) and the cranial nerve of vestibuloacoustic complex VIII, are of the plaque nature. The trigeminal ganglion V of the cranial nerve is of the spinous and placental nature (with neurons in the placental nature dominating the ventrolateral pole in the maxillomandibular region), but satellite cells of all cranial ganglia have been found to be fully squamous in nature.

In vitro experiments using both explanted and dissociated, neuronal enriched, sensory neurons of the spinal cord and cranial neurons showed that sensory neurons originating from the neuronal ridge respond to NGF, in contrast to neurons originating from the neuronal patch (excluding the ventrolateral portion). trigeminal ganglionic neurons, and the entire population of vestibular, knee, mandibular, and nodular ganglionic neurons), essentially have no response during embryonic development of NGF. Investigating the differences and the need for NGF for them, it was found that (see Table 1.), and comb-like sensory neurons are comb-sensitive, are sensitive to BDNF, and affect their viability and neurite development (Lindsay et al., 1985, J. Cell Sci. Supp., 3, 115-129, Lindsay et al., 1985, Develop Biol., 112, 319-328, Kalcheim and Gendrean, 1988, Develop Brain Res., 41, 79-86. ).

Table 2.

List of neurons responsive and non- responsive to BDNF

A. Responsive neurons

I. Chicken sensory neurons derived from the nerve ridge,

(a) dorsal basal ganglia,

(b) in the neck ganglion,

(c) in the dorsomedial triangular ganglion,

(d) in the mesencephalic triple nucleus **.

II. Chicken sensory neurons derived from the ectodermal patch,

(a) Nodular ganglia

(b) vestibular ganglion

(c) in the incisal ganglion

(d) in the ganglia of the knee

(e) in the ventrolateral triangular ganglion.

III. Rat retinal ganglion cells

IV. Chicken retinal ganglion cells

B. Non-responsive neurons

I. Chick and rat sympathetic neurons

II. Chicken parasympathetic ciliary neurons * - Barde et al., 1987, Prog. Brain Res., 71, 185-189 * - Davies et al., 1986, Nature, 319, 497-499 *** - Rodriguez-Tebar et al.

296-303

1989, Dev. Biol., 136,

Tebar and Barde (1988, J. Neurosci., 8, 3337-3342) investigated radioactively labeled BDNF binding parameters with dorsal basal ganglion neurons of a chicken embryo; these results confirm the existence of two classes of BDNF receptors, one with high affinity and the other with low affinity for BDNF. No high affinity receptors were found in sympathetic neurons.

Barde et al. (1987, Prog. Brain Res., 185-189) further investigated known targets of BDNF neurons. Prior to the present invention, it was not possible to identify cells that synthesize BDNF because of the absence of antibodies or nucleic acids specific for BDNF. Attempts to obtain monoclonal or polyclonal antibodies were unsuccessful. This interfered with the cloning of BDNF, the determination of the physiological effect in vivo, the isolation of developing neurons from BDNF, the quantitative determination of BDNF in tissues by immunoassays, and the localization of BDNF by immunocytochemistry.

2.2.3. Cloning of a gene encoding brain neurotrophin factor oligonucleotides

The cloning of the BDNF gene was first performed and described in U.S. Patent Application Serial No. 07 / 400,591, issued August 30, 1989, which is incorporated herein by reference in its entirety. Brief description of this patent: Very small amounts of the BDNF protein were isolated from porcine brain, amino acid fragments of this protein were identified and used for synthesis. These were used in a chain polymerase reaction as a matrix derived from cells producing BDNF. The PCR products were used as probes for the cloning of complete cDNA and / or genomic BDNF genes of various animals (human, porcine, rat, mouse) and sequencing of these genes was performed. Expression of recombinant BDNFs was performed in COS cells.

contained synthetic primers (PCR) corresponding to the residue sequences with cDNA,

3. Brief Description of the Invention

The present invention is about neurotropin-3 (NT-3), a recently discovered member of the BDNF gene family. The present invention is based, in part, on the identification of BDNF and NGF nucleic acid sequence homology (U.S. Patent 07/400591, supra). In accordance with the present invention, these domains of homology can be used to identify new members of the BDNF / NGF family, such methodology being used to identify NT-3. In accordance with the present invention, genes associated with BDNF / NGF neurotrophic factors and their products can be obtained which are identified by this method.

The present invention is, in part, recombinant DNA molecules encoding NT-3. Specifically, in one embodiment of the present invention, DNA encoding NT-3 can be obtained by isolation from human DNA, or guinea pig DNA or rat DNA. The present invention provides vectors for recombinant DNA expression which contain at least a portion of the nucleic acid sequences, such as those described in FIGS. 2 (guinea pig NT-3), FIG. 7 (rat NT-3). The present invention provides vectors for recombinant DNA expression which can be used to produce recombinant NT-3 protein and peptides of this protein.

Alternatively, the present invention provides NT-3 proteins and corresponding peptides, and may also synthesize and purify these compounds according to the methods of the present invention. The present invention also encompasses antibodies against NT-3 proteins and peptides.

In accordance with the present invention, NT-3 may be used in the diagnosis and / or treatment of neurological disorders such as, but not limited to, peripheral neuropathy such as diabetic neuropathy, toxic and nutritional neuropathies, hereditary neuropathies and neuropathies associated with AIDS, and degenerative diseases. , such as Alchaimeir disease. NT-3 was found to support the viability of dopaminergic neurons; accordingly, in a most preferred embodiment of the invention, NT-3 can be used in the treatment of Parkinson's disease. Because NT-3 has been found to possess a spectrum of activities different from BDNF or NGF, NT-3 can be used to stimulate and repair central nervous system growth.

Thus, the present invention provides an amino acid sequence of neurotrophin-3 (NT3) having the following amino acid sequence:

<td>Tyr</td><td>Ala</td><td>Glu</td><td>His</td><td>Lys</td><td>Ser</td><td>His</td><td>Arg</td><td>Gly</td><td>Glu</td><td>Tyr</td><td>Ser</td><td>Or</td><td>Cys</td>

<td>Asp</td><td>Ser</td><td>Glu</td><td>Ser</td><td>Leu</td><td>Trp</td><td>Or</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Ala</td><td>lle</td>

<td>Asp</td><td>lle</td><td>Arg</td><td>Gly</td><td>His</td><td>Gln</td><td>Or</td><td>Thr</td><td>Or</td><td>Leu</td><td>Gly</td><td>Glu</td><td>lle</td><td>Lys</td>

<td>Thr</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Pro</td><td>Or</td><td>Lys</td><td>Gln</td><td>Tyr</td><td>Phe</td><td>Tyr</td><td>Glu</td><td>Thr</td><td>Arg</td>

<td>Cys</td><td>Lys</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Pro</td><td>Or</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Gly</td><td>lle</td>

<td>Asp</td><td>Asp</td><td>Lys</td><td>His</td><td>Trp</td><td>Asn</td><td>Ser</td><td>Gln</td><td>Cys</td><td>Lys</td><td>Thr</td><td>Ser</td><td>Gln</td><td>Thr</td>

<td>Tyr</td><td>Or</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Ser</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Or</td><td>Gly</td>

<td>Trp</td><td>Arg</td><td>Trp</td><td>lle</td><td>Arg</td><td colspan="3">[lle Asp Ί</td><td>'Mr.</td><td>Ser</td><td>Cys</td><td>Or</td><td>Cys</td><td>Ala</td>

<td>Leu</td><td>Ser</td><td>Arg</td><td>Lys</td><td>lle</td><td>Gly</td><td>Arg</td><td>Thr,</td><td></td><td></td><td></td><td></td><td></td><td></td>

or a functionally active protein substantially homologous to said NT-3 protein, or a subset of said NT-3 protein or a homologue thereof comprising a functionally active peptide for use in the treatment of a disease or disorder of the human or animal nervous system.

The invention also provides a pharmaceutical composition 1 comprising an amino acid sequence of a neurotrophin-3 protein:

<td>Tyr</td><td>Ala</td><td>Glu</td><td>His</td><td>Lys</td><td>Ser</td><td>His</td><td>Arg</td><td>Gly</td><td>Glu</td><td>Tyr</td><td>Ser</td><td>Or</td><td>Cys</td>

<td>Asp</td><td>Ser</td><td>Glu</td><td>Ser</td><td>Leu</td><td>Trp</td><td>Or</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Ala</td><td>lle</td>

<td>Asp</td><td>lle</td><td>Arg</td><td>Gly</td><td>His</td><td>Gln</td><td>Or</td><td>Thr</td><td>Or</td><td>Leu</td><td>Gly</td><td>Glu</td><td>lle</td><td>Lys</td>

<td>Thr</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Pro</td><td>Or</td><td>Lys</td><td>Gln</td><td>Tyr</td><td>Phe</td><td>Tyr</td><td>Glu</td><td>Thr</td><td>Arg</td>

<td>Cys</td><td>Lys</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Pro</td><td>Or</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Gly</td><td>lle</td>

<td>Asp</td><td>Asp</td><td>Lys</td><td>His</td><td>Trp</td><td>Asn</td><td>Ser</td><td>Gln</td><td>Cys</td><td>Lys</td><td>Thr</td><td>Ser</td><td>Gln</td><td>Thr</td>

<td>Tyr</td><td>Or</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Ser</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Or</td><td>Gly</td>

<td>Trp</td><td>Arg</td><td>Trp</td><td>lle</td><td>Arg</td><td>lle</td><td>Asp</td><td>Thr</td><td>Ser</td><td>Cys</td><td>Or</td><td>Cys</td><td>Ala</td><td>Leu</td>

<td>Ser</td><td>Arg</td><td>Lys</td><td>lle</td><td>Gly</td><td>Arg</td><td>Thr,</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

a therapeutically effective amount of either a functionally active protein substantially homologous to said NT-3 protein or a subset of said NT-3 protein or a homolog thereof comprising a functionally active peptide, and a second cytokine agent, preferably NGF or BDNF, together with a pharmaceutically acceptable carrier. .

The invention also provides an antibody, antibody fragment or derivative thereof which recognizes a neurotrophin-3 protein (NT-3) having the amino acid sequence shown above, or a fragment of this peptide, or a derivative thereof.

The invention also provides a method of producing an antibody as described above comprising the steps of:

(a) immunizing an animal (non-human) with a neurotrophin-3 or peptide comprising a neurotrophin-3 antigenic determinant to produce an antibody reactive with neurotrophin-3;

(b) isolating the antibody produced in step (a) from the animal.

The invention also provides an in vitro method of diagnosing a nervous system disorder or disorder, comprising:

(a) contacting the tissue with a labeled detectable tag of an antibody molecule that can bind to a neurotrophin-3 protein having the sequence set forth above, or a fragment or derivative of that peptide, under conditions where such binding is possible; and (b) determining whether such a merger has occurred.

The invention also provides a labeled detectable labeled antibody molecule that is capable of binding to a neurotrophin-3 protein having the above amino acids, fragments of this peptide, or derivative thereof, for use in diagnosing an in vivo disease or disorder of the nervous system.

The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a neurotrophin-3 protein or a subset thereof comprising a functionally active peptide, wherein said neurotrophin-3 protein has the following amino acid sequence:

<td>Tyr</td><td>Ala</td><td>Glu</td><td>His</td><td>Lys</td><td>Ser</td><td>His</td><td>Arg</td><td>Gly</td><td>GLu</td><td>Tyr</td><td>Ser</td><td>Or</td><td>Cys</td>

<td>Asp</td><td>Ser</td><td>Glu</td><td>Ser</td><td>Leu</td><td>Trp</td><td>Or</td><td>Thr</td><td>Asp</td><td>Lys</td><td>Ser</td><td>Ser</td><td>Ala</td><td>Ile</td>

<td>Asp</td><td>Ile</td><td>Arg</td><td>Gly</td><td>His</td><td>Gln</td><td>Or</td><td>Thr</td><td>Or</td><td>Leu</td><td>Gly</td><td>Glu</td><td>Ile</td><td>Lys</td>

<td>Thr</td><td>Gly</td><td>Asn</td><td>Ser</td><td>Pro</td><td>Or</td><td>Lys</td><td>Gln</td><td>Tyr</td><td>Phe</td><td>Tyr</td><td>Glu</td><td>Thr</td><td>Arg</td>

<td>Cys</td><td>Lys</td><td>Glu</td><td>Ala</td><td>Arg</td><td>Pro</td><td>Or</td><td>Lys</td><td>Asn</td><td>Gly</td><td>Cys</td><td>Arg</td><td>Gly</td><td>Ile</td>

<td>Asp</td><td>Asp</td><td>Lys</td><td>His</td><td>Trp</td><td>Asn</td><td>Ser</td><td>Gln</td><td>Cys</td><td>Lys</td><td>Thr</td><td>Ser</td><td>Gln</td><td>Thr</td>

<td>Tyr</td><td>Or</td><td>Arg</td><td>Ala</td><td>Leu</td><td>Thr</td><td>Ser</td><td>Glu</td><td>Asn</td><td>Asn</td><td>Lys</td><td>Leu</td><td>Or</td><td>Gly</td>

<td>Trp</td><td>Arg</td><td>Trp</td><td>Ile</td><td>Arg</td><td>Ile</td><td>Asp</td><td>Thr</td><td>Ser</td><td>Cys</td><td>Or</td><td>Cys</td><td>Ala</td><td>Leu</td>

<td>Ser</td><td>Arg</td><td>Lys</td><td>Ile</td><td>Gly</td><td>Arg</td><td>Thr,</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

or a functionally active protein substantially homologous to said NT-3 protein, or a subset of said NT-3 protein, or a homolog thereof comprising a functionally active peptide, together with a pharmaceutically acceptable carrier adapted for intradermal, intraruminal, intraperitoneal, intravenous, subcutaneous, intraventricular or for intrathecal administration, or delivery through an implanted device or catheter.

4. Description of figures

FIG. 1. Comparison of nucleic acid sequences of BDNF and NGF in different animals. Sequence analysis and amino acid residue analysis of the gene encoding BDNF revealed that this protein has many structural similarities to NGF. According to the primary sequence of mature BDNF, as well as the overall structure and similar processing, it can be reliably stated that NGF and BDNF genes can be derived from a single common primary gene. Inside the mature polypeptides, if only three spaces are introduced in the NGF sequence for optimization of identity, then the 51 amino acid residue is the same in all of the various NGFs known to date and porcine, human BDNF. These homologous domains contain all 6 cysteine ​​residues, judging that NGF and BDNF have similar secondary structure. In addition, four segments of six or more amino acid residues are also seen in this drawing, in which all types of NGF and porcine BDNF are identical or differ by no more than one conservative amino acid residue. As such, NGF and BDNF can be said to be close members of the gene family.

FIG. 2. Sequence of the guinea pig NT-3 gene and amino acid residues. The amino acid sequence starts with the first ATG codon, which is after the 3 'frame stop codons. The underlined sequences indicate the location of the PCR primers used. The single consensus sequence for N-glycosylation is underlined twice and the arrow indicates the putative start of transcription of mature NT-3.

FIG. 3. Comparison of amino acid residue sequences of mature mice NT-3, NGF (Scot et al., 1983, Nature, 302, 538-540) and BDNF (Leibrock et al., 1989, Nature, 341, 149-152). The mature mouse BDNF sequence presented here is 100% identical to the porcine BDNF sequence. The arrows in bold and shaded amino acid residues, identical to all three proteins, indicate cysteine ​​residues. Asterisks indicate spaces made for structure optimization. V1-V4 indicate variable domains containing more than three close amino acid residues.

FIG. 4. Distribution of NT-3 iRNA in mouse tissues. Twenty micrograms of total RNA was added to each lane and hybridized with labeled 32P double-stranded DNA. (A) A single band of approximately 1.4 kb (thousands of bases) is visible in all tissues, with the weakest signal in the lungs and the strongest in the heart. The skeletal ra-umuo was taken from the thigh. (B) In brain studies, the strongest signal was obtained in the Ammonium rage and the cerebellum.

FIG. 5. Viability of sensory neurons isolated from nodular ganglia derived from an 8-day-old chicken embryo. 5,000 cells were plated on a polyiornithine-laminin substrate, and live neurons were counted after 24 hours. The BDNF concentration used was twice the minimum concentration required for maximum viability. No living neurons were left untreated with BDNF, either in conditioned medium used at a 1:50 dilution with untransfected cells or cells transfected with control DNA.

FIG. 6. (A) PCR product obtained using IB and 2C primers (designated R1B / 2C) which can identify a novel gene, NT-3, as well as NGF and BDNF genes in rat genomic DNA. (B) Restriction map of rat NT-3 genomic clone. Two independent bacteriophage clones that specifically hybridized with R1B / 2C probes were isolated from the genomic rat library. A restriction map of a single clone with a 19.5 kb insert is presented schematically. The bold line represents the NT-3 open reading frame (ORF) (see Fig. 7A). The R1B / 2C location is shown.

FIG. 7. Rat NT-3 sequence and its homology to rat NGF and rat BDNF. (A) Nucleotide and amino acid residue sequence of NT-3. The ORF comprising the DNA sequence encoding the NT-3 gene with translation into the amino acid residue sequence over the DNA sequence has an asterisk marked the beginning and end of the open reading frame. The amino acid residues are numbered from the +1 position of LT 4063 B, the first amino acid residue in the mature NT-3 protein (119 amino acid residues). The cleavage region of the mature NT-3 protein is framed, as well as the glycosylation region that precedes this cleavage region. Another potential cleavage domain, similarly located to the intermediate processing domain of NGF (Darling et al., 1987, Cold Spring Harb. Spring Quant Bio, 1, 427-34), but which is not passivated by the BDNF protein, is defined by the frame and? Cleave. Six cysteine ​​residues in the mature NT-3 protein are underlined. The methionine codon (position-139) of probaltyme NT-3 transcription initiation is the start region B is also underlined. This figure shows the site acceptor boundary (the intron opposite the start codon B). B) Comparative analysis of rat NT-3 with rat NGF and rat BDNF. MacVektor sequencing software (purchased from International Biotechnologies Ine.) Was used to analyze rat NT-3 ORF with NGF and BDNF gene ORF (using a window size of 20 with a maximum overlap of 20%). Significant overlaps are seen in the diagonal of this matrix, a schematic representation of the product NT-3 protein is shown below; the two domains of homology are upstream of the mature NT-3 protein and are seen in alignment with NGF and BDNF, labeled I and II. As shown in this drawing, Area I precedes B Starting Area, from which a short test probe of the NT-3 is synthesized, confirming that a longer probation is also synthesized. C) Estimation of NT-3, NGF, and BDNF homology in domains I and II. The sequences in these areas are arranged so as to have maximum homology, with fractures in them. The areas of BDNF or NGF overlapping NT-3 indicated stars, overlaps between NGF and BDNF are indicated by dots in the NGF sequence. + top sequences indicate residues that are fully conserved between rat NT-3, NGF and BDNF sequences and other animals tested. Shown in the NGF sequence identified previously in the BDNF sequence, provided herein in the NT-3 sequence, are the following links: signal sequence cleavage region (Edwards et al., 1988, Mol. Cell Biol. 8: 2456-64); an intermediate NGF cleavage region not present in the BDNF sequence but in the NT-3 sequence; glycosylation domain; a site of proteinolysis that elicits mature factors. D) Comparison of mature NT-3, NGF, and BDNF protein sequences. The conservative cysteine ​​residues are marked with a thick square. Characters:

and - have the same meanings as C). The cleavage link at the C end, which is only in the NGF sequence, is also indicated.

comparison day) basal ganglia

FIG. 8. Cultures of NGF, BDNF, and NT-3 in explanted chicken embryo (8 ganglia. Microphotography of dorsal ganglia (DRG) (photo AD), nodose (photo EH), and ganglion sympathetic chain (SG) (photo IL)) were performed on 24 hours (DRG and NG) or 48 hours (SG) without any neurotrophic factor (Controls: A, E, I) and in the presence of COS cell supernatant containing NGF (B, F, J) or BDNF (C, G, K) or NT-3 (D, H, L). Almost no evidence of neurite stunting in control cultures (500 μΐ of cell supernatant from mocL-transfected cells). NGF (10? 1 COS cell supernatant) induced a strong regrowth of DRG and SG, but not NG. Increasing the amount of NGF COS cell supernatant from 20 to 500 μΐ showed no effect on NG. BDNF (10 μΐ COS cell supernatant) induced sprout regrowth in DRG and NG but not SG, the increase (20 to 500 μΐ) had no effect on SG. NT-3 (20 μΐ COS cell supernatant) DRG and NG, and 200 μΐ SG) induced sprout regrowth in all three types of ganglia, although sprouting initiation was slower and weaker than SG. Ganglia were cultured as explants on a halogen gel (Lindsay RM and Rohrer M., 1985 Dev. Biol., 112, 30-48) in F14 medium plus

5% horse serum as described (Lindsay et al., 1985,

Dev. Biol., 112, 319-28). Scale size is 200 pm.

FIG. 9. NT-3 maintains viability and stimulates neurite outgrowth in high concentration DRG neuronal cultures. Photographs of dissociated DRG cultures of chicken embryo (8 days) enriched in neurons (more than 95% neurons) exposed for 48 hours to either (A) mocktransfected COS cell supernatants (500 μΐ) or (B, C) NT-3 transfected cell supernatants (50 μΐ). A and B - dark field photography; A (control culture) survived less than 5% of neurons per plate; B - the amount of neurons remaining after this process was about 60% of the amount of neurons added to the plate. From the magnitude of the response from the volume curve, it was found that the maximal effect of NT-3 was achieved using chick E8 DRG neurons. (C) Contrast microphotography of the same culture as (B) but at higher magnification. Note the large number of bright neuronal cells and that there are no other non-neuronal cells. Cultures were obtained as described (Lindsay et al., 1985, Dev. Biol. 112, 319-28). Scale size 150 pm. (C)

107, 303-14) .

sources were

FIG. 10. Comparison of NT-3, NGF and BDNF expression in rodent tissues as assessed by Northern blotting. RNA was isolated from the indicated rat tissues (left photograph) or mouse tissues (right photograph) (Auffray C. and Rrougeon T. 1980, Eur. J. Biochem.) Ten micrograms of RNA from the indicated were fractionated on a 1% agarose gel and transferred to nylon. membrane 10x SSC; triple blotting and hybridization were performed (Mahmond M. and Lin V. H., 1989, Biotechniques, 7, 331-3) at 68 ° C with 32P-labeled (Fleingerg AP and Vogelstein, 1984, Ana. Biochem., 137, 266-7) DNA fragments of rat NT-3, rat BDNF and rat NGF. followed by washing at 68 ° C with 2x SCC, 0.1% SDS. NT-3, BDNF, and NGF DNA fragments were obtained from expression systems that contained the genes for these proteins in pCDM18, Xhol inserts, about 775 bp in size separated on the gel prior to radiolabeling. Also in this drawing. embedded photo of a gel enhanced with ethidium bromide allows the total DNA content of the samples to be estimated.

FIG. 11. Comparison of human and rat NT-3 gene DNA sequences. The predicted broadcast start link is labeled MATURE. The mature human and rat NT-3 proteins have identical amino acid residue sequences, whereas the prepro domains differ at 11 positions, underlined.

FIG. 12. Expression of human NT-3 using metabolic tagging. After 5x10⁵ COS-M5 cells were plated in 60-mm Petri dishes and grown overnight in 37 ° C complete DMEM with 10% fetal bovine serum (FBS). These cells were transfected (using the calcium phosphate method described by Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-52) with 20 µg of the plasmid pC8-hN3 (PI), which contains the human NT-3 gene with cytomegaly. virus-regulated promoter, or mocktransfected (without plasmid DNA). After 48 hours, cells were rinsed and incubated for 1 hour in 1 ml DMEM medium without methionine and cysteine ​​plus 1% bovine serum. each culture was supplemented with a mixture of 35Smethionine and 35S-cysteine ​​(100 CCi each, New England Nuclear), and cells were then incubated for 4 hours at 37 ° C in this medium. After 50 μΐ samples were mixed with 25 μΐ double concentration sample buffer containing sodium dodecyl sulfate (SDS), boiled for 5 minutes and subjected to electrophoresis under 15% polyacrylamide gel under denaturing conditions (SDS PAGE) Laemmli, 1970, Nature, 227, 680-685 ). These proteins from polyacrylamide gel after electrophoresis were transferred (3 hours, 100 mA) onto a nylon * membrane (Immobilon, Millipore) using buffer solutions as described by Tavbin et al., 1979, Proc. Natl. Acad. Sci. USA, 76, 4350-4354. The membranes were air-dried, and the labeled proteins were detected by autoradiography (16 hours, at room temperature, using Kodak X-AR photographic film and Cronex, Du Pont amplification screen).

FIG. 13. Diagram showing the remaining live tyrosine hydroxylase cells without NT-3 supernatant (0) and with NT-3 supernatant at dilutions of 1: 300, 1: 100, 1:50 and 1:25.

FIG. 14. A diagram analogous to that described in FIG. 13, except that the cell concentration was 9,000,000 per plate.

FIG. 15-A. Comparison of NT-3, BDNF, and NGF synthetic transcripts. Dothybridization using radioactive oligonucleotides homologous to reverse sequence at the 5 terminus, all three transcripts were controlled for equal concentrations (2ng) of synthetic NT-3, BDNF, and NGF transcripts by spectrophotometric evaluation.

FIG. 15-B. Estimation of NT-3, BDNF, and NGF iRNA levels in total RNA isolated from mature rat brain relative to RNA standards. 10μ total RNAs isolated from mature rat brain and 4, 10 and 20 pg synthetic transcripts corresponding to each neurotrophin were blotted with radioactive probes specific for each neurotrophin.

FIG. 16. NT-3, BDNF and NGF gene expression and total RNA (10 µg / lane) isolated from rat.embryonic (A), developing rat brain (B), and perinatal and adult animal tissues (C). Tissues: A - BR: Adult animal brain, standardized, Figs. IB; Plac: placenta, EMB: whole embryo, SPC: spinal cord; THY: thyme, LTV: liver; HRT: Heart; BR: The brain. The size of the transcripts is indicated in thousands of bases (kb) to the right.

FIG. 17. NT-3, BDNF and NGF gene expression and total RNA (10 µg per lane) isolated from neonatal discrete nervous system regions (A) and adult animals.

Areas: BR: Adult animal brain, standardized as Figs. IB; CBL: Cerebellum; HBR: posterior brain; MBR: midbrain; DAY: Intermediate Brain; STR: stralum horn; HIP: ammon horn CTX: neocortex; ADR: adrenal glands; RET: retina; SCN: Sciatic nerve; SPC: Spinal cord.

FIG. 18. Quantification of NT-3, BDNF, and NGF transcripts in CNS and peripheral tissues, neonates, and adults. The resulting blotting products, including those obtained in Figs. 16,17,19 and densitometric scanning of Mesohpierre (Maisorpierre et al., 1990, Science 247, 1446-1451). All levels were normalized to neurotrophin levels in the adult brain; in the adult brain, the same for all neurotrophins (see. text) and accept for one unit for each neurotrophin. Values ​​greater than the scale are plotted above the fractured histograms. Neural and non-neural samples are marked in the drawings. Samples: BRN cerebellum, no cerebellum; CBL: Cerebellum; HBR: posterior brain; MBR: midbrain; DAY: Intermediate Brain; STR; stralum; HIP: Ammon Horn; CTX: neocortex; OLF: Smokehouse; SPC; spinal cord; SCN sciatic nerve; RET; the retina; ADR: adrenal glands; HRT: Heart LTV: Liver; THY: thyme, SKN: leather; MUS: skeletal muscle; LNG: lungs, INT: intestine; KID: kidney; SPL: Spleen.

FIG. 19. Gene expression of NT-3, BDNF, NGF and NGFR during development of spinal cord (A, E), cerebellum (B, F) and ammonia horn (C, G). 10? g total RNA isolated from different indicated developmental times was compared with the expression of various transcripts. Quantitative densitometry data for neurotrophic transcripts are presented in E, F, G.

5. Detailed Description of the Invention.

The present invention relates to neurotrophin-3, a novel neurotrophic compound of the BDNF / NGF family, and to other members of the BDNF / NGF family that can be identified by analogous methods. For a more detailed description, but not a limitation, the detailed description of the invention is divided into the following parts:

1. identification of additional BDNF / NGF family members,

2. cloning of neurotrophin-3,

3. expression of neurotrophin-3,

4. biological activity test,

5. neurotrophin-3 genes and proteins,

6th obtaining antibodies against neurotrophin-3; and

7th application of the invention.

5.1. Identification of additional BDNF / NGF family members

NGF and BDNF are alkaline proteins that are composed of

120 amino acid residues with 50% homology, including 6 absolutely conservative cysteine ​​residues that form three disulfide bridges in active NGF, as shown in Bradshaw A., 1978, Ann. Rev. Biochem., 191-216; Leibrock et al., 1989, Nature, 341, 149-52. Comparison of evolutionarily divergent NGF sequences has shown that amino acid residues flanking cysteine ​​residues form the most conservative regions of the molecule (Meier et al., 1986, EMBO, 5, 1489-93; Selby et al., 1987, J. Neurosci. Res. , 18, 293-8). Surprisingly, these domains are most similar between BDNF and NGF (Leibrock et al., 1989, Nature, 341, 149-52).

Rational search for additional BDNF / NGF family members can be performed by conservative segments in homologous NGF and BDNF sequences. For example, additional members of the BDNF / NGF family may be identified by selection, by selecting from nucleic acid sequences those sequences that are homologous to BDNF and NGF, and further identifying selected sequences that are non-homologous to BDNF and NGF. The term non-homologous may be used for regions having at least six nucleotides in which at least two nucleotides are different from NGF and BDNF sequences.

The present invention is recombinant DNA which is homologous to BDNF and NT-3, or alternatively NGF and NT-3, but which contains domains, respectively, non-homologous to BDNF and NT-3, or NGF and NT-3. These additional members of the BDNF / NGF gene family have been identified using probes that are homologous to regions. Further analysis revealed that the family sequences of these BDNF / NGF / NT-3 genes differ from known BDNF / NGF / NT-3 gene member sequences. For example, the most favorable implementation is performed as follows. The four conservative segments may be consistent were members of the family of this invention

For each of the (frames) specified in the table [see. below), oligonucleotides of 10-20 nucleotides in size can be synthesized that encode all possible amino acid residue sequences found in NGF or BDNF with three or seven contiguous codons. Numbered from the N terminus of the mature polypeptides (such that the His (134) protein in the preproBDNF becomes His (l) in the mature protein), the following four frames can be characterized as follows (amino acid residue numbering in mature human proteins):

<td>Table 3.</td><td></td><td></td><td></td>

<td>Frame 1:</td><td>NGF</td><td>Gly (10)</td><td>- Ser (19)</td>

<td></td><td>BDNF</td><td>Gly (8) -</td><td>Ser (17)</td>

<td>Frame 2:</td><td>NGF</td><td>Lys (50)</td><td>- Cys (58)</td>

<td></td><td>BDNF</td><td>Lys (50)</td><td>- Cys (58)</td>

<td>Frame 3:</td><td>NGF</td><td>Gly (67)</td><td>- Asp (72)</td>

<td></td><td>BDNF</td><td>Gly (67)</td><td>- Asp (72)</td>

<td>Frame 4: NGF</td><td>NGF</td><td>Trp (99)</td><td>- Cys (110)</td>

<td></td><td>BDNF</td><td>Trp (100)</td><td>- Cys (111)</td>

<td>Oligonucleotides,</td><td>to synthesize</td><td colspan="2">by pairs of frame sequences</td>

<td>(Table 3),</td><td>can be</td><td>used ami</td><td>primers</td>

for amplification by PCR, a template using RNA or DNA isolated from sources of interest. This source may contain iRNA or cDNA or genomic DNA for the expression of any eukaryotes capable of expressing a polypeptide related to BDNF or NGF. Only six PCRs (Frame 1 and 2 primer; Frame 1 and 3; Frame 1 and 4; Frame 2 and 3; Frame 2 and 4; Frame 3 and 4) can find a gene or gene product containing two of the four conservative sequences of NGF and BDNF indicated segments. By synthesizing several different primers for each frame, you can perform a full search with less PCR. It is also possible to vary the stringency of hybridization during PCR to vary nucleotide sequences relative to an unknown NGF or BDNF gene. If the resulting segment is a previously unknown segment of the BDNF / NGF gene family, then the segment can be cloned and sequenced and used as a probe to isolate a complete cDNA or genomic clone. The sequence of the unknown gene can then be determined, its expression analyzed, and the gene product used to form a functional assay.

modeling (Hopp Sci. USA, 78, recombinant

In addition, the present invention makes it possible to construct novel recombinant molecules which are BDNF / NGF gene families but are not naturally occurring using the BDNF / NGF homologous sequences. For example, but not by way of limitation, the present invention may construct a recombinant molecule by combining parts of the NGF and BDNF genes. Such a molecule may contain both NGF and BDNF and exhibit novel biological activities including agonistic and antagonistic activities. Primary BDNF and NGF sequences can also be used to predict the tertiary structure of molecules using Woods, 1981, Proc.

3824-28); BDNF / NGF genes can computer Natl. Acad. chimeric genes may be constructed from the tertiary structure and correlation of biological activity, and chimeric genes comprising portions of any one or more members of the BDNF / NGF family may be constructed.

5.2 Cloning of neurotrophin-3

From any organism, the NT-3 gene can be identified using regions of homology between BDNF and NGF using the methods described above. In the two most preferred, specific embodiments of the present invention, the gene can be identified and cloned as follows:

the rest at 36.

In the first case, a single sense (or 5 ') primer with the sequence (using IUPAC nomenclature) is - GGGGATCCGC GG1 TGU MG1 GG1 ATH GA, (primer 1 containing BamHI and SacII restriction enzyme cleavage sites) and anti-sense (or 3') primer which has the nucleotide sequence TCGAATTCTAG AT 1CK 1AT RAA 1CK CCA (primer 2 containing EcoRI and XbaI cleavage sites) can be used in PCR (Saiki et al., 1985, Science 230, 1350-1354) and is performed using commercial thermal cycler (for example, Perkin-Elmer Cetus) and a thermostable DNA polymerase from Thermus aguaticus (Taq polymerase). After approximately four cycles of renaturation at 45 ° C, the cycles can be performed at 49 ° C. The resulting DNA amplification products are purified by electrophoresis on a polyacrylamide gel, and the product of the expected size (about 137 base pairs) is eluted from the gel and then double digested. one cleaving the NGF gene inside the amplified segment of the organism used to amplify the segment and the other cleaving the BDNF gene. Uncleaved DNA (which is presumed to encode neither NGF nor BDNF) can be isolated by DNA electrophoresis on a polyacrylamide gel, eluted and asymmetrically amplified (Innis et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 94369440). ) and sequenced (Sanger et al., 1979, Proc. Natl. Acad. Sci. USA, 72, 3918-3921) using, for example, primers 1 and 2.

Additional sense and anti-sense primers may be constructed from the sequence thus obtained to more accurately represent the sequence of the novel gene. For example, additional sense primers such as GGGATTGATGACAAA (primer 3), and ACT CTC AGT GCA AAA CTT CGC (primer 4), and antisense (5 '), the primer CGG ATC CGA ATT CTG CAG (T) 12V (primer), can be used. 5). RNA isolated from tissues capable of producing neurotrophin activity, such as the brain (using any standard method such as described in Okayama 1987, Meth. Enzymol. 154, 3-28), can be transcribed in reverse order, such as primer 5, which is coincident with the 3 'poly (A) terminus and utilized, constructed with cleavage sites for BamHI, EcoRI and Pstl (Leibrock et al., 1989, Nature, 341, 149-152). The resulting cDNA can be amplified by PCR using primers 4 and 5. Southern blotting can then be performed using the final reaction products and hybridized with the oligonucleotide sequence labeled 32P, which coincides with the downstream sequence of primers 3 and 4. The DNA fragments thus identified can be cloned into a suitable vector and the resulting longer insert can be used for screening the genomic library of the test organism. Positive clones thus identified can be analyzed by standard restriction analysis and nucleotide sequencing.

In another most preferred embodiment of the present invention, oligonucleotides corresponding to four protein segments which are conservative between NGF and BDNF can be synthesized. For example, the following amino acid residue sequences (presented in Figure 7D) may be:

1. Gly-Gly- (Tyr / Phe) -Ser-Val-Cys-Asp-Ser,

2. Lys-Gly-Tyr-Phe- (Tyr / Phe) -Glu-Thr-Lys-Cys,

3. Glu-Cys-Arg-Ile-Asp ,. Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr- (Ser / Ala) -Cys-ValCys.

PG reactions can use sense and anti-sense oligonucleotides (15-26 nucleotides in length, corresponding to 5-9 amino acid residues of the above sequences, as well as the anti-sense end

Amplification of sense and direction as well as non-degenerate restriction recognition. areas).

reactions between the pairs above the anti-spasmodic primers may be performed under standard conditions or preferably under optimal conditions for each pair of primers selected experimentally. For example, a sense primer (corresponding to 1 amino acid residue sequence, see above) and a 5 '-CAC TCG ACT CGA CAT CC-GTNTGY-GAY-WST-RTN-WS-3' and anti-sense primer (corresponding to the 2 amino acid residue sequence, see FIG. above) 5′-CCA AGC TTC TAG AAT TC-CA-YTT-NGT-YTC-RWARAA-RTA-YTC-3 ′ can be used in an amplification reaction using template genomic DNA or cDNA from a suitable source. Amplification reaction products obtained with primer pairs upstream sense and downstream antisense probes can be used in Southern blotting probes to hybridize with genomic DNA to identify a PCR product that hybridizes to sequence containing genomic DNA, as well as regions. such as non-NGF and non-BDNF sequences. A PCR product that identifies a novel genomic DNA sequence can be used to screen for genomic library DNA or cDNA as well as for the selection of clones encoding new members of the NGF / BDNF family.

5.3. Expression of neurotrophin-3

The nucleotide sequence encoding the NT-3 protein, or a portion thereof, may be inserted into an appropriate expression vector, i.e., a vector containing the necessary transcriptional and translational elements required for expression of the inserted sequence. The required transcriptional and translational elements may be taken from the native NT-3 gene and / or its flanking regions. various host-vector systems can be used to express protein coding sequences. These systems may include, but are not limited to, systems of mammalian cells infected with viruses (such as cow pox, adenovirus, etc.); insect cell systems infected with viruses (such as baculovirus); microorganisms: yeasts with yeast vectors, or bacteria transformed with DNA bacteriophages, plasmid DNA or cosmid DNA. In a most preferred embodiment of the present invention, the expression vector may comprise a CMV promoter (Stephens and Cocket, 1989, Nucl. Acids Res., 17, 7110) and SV40 replication source. The expression elements of these vectors differ in strength and specificity. Depending on the vector-host system used, any suitable transcription and translation elements may be used.

which contains controls

Any of the DNA fragment insertion methods described above can be used to construct expression vectors containing the chimeric gene, the appropriate transcription / translation elements, and the sequence encoding the protein. These methods may include in vitro and in vivo recombination (genetic recombination). Expression of the nucleic acid sequence encoding the NT-3 protein or peptide fragment may be controlled by another nucleotide sequence such that the NT-3 protein or peptide is expressed in a host transformed with recombinant DNA. For example, expression of NT-3 can be controlled by a known element promoter / exhanser. Promoters that may be used to control expression of NT-3 protein include, but are not limited to: the SV40 promoter region (Bernoist and Chambon, 1981, Nature, 290, 304-310), a promoter located in the long 3 'terminal repeat of the Sarcoma virus Rous ( Tamamoto et al.,

1980, Cell, 22, 787-797), herpesvirus thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.

Sci. USA, 78, 1444-1445), regulatory sequences of the metallothionine gene (Birnster et al., 1982, Nature,

296, 39-42), prokaryotic expression vectors such as the β-lactamase promoter (Willa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA, 75, 3727-3731), or the tac promoter (DeBar). et al., 1983. Proc. Natl. Acad. Sci. USA, 80, 21-25), see also Useful proteins from recombinant bacteria - Scientific American, 242, 74-94 (1980); plant-derived expressions include nopaline synthetase domain et al., Nature, 303, 209-213) or cauliflower mosaic virus (Gardner et al., 1981, Nucl. Acids Res., 9, 2871), and the ribulose-diphosphate carboxylase promoter of the photosynthetic enzyme (Herrera et al., Nature, 310, 115-120), promoter elements from yeast or other fungi such as: the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerol kinase) promoter, the alkaline phosphatase promoter, and animal transcriptional control regions that are characterized by vectors containing (Herrera-Eqtrella and the promoter 35S RNA specificity in transgenic animals: a gene that is active and the control domain of elastase I in pancreatic acinar et al., 1985, 1987, Mol.

cells (Swiff et al., 1984, Cell, 38, 639-646, Ornitz et al., 1986, Cold Spring Harbor Symp. Quant Biol. 50, 399-409; MacDonanld, 1987, Hepatology, 7, 425-515). ), an insulin gene regulatory domain active in pancreatic β-cells (Hanalan, 1985, Nature, 315, 115-122), an immunoglobulin gene regulatory domain active in lymphoid cells (Grosschedl et al., 1984, Cel, 38, 647-658, Adanus and

Nature, 318, 533-538, Alexander et al.

Cell Biol., 7, 1436-1444), a murine mammary tumor virus control domain active in testis, breast, lymphoid, and germ cells (Zeder et al., 1986, Cell 45, 485-495), albumin gene management domain, active in the liver (Pinkert et al., 1987, Genes and Devel., 1, 268-276), the control domain of the αΐ-fetoprotein gene, active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol., 5, 1639-1648, Hammer et al., 19987, Hammer, 235, 235, 53-58), an area of ​​control of the αΐ-antitrypsin gene active in the liver (Kelsey et al., 1987, Genes and Devel., 1, 161-171), a domain of β-globin gene management active in myeloid cells (Mogram et al., 1985, Nature, 315, 228-340 ;; Kollias et al., 1986, Cell, 46, 89-94), an alkaline protein-myelin-gene control region active in cerebral oligodendritic cells (Readhead et al., 1987, Cell, 48, 703-712), myosin light chains. -2 gene control domain active in skeletal muscle (Sani, 1985, Nature, 314, 283-286) and gonadotropic hormone gene control domain active

<td>in the hypothalamus 1378).</td><td>(Mason and</td><td>et al., 1986,</td><td>Science,</td><td> 234,</td><td> 1372-</td>

<td>Expression</td><td>vectors,</td><td>in which</td><td>is NT-3</td><td>gene,</td><td>can</td>

<td colspan="2">to be identified</td><td colspan="2">in three commons</td><td>Ways:</td><td>a)</td>

DNA-to-DNA hybridization, (b) presence or absence of marker gene function, and (c) expression of inserted sequences. A foreign gene inserted in an expression vector in the first way can be detected by DNA-DNA hybridization using sequences that are homologous to the inserted NT-3 gene. In the second case, the recombinant vector / host system can be identified and selected by the function of the marker gene (e.g., thymidine kinase activity, antibiotic resistance, transformation phenotype, baculovirus insertion, etc.). For example, if an NT-3 gene is inserted inside a marker sequence, recombinants containing the NT-3 gene may be identified by the absence of marker gene function. In the third case, recombinant expression vectors can be identified by assaying the product of the inserted foreign gene. Such studies may be based, for example, on the physical or functional properties of the NT-3 gene product in bio-experimental systems.

Once a particular recombinant DNA molecule has been identified and isolated, several known methods can be used to amplify it. Recombinant expression vectors can be amplified once the culture conditions and the appropriate recipient are established. As discussed above, expression vectors or derivatives thereof may be used, but are not limited to: human and animal viruses such as cow pox or adenovirus, insect viruses such as baculovirus, yeast vector, bacterophage vector (e.g., lambda), and plasmid plasmids. and cosmid vectors, and so forth.

In addition, the recipient strains may be selected to modulate the expression of the inserted sequences, or to modulate and produce a gene product in a desired specific manner. The use of certain promoters can increase expression by inducers; in this way, expression of the engineered NT-3 protein can be controlled. In addition, various recipients exhibit characteristic and specific translation and posttranslational processing and modification mechanisms (e.g., glycosylation, cleavage of proteins). Cell lines or system recipients may be selected such that desired modifications of the expressed protein and desired processing thereof are obtained. For example, expression in bacteria can be used to obtain a non-glycosylated product. By pressing on the yeast, a glycosylated product can be obtained. Expression in mammalian cells provides a native glycosylated heterologous NT-3 protein. In addition, reactions such as various proteinolysis can be influenced by the vector / host of different systems.

In a specific embodiment of the present invention, the DNA encoding prepro-NT-3 can be cloned into the plasmid pCMV, amplified and then used for transfection of COS cells by the calcium phosphate method (Chen and Okayama, 1987, Mol. Cell Biol. 7, 2745-2752). , and the NT-3 protein is isolated from the culture fluid (see Examples 6 and 7 below).

It has been concluded that MGF is synthesized as two different forms of probaltim, long and short (Darling et al., 1983, Cold Spring Harbor Symp. Quant. Biol. 48, 427-483). The short form is analogous to the prepro BDNF and NT-3 forms. Expression systems containing DNA encoding an analogous long form of BDNF or NT-3 may be useful in the present invention. The DNA encoding these long forms of probalt can be identified by sequencing the regions of the cDNA genomic DNA upstream of those encoding mature BDNF or NT-3 and by identifying open reading frames. Comparison of the NT-3 gene sequence with the NGF and BDNF sequences predicted the existence of long and short NT-3 protein probes (Fig. (Fig.7B). 7B). The expression efficiency of mature BDNF or NT-3 proteins from the long or short form of their probaltation may depend on the expression system used and may vary in different cell lines.

5.3.1. Identification and purification of the gene expression product

Once the recombinant that expresses the NT-3 gene is identified, the gene product can be analyzed. The analysis is based on the physical and activity characteristics of the product, with radioactive isotope labeling followed by electrophoresis.

Once the NT-3 protein is identified, it can be purified by standard methods: chromatography (e.g., ion exchange, affinity, gel chromatography), centrifugation, fractional precipitation, or another standard protein purification method. Product activity can be determined using, but not limited to, appropriate assays for dorsal basal neurons of the chicken embryo, sympathetic ganglia, or ganglion derived from the neural patch.

5.4. A study of the biological activity of neurotrophin-3

Any system capable of detecting NT-3 activity can be used according to the present invention. NGF,

BDNF Affects Quantitatively or Qualitatively Because NT-3, unlike neurites in both ganglia and sympathetic ganglia, regrowth, any of these systems can be used to determine activity in addition to dorsal basal (DRG). DRG assays can be performed as described by Barte et al. (1980, Proc. Natl.

Acad. Sci. USA, 77, 1199-1203). Nodular ganglia can be used as described in 1985, Dev. Biol., 112, 319-328).

The sympathetic ganglia system can be used for research as described in Barde et al., 1982, EMBO, 1,

549-553) .

ganglion culture NT-3 biological system for research Lindsay et al.

5.5. Neurotrophin-3 genes and proteins.

Using the methods described above (see also Chapters 6 and 7), nucleotide sequences and amino acid residue sequences were derived from them. The mouse genomic NT-3 sequence was identified, which is shown in Figs. 7. The sequence of human genomic NT-3, which is presented in Figs. 11, the figure also shows the rat DNA sequence. Each sequence or functional equivalents thereof may be used in the present invention. The invention further relates to genes and proteins that can be isolated from a pig, a bull, a cat, a bird, a horse or a rabbit, as well as from primates and other animals having NT-3 activity. The invention relates to NT-3 amino acid residue sequences comprising at least 10 nucleotides, as well as to sub-sequences which hybridize to NT-3 sequences used, for example, by hybridization, in Southern and Northern bolates; etc. The present invention provides an NT-3 protein, fragments thereof, derivatives having the amino acid residue sequences set forth in FIGS. 2, 7 and 11, or functional derivatives of these compounds. The present invention provides fragments of the NT-3 protein, or derivatives containing an antigenic determinant (or determinants), or derivatives having functional activity. As used herein, the term functionally active refers to having positive NT-3 protein activity in assays, for example, in chicken embryonic DRG, nodose or sympathetic ganglia.

For example, the nucleic acid sequences shown in Figs. 2, 7 and 11 may be substituted by amino acid substitution, addition or deletion, but these modifications retain functionally equivalent molecules. Due to degeneracy of the coding nucleotide sequence, other DNA sequences encoding substantially the same amino acid residue sequences shown in FIG. 2, 7 and 11 can be used in practice in accordance with the present invention. These include, but are not limited to, nucleotide sequences that contain all or part of the NT-3 gene shown in Figs. 2, 7, and 11, which contain various codon substitutions encoding functionally equivalent amino acid residues in these sequences, and producing silent mutations. Analogously to NT-3 proteins, or derivatives not limited to the present invention, such proteins having all or part of the primary amino acid residues are shown in Figs. 2, 7 and 11, comprising altered amino acid residue sequences wherein the amino acid residues are replaced by functionally equivalent ones resulting in a silent mutation.

or their fragments belong, but by that

For example, one or more amino acid residues in the middle of a sequence may be replaced by others of similar polarity, which is the functional equivalent by making silent sequence changes. Changes can be made within the types to which the amino acid residue to be substituted belongs. For example, nonpolar (hydrophobic) amino acid residues include alanine, leucine, valine, proline, phenylalanine, tryptophan and methionine. Polar neutral amino acid residues include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positive amino acid residues (alkaline) - arginine, lysine and histidine. Negative (acidic) - aspartic and glutamic acids. The present invention encompasses NT-3 proteins, or fragments thereof, or derivatives thereof that are differentially modified during or after translation, e.g., by glycosylation, proteinolysis, binding to antibodies or other cellular ligands, and the like.

In addition, the NT-3 protein may be further mutated in vitro or in vivo to create and / or disrupt a translation, initiation and / or termination sequence, or to create coding region variants and / or to create or remove existing restriction enzyme cleavage sites. to facilitate further in vitro modification. Any of the methods of mutagenesis can be used, including but not limited to in vitro directed mutagenesis (Hutchinsen et al., 1978, J. Biol. Chem., 253, 6551) using TAB® linkers (Pharmacia) et al.

5.6. Generation of anti-neurotropin-3 antibodies

In accordance with the present invention, the NT-3 protein, as well as fragments or derivatives thereof, can be used as antigens in the generation of anti-NT-3 antibodies.

To enhance the immune response, the sequence of NT-3 and NT-3 amino acid residues can be analyzed to determine the immunogenic regions of the molecule. For example, the amino acid sequence may be analyzed by computer for the determination of surface epitopes according to the method described by Weals, 1981, Proc. Natl. Acad. Sci. USA, 78, 3824-3828, which has been successfully applied in the identification of antigenic peptides in Hepatitis B surface antigen. Alternatively, the NT3 protein sequences of various animals may be analyzed by detecting relatively non-homologous regions of the sequence; these non-homologous sequences will be immunogenic to different species of animals with higher confinement.

Any method by which stable cells producing monoclonal antibodies can be obtained can be used to obtain the monoclonal antibody against NT-3. For example, the hybridoma technology developed by Kohler and Milstein (1975, Nature, 256, (495-487), as well as the triome technology of human B-cell hybridoma (Kozbor et al., 1983, Immunology today, 4, 72), EBV - hybridoma technology for the synthesis of human monoclonal antibodies (Cole et al., 1985, Kn. Monoclonal Antibodies and

Cancer Therapy, Alan R., Ziss Ine., P. 77-96) and other similar methods which encompass the present invention.

Monoclonal antibodies for therapy may be human monoclonal antibodies or chimeric human-mouse (or other animal species) monoclonal antibodies. Human polyclonal antibodies can be obtained by any known method (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. USA, 80, 7308-7312; Kozbor et al., 1983, Immunology Today, 4, 72-7). 79; Olsson et al., 1982, Meth. Enzymology, 92, 3-16).

Chimeric antibody molecules may be from the mouse antigen-binding domain and the conservative human portion (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81, 6851; Takeda et al., Nature, 314-452).

Various known procedures can be used to generate polyclonal antibodies with epitopes on NT-3 proteins. For the production of antibodies, various animals may be immunized by injection of NT-3 protein, or fragments or derivatives thereof, into, but not limited to, rabbits, mice, rats, and the like. Various adjuvants may be used to enhance the immune response, depending on the species, adjuvants may include but are not limited to Freud's mineral oils (complete and incomplete adjuvant), aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanionites, peptides, oil emulsions, snail lymph haemocyanins, dinitrophenol and potentially useful human adjuvants - BBC (Bacille Calmette Guerin) and Corynebacterium parvum.

constructed monoclonal

The molecular antibody clone for the NT-3 protein epitope can be selected by known methods. Recombinant DNA technology (see, for example, Maniatis Laboratory Manual for Molecular Cloning, 1982, Cold Spring Harbor, NJ) enables nucleic sequences to encode antibody molecules or antigen-binding regions of these molecules.

Antibody molecules can be purified by known methods such as immunoaffinity or immunoadsorption chromatography, other types of chromatography, such as HPLC (high performance liquid chromatography), or by combining various types of chromatography, and so forth.

Antibody fragments containing the molecule epitope can be generated by known methods. For example, such fragments may include, but are not limited to, the F (ab ') 2 fragment, which may be obtained by cleavage of antibody molecules with pepsin; Fab 'fragment which can be generated by disruption of disulfide bridges in F (ab') 2 fragment and 2Fab or Fab fragment obtained by papain digestion of an antibody molecule and reduction with a reducing agent.

5.7. Application of the invention.

sufficient for use.

The present invention relates to NT-3 nucleic acid sequences, purified NT-3 protein, peptide fragments or derivatives. NT-3 can be obtained in quantities for diagnostic and therapeutic purposes. Analogously, anti-NT-3 antibodies and NT-3 nucleotide probes can be used for diagnostic and therapeutic purposes. In most cases, the use of NT3 genes or gene products for the diagnosis and therapy of the same animal species is desirable, although in specific cases cross-usage of NT-3 may be beneficial.

5.7.1. Application in diagnostics.

The present invention, which relates to nucleic acid sequences encoding an NT-3 protein, an NT-3 protein, peptide fragments or NT-3 derivatives, as well as antibodies to an NT-3 protein, peptide or derivative, can be used to treat diseases or nerve disorders. diagnostics of system disorders associated with alterations in NT-3 expression.

In various embodiments of the invention, the NT-3 genes and their corresponding nucleic acid sequences and subsets, including complementary sequences, can be used for diagnostic hybridization. NT-3 nucleic acid sequences and subsets of approximately 15 nucleotides may be used. by hybridization with probes. Hybridization can be used to evaluate, predict, diagnose, or monitor conditions or conditions associated with alterations in expression of NT-3, including, in particular, conditions that cause sensory neuronal damage. Such diseases and conditions include, but are not limited to, CNS injuries, infraction, infection, degenerative nerve disease, tumors or postoperative lesions including but not limited to Alzheimer's disease, Parkinson's disease, Chantington's chorea. For example, total RNA from a patient's tissue can be examined to determine if there is an NT-3 protein iRNA, a change in the amount of NT-3 iRNA will indicate neuronal degeneration.

In an alternative embodiment of the present invention, antibodies to the NT-3 protein, peptide fragments or derivatives can be used to diagnose disorders and diseases of the nervous system, in particular, sensory abnormalities and retinal degenerative disorders, as well as the abovementioned disorders. The antibodies of the present invention may be used, for example, for in situ hybridization using patient tissue if such assay is required. In another example, the antibodies of the present invention may be used in an ELISA to detect and / or estimate the amount of NT-3 in solutions; similarly, the antibodies of the present invention can be used for Western blotting to detect and / or evaluate NT-3 in tissues or solutions.

In a further embodiment of the present invention, the NT-3 protein, peptide fragments, or derivatives may be used in the diagnosis of neurological damage and disease. In particular, but not limited to, labeled NT-3 proteins or peptide fragments may be used to identify tissues or cells having NT-3 receptors to identify aberrations in expression of NT3 receptors and subsequently to evaluate potential tissue abnormalities or cellular responses to NT-3.

5.7.2. Application in therapy.

The present invention relating to nucleic acid sequences encoding NT-3 proteins, proteins, peptide fragments or derivatives, as well as antibodies to NT-3 protein, peptides or derivatives, can be used in the treatment of diseases and disorders of the nervous system associated with NT-3. Expression of protein 3 or which may result from treatment of NT-3 with the body or with NT-3 antibodies.

In various embodiments of the present invention, the NT-3 protein, peptide fragments, or derivatives may be administered to patients whose nervous system is damaged by trauma, surgery, ischemia, infection, metabolic disorders, starvation (food deprivation), tumors, or poisoning. In various specific embodiments of the present invention, NT-3 may be administered topically to sensory neurons that have been excised, including but not limited to dorsal basal ganglion neurons, or in any tissue: knee, maxillary, or nodular ganglia; in the vestibuloacoustic complex in the ventrolateral polyus portion of the trigeminal ganglion, the mesencephalic triple nucleus 1, and the sympathetic ganglia. It may be desirable to introduce NT-3 peptides or NT-3 protein sorbed on a membrane, such as a silastic membrane, which can be implanted close to a damaged nerve. The present invention may also be used, for example, in patients with diabetic neuropathy, e.g.

VIII for faster recovery of cranial nerve, maxillomandibular multiplex mononeuropathy and diabetic peripheral neuropathy.

In addition, in the case of diabetic neuropathy, NT-3 peptides or NT-3 proteins may also be used to treat other peripheral neuropathies, including but not limited to: neuropathy associated with viral infection, including acquired immunodeficiency syndrome (AIDS), infectious moncr.ukleosis. with polyneuritis, viral hepatitis with polyneuritis, syndrome (polyneuritis); neuropathy, botulism, toxic polyneuropathies, neuropathies associated with alcohol poisoning: nutritional neuropathies, including subacute combined degeneration; angiopathic neuropathies, including neuropathies associated with systemic lupus erythematosus (lupus erythematosus); neuropathies associated with sarcoid diseases; carcinomatous neuropathy; Compressive neuropathy (for example, carpal tunnel

Geine-Bare associated with including lead or syndrome) and hereditary neuropathies, neuropathies that can be treated with NT-3 proteins may be associated with musculoskeletal disorders

Inherited

NT-3 or fibular atrophy, familial dystonia, and progressive hypertonic neuropathy.

In other embodiments of the present invention, the NT-3 protein, or fragments or derivatives, may treat congenital conditions including, but not limited to, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, and Huntington's chorea.

peptide utilized including,

Parkinson's be or any disease,

In a specific embodiment of the present invention, administration of the NT-3 protein or peptide fragments or derivatives of this protein may be performed by surgical tissue implantation in the treatment of Alzheimer's disease and / or

Parkinson's disease. As shown in paragraph 11, see FIG. below, NT-3 maintains the viability of dopaminergic neurons; dopaminergic neurons are killed in Parkinson's disease, NT-3 can be used in the treatment of Parkinson's disease, and therapeutically effective amount of NT-3 is administered to the patient, if necessary. About 35% of patients with Parkinson's disease have been shown to have Alzheimer's-type dementia; NT-3 obtained according to the present invention can be used in the therapeutic treatment of Alzheimer's disease with Down syndrome. In addition, as illustrated in paragraph 12, (cf. below) large amounts of NT-3 are experimented with in the development and differentiation of the nervous system; accordingly, NT-3 may be used to treat abnormalities in the development of the nervous system, such as Down syndrome, as well as in the treatment of abnormalities associated with cell dedifferentiation, such as tumors or in lesions that occur during regeneration of the nervous system. NT-3 obtained according to the present invention can be used in the treatment of various dementias as well as in the treatment of congenital switching disorders.

In a further embodiment of the present invention, the NT-3 protein, fragments and derivatives thereof can be used in combination with other cytokines to achieve the desired neurotropic effect. For example, but not limited to, NT-3 according to the present invention may be used with another agent, such as BDNF or NGF, to achieve a synergistic stimulating effect on neutron growth and / or viability, and either to maintain or enhance function, the synergistic effect in the sense that combinations of an NT-3 protein or peptide moiety or derivative with another agent produce an effect that is more potent than using these agents alone. NT-3 can act synergistically with other peptide-derived CNS-derived factors that are not fully characterized during growth, development, maintenance of differentiated functions, and maintenance of the viability of various subpopulations of neurons in the central nervous system. Alternatively, the effects of NT-3 and a second neurotropic agent may be additive.

Based on the full characterization of the NT-3 molecule, novel peptide fragments, derivatives, or mutants of the NT-3 protein may be constructed according to the present invention, which may act as antagonists to all or some of the NT-3 functions. Such NT-3 antagonists can be used to selectively remove sensory neurons, for example in the treatment of chronic pain syndrome. In another embodiment of the present invention, antibodies to the NT-3 protein or peptide fragment or derivatives thereof can be administered to patients with a variety of neurological disorders. For example, such treatment may be used in patients with excess NT-3 production. Anti-NT-3 antibodies can be used to prevent aberrative regeneration of sensory neurons (for example, after surgery), or as mentioned above in the treatment of chronic pain syndrome.

The distribution of NT-3 in tissues as described in sections 6 and 7 below indicates that higher levels of NT-3 iRNA are expressed in the brain, kidney, heart and spleen compared to other tissues. The NT-3 protein produced in non-nerve tissue may be identical or non-identical to the expressed brain. Individual forms of NT-3 may function in both nerve tissue and non-nerve tissue; Changes in NT-3 expression may indicate diseases affecting the nervous system and other organ systems. Alternatively, cognate NT-3 family molecules may perform a variety of functions in nerve and non-nerve tissue. Therefore, NT-3 according to the present invention can be used for the treatment of diseases involving the nervous system as well as diseases of the nervous system regulating non-nervous tissues, including the heart, hemopoietic, renal and reticuloendothelial systems.

In addition, as noted in section 6 below, NT-3 expression is enhanced in immature animals compared to adults. In accordance with the present invention, NT-3 may be useful in the treatment of developmental abnormalities or, alternatively, in the stimulation of nervous system regeneration following CNS lesions.

5.8. Pharmaceutical forms.

The active compositions of the present invention may contain all or only part (s) of the NT-3 gene product, including a protein, peptide fragments or derivatives thereof, or antibodies (or antibody fragments) against the NT-3 protein, peptide fragments or derivatives thereof, or in combination with at least one other agent such as NGF or BDNF; these compositions may be administered with any sterile biocompatible pharmaceutical carrier, including but not limited to saline, saline buffer, dextrose, and water.

The amino acid residue sequence or subset of an NT-3 protein, peptide fragment or derivative may be substantially as set forth in FIGS. 2, 7 and 11; it may be desirable to use the NT-3 protein, from 140 to about 258 amino acid residues, as shown in Figs. 2, 1 to about 119 amino acid residues, as shown in Figs. 7, or from the 1 amino acid residue designated mature in Figs. 11 to the end of the peptide chain of FIG. 11, or containing a functionally equivalent sequence, since this subset is believed to be a functional preferred moiety of the NT49 standard. The NT-3 sequence may be the NT-3 gene sequence of any suitable animal, including, but not limited to, human, porcine, rat, chicken, cow, dog, sheep, goat, cat, rabbit, and the like. The amounts of NT-3 protein, peptide fragment, derivative or antibody used that are effective in treating the disease or condition will depend upon the nature of the lesions and conditions and may be determined in clinical trials. When possible, quantify response at baseline in the in vivo NT-3 assays described above, then in animal models, and only after these assays in humans. According to the data, in vitro, in a specific dosage form of the present invention, which according to the present invention effectively maintains the viability of sensory neurons, can maintain a local concentration of NT-3 protein of about 0.1 to 10 µg / ml.

in vitro dependence, such as those obtained in an embodiment, may be intracutaneous, intravenous, subcutaneous,

Methods of administration include, but are not limited to, intramuscular, intra-abdominal, oral, and intranasal. In addition, it may be useful to administer the medicament of the present invention to the central nervous system by any suitable route, including intragastric and intrarectal;

intragastric delivery may be accomplished with the aid of a catheter, for example, attached to a reservoir such as Ommaya.

It may be advantageous to administer the dosage forms of the present invention locally to areas requiring treatment, such as, but not limited to, topical infusion during surgery, catheter injection or implantation, the implant may be porous or non-porous or floral, Including membranes such as sialastic membranes or fibers.

According to the present invention, pharmaceutical compositions containing NT-3 proteins, peptide fragments or derivatives can be administered in the form of liposomes, microparticles or microcapsules. In various embodiments of the present invention, it may be useful to use such dosage forms to achieve sustained release of NT-3 or related products.

The present invention provides for the administration of cells that are active in the production of NT-3, cognate NT-3 substances, NT-3 antagonists or anti-NT-3 antibodies in areas that require increased or decreased NT3 concentration.

6th An example. Cloning and characterization of the mouse neurotrophin-3 gene

6.1. Materials and Methods

6.1.1. Polymerization Chain Reaction

Two oligonucleotide primers have been synthesized according to two amino acid residue sequences that are conservative in BDNF and all known NGF (Leibrock et al., 1989, Nature, 341, 149-152). Meaningful (or

5 ') primer sequence: GGG GAT CCG C GG1 TGY MGY GG1 ATH GA (Primer 1, UPAC nomenclature, l = Inosine), this primer contains BamHI and SacII cleavage regions; antiprasmin (3 ') primer sequence: TCG AAT TCT AG AT 1CK 1AT PAA 1CK CCA (primer 2), this primer contains EcoRl and Xbal cleavage regions.

The PG reaction (Saiki et al., 1985, Science 230, 1350-54) was carried out using a mouse mouse genomic DNA reaction in a Perkin-Elmer Cetus thermocycler with Ta-polymerase (Gene Amp). After 4 cycles at 45 ° C, the remaining 36 cycles were performed at 49 ° C. The resulting amplification products were of the expected size of 137 base pairs, eluted from the polyacrylamide gel, re-amplified and digested with HindIII and Arai, the first cleaving with murine NGF and the second with murine BDNF. Uncut DNA was eluted and asymmetrically amplified (Janis et al., 1988, Proc. Natl. Acad. Sci. USA, 85, 9436-9440) and sequenced (Sanger et al., 1979, Proc. Natl. Acad. Sci. USA, 72, 3918-3921) using primers 1 and 2. Based on the sequence thus obtained, two additional sense primers corresponding to nucleotides 808-822 (primer 3) and 824-844 (primer 4) were synthesized by addition of SalI and PstI cleavage sequences. RNA was extracted from the brain, liver and muscle of an adult mouse (Okayama et al., 1987, Meth. Enzymology, 154, 3-28) and reverse transcriptase was transcribed using an atypical (3 ') primer: CGG ATC CGA ATT CGT CAC (T) 12V (primer 5) constructed to align with the 3' poly (A) terminus, containing the BamHI, EcoRI and Pstl cleavage domains required for cloning (Leibrock et al., 1989, Nature, 341, 149-152). These iRNAs were PCR amplified using primers 3 and 5, and re-amplified using primers 4 and 5. Southern blotting was performed with the final reaction products and hybridized with the 32P-labeled back oligonucleotide corresponding to the fragment 879 to 893. The DNA fragments thus identified were cloned into Bluescript® SK⁺ vector (Stratagene) and the longer insert obtained (460 base pairs) was used to screen the EMBL3 mouse genomic library (Klontex). Two positive clones were identified and DNA from one clone was digested with various restriction enzymes. The restriction fragments were probed with 460 base pair inserts: 700 bp Hindlll and 4000 bp PstI were subcloned into Bluescript® SK⁺ vector. The full Hindlll sequence shown at the 3 'and 5' ends was extended using the PstI fragment.

6.1.2. Northern blotting.

Total RNA was extracted from an adult female (Okayama et al., 1987, Meth. Enzym. 154, 3-2) by electrophoresis on a 1.3% formaldehyde-crosslinked agarose gel (Hetrach et al., 1977, Biochem., 16, 4743-4751). ). RNA was transferred onto a nylon membrane (Hydond-N, Amerscham) and hybridized overnight at 42 ° C in 1 ml 200 mM sodium phosphate buffer (pH 7.2) containing 40% formamide, 5x Denchardt solution and 200 µg / ml salmon sperm DNA. . The random double-stranded probe used was designated 32P (Feinberg et al., 1979, Anai. Biochem., 137, 266-267) corresponding to the sequence from nucleotide 319 to 1093 (Fig. 2). Specific activity was 1.3x10 cpm / µg, 10 cpm was added to the hybridization buffer. The rinse was 60 min. with 0.1xSSC buffer with 0.5% SDS. The filters were exposed for 5 days at - 70 ° C with a magnifying screen.

6.1.3. Expression of neurotrophin-3

The oligonucleotide primers were synthesized according to the first 19 nucleotides (plus the EcoRI cleavage region) and the last 19 nucleotides (plus the BamHI cleavage region in the open reading frame in Figure 2). After PCR amplification using the matrix of FIG. 2 PstI genomic fragment, the resulting product was cloned into the EcoRI-BamHI cleavage region in the expression vector pCMV (Anderson et al., 1989, J. Biol. Chem. 264, 8222-8229). It was fixed

NT-3 insert sequence in vector pCMV. COS-1 cells were transfected using the calcium phosphate method (Chen and Okayama, 1987, Mol. Cell. Biol. 7, 2745-2752), and the culture medium was harvested as described above (Leibrock et al., 1989, Nature 341). , 149-152). Control medium was obtained by treating COS-1 cells with calcium phosphate, or the pCMV / NT-3 construct from which the stop codon of the NT-3 gene was deleted. Both media showed no bioactivity at 1:50 dilution. Dissociated nodose ganglia and neurons were cultured in 24-well plates (Lindsay et al., 1985, Dev. Biol. 112, 319-328), BDNF was isolated from porcine brain (Hofer and Barde, 1988, Nature, 331, 261-262). ).

6.2. Results and discussion.

The main problem in characterizing neurotrophic factors is their low content. Like NGF, BDNF was characterized using protein purification methods (Cohen et al., 1960, Proc. Natl. Acad. Sci. USA, 46, 302-311; Barde et al., 1982, EMBO, JI, 549- 553). Such a method is suitable for NGF because its levels in the adult mouse forearm are very large, as is BDNF, since enough porcine brain can be used for experiments. The sequence homology established by NGF and BDNF suggests that various strategies may be employed to characterize other members of this gene family (Leibrock et al., 1989, Nature, 341, 149-152). For detailed analysis of the murine NGF and BDNF amino acid sequences (as), two as regions of 6 amino acid residues (arl) were identified and underlined in Figs. 2, these areas are very convenient as primers for PCR (Saiki et al., 1985, Science 230, 1350-1354). The mouse genomic matrix was used because the NGF and BDNF genes do not contain introns interrupting exonic coding of biologically active proteins. This technique resulted in amplification of NGF and BDNF sequences which were then digested with restriction enzymes. The remaining non-cleaved DNA fragment was sequenced and a sequence was found which is neither BDNF nor NGF. Two specific sense (5 ') primers were synthesized for further PG reactions, the template used was transcription of complementary DNA synthesized by reverse RNA extracted from mouse brain, muscle and liver; The 3 'primer was constructed to overlap the poly (A) sequences of FIG. 2). Amplification of samples from three tissues yielded products of similar size, which were cloned and used for screening the mouse genomic library. The single genomic clone thus identified was sequenced in Figs. 2). In the open reading frame, a potential protein containing 258 arl (starting with the first residue of methionine found after the 3 'stop codon in the frame) was named this neurotrophin-3 (NT-3). In all respects, the overall structure of the putative protein is similar to NGF and BDNF, with a putative signal chain of 18 arl (of which 5 and 9 arl are identical to BDNF and NGF, respectively) extending prosecution from 121 arl Such a sequence is believed to be involved in protein structuring and formation of regular disulfide bridges (Edwards et al., J. Biol. Chem. 263, 6810-6815), as well as both NGF (103 arl) and BDNF (112 arl) were detected. The only potential region for N-glycosylation is 9 arl (BDNF and NGF8 arl) upstream of the proteinase labile region consisting of basic amino acid residues at which the mature NT-3 protein begins (Fig. 2, arrow). The mature NT-3 protein is thought to be composed of 119 amino acid residues (relative molecular weight 13.625 D, pi = 9.3). Comparison of murine NGF, BDNF, and NT-3 revealed 54 arl homology (Fig. (Fig.2). 2). All six cysteine ​​residues, known as NGF and BDNF, all form disulfide bridges (Leibrock et al., 1989,

Nature. 341, 149-152, Angeletti 1973. Biochem., 12,

100-115), and they are in the conservative portion of the molecule (Fig. 3, arrow). It should be noted that when compared to the NGF and BDNF sequences, NT-3 contains less than 7 identical amino acid residues (compared to 61 identical amino acid residues in murine NGF to BDHF). Thus, nearly 50% of the protein's primary structure is conservative, which is apparently required for the formation of a basic tertiary structure common to all three proteins. In addition, these three sequences contain 4 variable domains, each of which contains 7 to 11 amino acid residues (designated YI-Y4 in Figure 3), apparently defining neuronal specificity.

For the expression of NT-3 gene expression, RNA was extracted from various mouse tissues and analyzed by NT-3 specific probe (Fig. (Fig.4). 4). In all tissues tested, only 1.4 thousand were identified. bases size bar. But the size of the iRNA expression was highly variable. Expression was variable in the cerebellum (Fig. 4B), with the highest expression of iRNAs in the cerebellum and ammonia horn. These results demonstrate that tissue distribution of NT-3 is different from that of BDNF and NGF. Indeed, NGF iRNA is found predominantly only in the brain, whereas NGF iRNA is barely detectable in tissues such as the liver or skeletal muscle (Heumann et al., 1984, EMBO, 3, 31833189). Interestingly, ammonium rage expressing NGF iRNA (Korshing et al., 1985, EMBO, 4, 1389-1393) also expresses BDNF and NT-3 iRNA at higher levels than in other brain regions (Figs. .4B).

To verify that the NT-3 protein is biologically active and secreted, the sequence encoding the entire protein was cloned into an expression vector (pCMV), which was used for transfection of COS cells (monkey kidney cells) (Fig. 5). Knowing that NT-3 iRNAs are present in peripheral tissues, various chick embryo neurons have been cultured which extend into these tissues and require trophic factors for their viability. Motion neurons isolated from porcine 6-day-old embryonic (E6) brain (Dohrman et al., 1986, Dev. Biol. 118, 209-221), ciliary neurons (E8), and dissociated sympathetic neurons (Eli) did not survive in the NT. -3. However, the response of sensory neurons isolated from E8 primary sensory ganglia has been observed. As depicted (Fig. (Fig.5). 5), NT-3 maintains the viability of 30% of neurons isolated from nodular ganglia. In addition, this effect, in addition to the action of BDNF synthesizing in central projection regions and known for its effect on a subpopulation of nodular neurons (Lindsay et al., 1985, Dev. Biol. 112, 319-328), and both most neurons (90%), cf. FIG. 5. It is important to note that NT-3 iRNA is found in the visceral targets of this ganglion, including the heart, liver and lungs (Fig. 4A). Populations of sensory neurons responsive to NT-3 were detected in E8 dissociated dorsal basal and trigeminal ganglia as well as in E8 sympathetic ganglion explants. Thus, NT-3 exhibits hitherto uncharacterized biological activity in some peripheral tissues, including the liver (Lindsay and Tarbit, 1979, Neuro. Sci. Lett., 12, 195-200) and skeletal muscle (Davies, 1986, Dev. . Biol., 115, 56-67) and maintains the viability of visceral and proprioreceptive sensory neurons that do not respond to NGF (for review, see Davies, 1987, Development, 101, 185-208).

Taken together, these facts indicate that NT-3 is a neurotrophic factor related in structure and function

NGF and BDNF, the first two neurotrophins. The proposed name is neurotrophin (NT) for this class of proteins and, by analogy, for interleukins, numbered in their discovery sequence.

Example: Cloning and characterization of rat neurotrophin-3.

Homology between NGF and BDNF was used in the cloning strategy to find additional members of this family. This describes the cloning of a gene encoding a third member of the BDNF / NGF family. This new biological activity and compared to NGF and BDNF.

named neurotrophin-3, a factor with defined speci

7.1. Materials and Methods

7.1.1. Polymerization Chain Reaction (PCR)

Degenerate oligonucleotides corresponding to four segments of the protein sequences that are conservative for NGF and BDNF were synthesized; the following protein sequence fragments, which may also be found in the NGF and BDNF sequences presented in (Figure 7D), are:

1. Gly-Glu- (Tyr / Pe) -Ser-Val-Cys-Asp-Ser;

2. Lys-Glu-Tyr-Phe (Tyr / Phe) -Glu-Thr-Lys-Cys;

3. Gly-Cys-Arg-Ile-Asp;

. Trp-Arg-Phe-Ile-Arg-Ile-Asp-Thr- (Ser / Ala) -Cys-ValCys.

The PG reactions used a series of degenerate sense and anti-sense oligonucleotides (degenerate region 15-26 nucleotides corresponding to 5-9 amino acid residues of the indicated protein in both the sense and anti-sense direction as well as the non-degenerate tail encoding the restriction enzyme cleavage region). The amplification reaction between the upper pores and the lower anti-spasmodic primers was performed under the conditions recommended by Perkin-Elmer-Cetus, except that the renaturation temperature, Mo.⁺⁺concentration and duration were varied to optimize amplification conditions for each pair of primers. The exact sequence of the IB sense primer (coding sequence for the protein from region 1 and above) was: 5'GAC TCG AGT CGA CAT CG-GTN-TGY-GAY-WSN-RTN-WS-3 'and 2C anti-sense primer (corresponding to the sense codons for the protein sequence) from 2 and above) the sequence was: 5′-CCA AGC TTC TAG AAT TC-CA-YTT-NGT-YTC-RWA-RAA-RTAYTG-3 ′ (abbreviations by IUPAC codes). Sequence analysis revealed that the IB oligonucleotide contained two mismatched nucleotides compared to the NT-3 sequence when the 2C oligonucleotide had one mismatched nucleotide. Radiolabeling The introduction was performed by Perkin-Elmor / Cetus amplification with the following modifications: 1-10 ng of template DNA on low melting agarose was added to a reaction mixture not labeled with dATP, aGTP and DTTP at a final concentration of 50 μΜ; was added 50 μΰί a³²P-dGTP (3000 Ci / mol) in 50 μϊ of reaction mixture and subjected to 7 cycles of amplification. The primers used for amplification were identical to the degenerate primers used in the original PCR.

7.1.2. Southern btolation of rat genomic DNA library using NT-3 probe.

The PG reaction product R1B / 2C was obtained by amplifying template genomic rat DNA using degenerate primers IB and 2C as described in Section 7.1.1. The resulting PCR product using degenerate primers IB and 2C (designated R1B / 2C) contains a novel gene, NT-3, as well as NGF and BDNF genes in rat genomic DNA. Genomic DNA was. derived from Fisher rat liver (Maniatis et al., 1982,

Molecular Cloning, Laboratory Manual), digested with EcoRI and fractionated in 10 µg of 1% agarose gel. This DNA was transferred onto nitrocellulose using 10xSSC buffer (Maniatis et al., 1982, Molecular Cloning,

Laboratory Manual) and hybridized (Mahmoud and Lin, 1989, Biotechniques, 7, 331-3) with 32P-labeled

The R1B / 2C PCR product 60C is washed with 2xSSC / 0.1% SDS

At 65 ° C. NT-3, NGF and BDNF bands were identified, and the positions of NGF and BDNF bands were predetermined using specific probes. Size is indicated in thousands of bases on the left.

7.1.3. Expression of neurotrophin-3

The express rat NT-3 system was constructed using PCR by amplifying the coding region of the short probe NT-3 probe size 3.2. kb, the rat PstI genomic fragment (Fig. 6B), which extends the NT-3 gene; the synthetic oligonucleotides used in the PCR had Xhol cleavage regions at their ends to allow the amplification of the coding region to be inserted into the Xhol cleavage region in the polynucleotide expression vector pCDM81 (Seed, 1987, Nature, 329, 840-42). The specific nucleotides used for amplification of the rat NT-3 coding region were: Upper sense primer 5'-CGG TAC CCT CGA GCC ACC ATG TCC ATC TTG TTT TAG GAG-3 '(underlined ATG corresponds to codon B in start region, downstream) from the ATG that overlaps the NT-3 sequence; upstream of the ATG is the synthetic Xhol cleavage region) and the lower anti-sense primer 5'-CGG TAC CCT CGA GAT GCC AAT TCA TGT TCT TCC G-3 '(underlined triplet is complementary to termination) for the codon NT-3 gene; this triplet flanked the exact antiprasmatic NT-3 sequence and the 5 'end of this primer is the Xhol cleavage region). The rat NT-3 expression plasmid was designated pC8-N3 (PI). A similar strategy was used to insert NGF and BDNF coding sequences into the Xhol cleavage region into the same vector by expression constructs pCDM8, NT-3, NGF and BDNF as described by Okayama and Berg (1982, Mol. Cell. 5: 1136). -42) COS-M5 cells, 5x10⁵ cells in a 60 mm plate and cultured 2.5. ml Dulbeco Modified Eagle High Glucose (4500 mg / ml) and 10% coarse fetal serum; supernatants were harvested after 72 hours.

7.2. Results

7.2.1. Cloning of the neurotrophin-3 gene was detected in cases also obtained by BDNF genomic DNA sequencing without PCR

Expected amplification products (as predicted from NGF and BDNF sequences) were obtained using various pairs of degenerate primers. These products were initially subjected to restriction analysis to quantify NGF, BDNF, or novel sequences. All ethidium bromide staining contained restriction fragments corresponding only to NGF sequences. But using the same PCR products as probes, hybridization of rat genomic DNA by Southern blotting revealed that a single product (labeled R1B / 2C, see (Fig. 6A), identified a new NGF and BDNF (Fig. 6A); by blotting, it was possible to identify rarely amplified sequences that were not detected by other methods. The R1B / 2C probe also detected new sequences in the genomic DNA of evolutionarily divergent species (including human, mouse, chick), and is believed to have a functional gene.

and xenopsus), identified

The R1B / 2C probe as well as specific NGF and BDNF probes were used to isolate and screen this gene (Maniatis et al., 1982, Molecular Cloning,

Laboratory Manual), the rat DNA genomic library (obtained from Ciontex laboratories, Ine, Palo Alto, CA), prepared from Sprague-Dawley rat DNA (partially digested with Sau3A restriction enzyme and cloned into the EMBL3 / SP6 / T7 bacteriophage vector) was screened. Two independent bacteriophage clones were identified that hybridized to the R1B / 2C probe but did not hybridize to the other two probes. Restriction mapping of rat genomic inserts revealed that they contained the same gene (Fig. (Fig.6B). 6B). The bacteriophage clone with the longest insert was labeled with &lt;)&gt; rN3 (GI). Upon sequencing the gene identified by the R1B / 2C probe, it was found to encode a novel NGF / BDNF family member (Fig. (Fig.7). 7) and was named Neurotrophin-3.

7.2.2. Sequencing of mature neurotrophin.

DNA sequencing was performed by the double-deoxynucleotide sequence termination method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA, 74, 5463-7) using the Sequenase version 2.0 produced by Biochemical Company USA using procedures recommended by the manufacturer. .

NGF has two distinct probaltimals, called the long (starting from the start region A) and the short (starting from the start region B), they differ in length and sequence at the N terminus (Darling et al. 1987, Cold Spring

Harbor Symp. Quant. Biol., 1, 427-34, Selby et al.

1987, Mol, Cell. Biol., 7, 3057-64, Edwards et al.

1988, Mol, Cell. Biol., 8, 2456-64). And from long and short probaltim, one can obtain proteinolysis mature

NGF protein, which is 120 amino acid residues from the N terminus of probes.

By sequencing NGF genes in several animal species, the major sequence from the N terminus was found to be in separate exons, with the exception of four codons (ValHis-Ser-Val) located at the 5 'end of the exon, which encodes a complete short probaltim (start region B). Previously, it has been shown here that two of the four codons (ValX-X-Val), including the binding region in the RNA splicing that precedes them, are conservative and are located above the conservative start region B in BDNF genes isolated from various animal species; this made it possible to assume that there was a long and short form of probation of BDNF. There is a conserved codon sequence Val-X-XVal as well as a spliced ​​binding region and the putative intron boundary is shown (Fig. 7A). This sequence analysis predicted the existence of coding exons in the NT-3 gene, which encodes the long probalt. The finding of a conservative sequence against starter region A further reinforced the existence of the long form of the BDNF probalt and revealed the important evolutionary role of this long probalt in all NGF family members. The predicted N-terminal sequence of mature NT-3 protein is a common proteinase cleavage region (Arg-Arg-Lys-Arg) and is similar to sequences present in NGF and BDNF proteins (Figs. 7A, C). In some animal species, two amino acid residues at the C terminus of NGF proteins are removed by proteinases. Unlike NGF, rat NT-3 lacks a potential proteinase cleavage region at the C terminus of protein (Fig. 7A, C), and it was concluded that, as with all currently studied BDNF proteins, NT-3 protein has no C protein terminus. modification.

According to these statements, the estimated size of the mature NT-3 protein is 119 amino acid residues, with an estimated pi of about 9.5. By size, total protein load, NT-3 protein is very similar to NGF and BDNF. Seven amino acid residues of the mature NT-3 protein. The C terminus is different from NGF and BDNF. Beginning with the eighth amino acid residue in the mature NT-3 protein, the optimal overlap requires a two amino acid residue in the BDNF protein and a single amino acid residue in the single-insert protein NGF (see Fig. 7D). The mature rat NT-3 protein has 57% sequence homology to rat NGF and 58% sequence homology to rat BDNF, 57 of the 120 amino acid residues (48%) being common to all three proteins (see (Fig. 7D). . The six cysteine ​​residues found in NGF and BDNF are conservative and are found in the NT-3 protein, with the highest homology domains in these three proteins concentrated around these cysteine ​​residues.

7.2.3. Neurotrophin-3 probaltim analysis

Above the expected cleavage region, which cleaves to give the mature NT-3 protein, is the total glycosylation region - (Asn-X-Thr / Ser) (see (Fig. 7A, C), this region was found in the same position in NGF and BDNF (Ullrich et al., 1983, Nature, 303, 821-5; Leibrock et al., 1989, Nature, 341, 149-52). Does this glycosylation domain play any role in the processing of NT-3, NGF, or BDNF probes? remains unexplained.

Further comparison of the probe sequence of NT-3 with that of NGF and BDNF probes reveals two regions of homology upstream of the mature NT-3 sequence (regions I and II (Fig. 7B, C).

NGF gene), which would be a probalt of 258 amino acid residues, similar in size to the short NGF probaltim (241

arl) and the short BDNF probalt (249 arl), claim that region B is synthesized in existence (the short start methionine codon, secretory signal sequence, and signal sequencing region of these three factor probes are conservative) (Fig. 7C). that the homology domain I precedes the start region B, it is possible to predict the existence of a long probe NT-3 whose transcription will start from the start codon A (see Fig. 7A, B, C). As found in NGF (Uilrich et al., 1983, Nature 303, 821-5, Selby et al

1987, Mol.Cell. Biol., 7,

3057-64), and presumably will be coded

BDNF, such a starter region, in additional exons upward to a single exon that encodes the entire short probalt.

In the additional domain of homology I and II, comparing the hydrophilic domains of NT-3, NGF, and BDNF proteins, the probaltim sequences above the mature proteins were found to have similar hydrophilicity.

7.2.4. Neurotrophin-3 exhibits neurotrophic activity

The remarkable homology of NT-3, NGF, and BDNF makes it highly likely that NT-3 may exhibit neurotrophic activity. NGF and BDNF maintain the viability of individual neuronal populations of the peripheral and central nervous system in vivo and in vitro (reviewed in Whiimore and Senger, 1987, Brain Res. Rev ,, 12, 439-64, Lindsay, 1988) The Making of the Nervous System, p. 149-65, Davies, 1988, Trends Genet. 4, 139-43). For example, the use of any of the above factors in avian embryo development prevents natural neuronal death in the peripheral ganglia (Fig. Hoffer and Barde, 1988, Nature 331, 261-2). Addition of explant ganglia to NGF and BDNF results in neurite outgrowth (Davies et al., 1986,

Neurosci, 6, 1897ganglion neurons

1904), and cultures, these factors maintain neuronal viability and initiate their differentiation (Lidsay et al., 1985, Dev.

Biol., 112, 319-328). In vitro studies using multiple chicken peripheral ganglia were used to detect differences in neurotrophic NGF and BDNF activity. Both of these factors affect populations of sensory neurons from the dorsal basal ganglia in the nerve ridge (DRG), and BDNF maintains nodular ganglion sensory neurons (MG) (Lindsay et al., 1985, Dev.

Biol., 112, 319-328). Unlike BDNF, NGF can maintain viability and promote growth in sympathetic ganglion neurons (SGs) (Barde et al., 1983, EMBOJ, 1,

549-553) .

To determine the potential biological activity of NT-3, the rat NT-3 gene was inserted into the pCDM8 vector (Seed, 1987, Nature, 329, 840-842), which was used to transiently express BDNF and NGF in mammalian cells. This construct was constructed for the expression of the short probalt form of NT-3, expressing short forms of NGF and BDNF probate forms to yield biologically active compounds (Edwards et al., 1988, Mol. Cell., Biol., 8, 2456-64, Leibrock et al. et al., 1989, Nature 341: 149-152). NT-3, NGF, and BDNF gene constructs were transfected into COS cells; The culture supernatant was harvested and initially tested at different concentrations for the ability of neurons from DRG explants to grow (see Fig. 8). The first evidence that the NT-3 gene encodes neurotrophic activity was that the product of this gene induces vigorous growth of neurites from DRG explants (Fig. (Fig.8). 8).

To determine whether NT-3 acts directly on neurons, the influence of this factor on concentrated dissociated DRG neuron cultures was investigated (Fig. 9). In the absence of Schwann cells and fibroblasts, NT-3 maintained viability and stimulated the growth of neurites in 60% of DRG neurons. Knowing that BDNF and NGF together support 100% DRG neurite viability (Lindsay et al., 1985, Dev.

Biol., 112, 319-328), it can be stated that NT-3 maintains cell viability of those that interact with one of two factors.

7.2.5. The neurotrophic activity of NT-3 differs from that of NGF and BDNF.

Further investigation of the neural specificity of NT-3 was investigated in NG and SG explants. As expected, the experiments confirmed that NGF produces SG neuritis regrowth, but not the NG of chick embryo E8 explants;

The BDNF summoned NG explorers. Interestingly, in exploratory neurites or in cultures from sample experiments, embryo E8 DRG, neurite outgrowth, but not SG that NT-3 induced NG and SG outgrowth (Fig. 8), it testifies to the broader specificity of NT-3 than NGF or BDNF. However, NT-3, like NGF and BDNF, did not maintain viability or induce regurgitation of dissociated neuronal enriched ciliary chicken ganglia. As demonstrated above, parasympathetic neurons present in this ganglion respond to rat GNTF, a neuronal factor not from the NGF / BDNF / NT-3 gene family. (Manthorpe et al., 1986, Brain Res. 367, 282-6; Stockli et al., 1989).

Nature, 342, 21-28). No reaction was observed in any of these experiments using supernatants of COS cells transfected with control vectors (Fig. (Fig.8). 8). Table IV shows the response of explanted chicken nodosa ganglia (NG) and paravertebral sympathetic ganglia (SG) to various concentrations of NT-3. The ganglia were cultured for the indicated period of time in the same manner as explants in 1 ml collagen gel as described by Lindsay and Rohrer, 1985, Dev. Biol., 112, 30-48).

Growth regrowth was scored from 0 to 5, with 5 maximal regrowth observed in the dorsal basal ganglia using saturated nerve growth factor (NGF) dose (1-10 ng / ml). Rat NT-3 was used from conditioned media of COS-M5 cells transfected with pC8-rN3 (GI) plasmid as described above. At all tested doses greater than 50 dilutions, NT-3 induced significant regrowth in all types of explanted ganglia, although at low concentrations this response was less pronounced in sympathetic ganglia. The highest sprout regression reaction using NT-3 was obtained for dorsal basal ganglia and was close to that of NGF and BDNF. The peak response of nodular ganglia was significantly higher with NT-3 than with BDNF. NGF did not act on nodose ganglia. The peak sympathetic ganglion response with NT-3 was lower than with NGF and was only observed at high NT-3 concentrations than was required for dorsal basal cultures or nodose ganglia.

Table IV

Recombinant rat NT-3 induces sprouting in the following explants:

Dorsal basal ganglia of E8 chicken embryo

DRG - E8 chick embryo dorsal basal ganglia

NG - E8 Chick embryo for nasal ganglia

<td>SG - E8 parasympathetic</td><td>chicken chains</td><td>of the embryo ganglia.</td><td colspan="2">paravertebral</td>

<td></td><td></td><td>Growth</td><td>regrowth</td><td>rating</td>

<td></td><td></td><td>DRG</td><td>NG</td><td>SG</td>

<td></td><td></td><td>24 or.</td><td>24 or.</td><td>24 or.</td>

<td>Control 500</td><td>μΐ mock-</td><td> 0-0.5</td><td> 0</td><td> 0</td>

transfected

<td></td><td>Of COS cells supernatant</td>

<td>NT-3</td><td>10 μΐ cells supernatant</td>

<td>NT-3</td><td>50 μΐ cells supernatant</td>

<td>NT-3</td><td>200 μΐ cells supernatant</td>

<td>NT-3</td><td>500 μΐ cells supernatant</td>

Evaluation of regrowth in case of 4-6 reps.

<td> 3</td><td> 2-3</td><td> 0-0.5</td>

<td> 4</td><td> 2-3</td><td> 0-0.5</td>

<td></td><td> 2-3</td><td> 0-1</td>

<td></td><td> 0-2</td><td> 1-2</td>

<td>was</td><td>done</td><td>each</td>

* At higher concentrations of NT-3, regrowth reduction is reduced, as determined by the length of regrowth in explants and their number in NG explants.

7.2.6. Rat neurotrophin-3 activity occurs in mammalian neurons.

Exploratory experiments using dorsal basal ganglia taken from 14-day-old rat embryos were repeated to determine the effect of rat NT-3 mammalian neurons (Table 5). Purified NGF was used as a control. The rat E14 explant dorsal basal ganglia (four ganglia per ml of culture) was cultured for 24 hours, almost the same as the chicken ganglion (Fig. 8, Tab. 4) without addition of neurotrophic factor (control), addition of nerve growth factor from mouse forebrain (NGF), or addition of recombinant rat NT-3 (codified COS-M5 cell, transfected with pC8-rN3 (PI) as described above). . In each ganglion, regrowth was assessed (See Table 4). The results presented above show that NGF and NT-3 induced severe regrowth in exploratory dorsal basal ganglia.

Table V.

Effect of NT-3 on growth of dorsal basal ganglion gland explants in rat embryos.

Growth Rate Assessment (E14 rat DRG)

Control 0

NGF (mouse, 5 ng / ml) 4

NT-3 20 μΐ, rats 3

NT-3 COS cell supernatant

Growth regression is graded from 0 to 5, presented separately for each ganglion.

Because DRG, NG, and SG explants respond to at least two of the three related neurotrophic factors, the maximum response to these ganglia depends on the factor used. For DRG, the response to saturated concentrations of NGF, BDNF and NT-3 was relatively equivalent. But NG's maximal response to NT-3 was stronger than that of BDNF; SG maximal response to NT-3 was significantly weaker and delayed compared to NGF.

7.2.7. Investigation of neurotrophin-3 synthesis domains.

It has been found that as neurons develop, their viability depends on the neurotrophic molecules involved. Long-term viability, even in adults, may require neurotrophic support. (Thansen et al., 1987, Ciba. Found. Symp. 126, 82-95). In other cases, the viability of mature neurons may be more independent of neurotrophic factor; but such factors have been found to strongly influence the neuronal differentiated phenotype (Lindsay and Harmar, 1989, Nature, 337, 362-364). Identifying the synthesis domains of the neurotrophic molecule would help to clarify their physiological role.

For the study of NT-3 fusion regions and to compare NT-3 expression with NGF and BDNF expressions, three-stranded RNA from various mature rat tissues, Southern blots, was hybridized with probes specific for each gene (Fig. (Fig.10). 10). As demonstrated previously (Neumann et al., 1984, EMBO, 3, 3183-9; Shelton and Reichardt, 1984, Proc. Natl. Acad. Sci. USA, 81, 79517955), NGF iRNA expression was highest in brain, heart and spleen; traces were found in other tissues examined. BDNF expression was more localized; the highest expression was found in the brain (Liebrock et al., 1989, Nature, 341, 149-152), significant amounts were found in the heart, lungs and muscles. As with NGF, the NT-3 transcript (1.4 kb) was detected in all adult tissues examined. But in all peripheral tissues, the expression level of NT-3 iRNA was at best the same as in the brain of adult animals and only in some cases (eg kidney, spleen) was higher.

The expression levels of NGF, BDNF, and NT-3 in the brain of newborn and mature mice were also examined.

In contrast to NGF and BDNF, expression levels of NT-3 iRNA in neonatal cerebellum were higher than in mature animals (Fig. (Fig.8). 8). In more detailed analysis, NT-3 iRNA expression levels in the central nervous system were found to be significantly higher during fetal development and then decreased to mature levels.

7.3. Discussion.

factor sequences, guess,

Structure analysis of NGF, BDNF, and the newest member of this family, NT-3, identified several conservative domains and concluded that these functional differences are determinable outside of the conservative domains, that there are long and short probalt forms of all three proteins, intriguing questions about the existence of these forms of probaltim in vivo. Long forms of probation can be processed more efficiently than short forms. But, vectors expressing short forms of probalt release biologically active substances in COS cells.

The finding that these three are expressed specifically in different tissues reinforces neurotrophic factors during developmental stages and reinforces the claim that neural development is differently localized in time and depends on discrete neurotropic activities. According to development-dependent expression of NT-3, this factor may be of importance in the early stages of development of the nervous system. Initial characterization of NT-3 neurotrophic activity in vitro, coupled with the predominance of NT-3 iRNA in adult animal brain and adult animal peripheral tissues, suggests that NT-3 may have a wide influence on neuronal function and / or maintenance of viability in adult animal neurons . The increased expression of NT-3 suggests that this factor also affects non-nervous system cells, as has been made for NGF protein (Otten et al., 1989, Proc. Natl. Acad. Sci USA, 86,10059-10063).

Although it has not been clearly demonstrated that neurons can respond to multiple neurotrophic factors simultaneously, the facts suggest that NGF and BDNF may act on overlapping populations of neurons. For example, introduction of NGF or BDNF can save most DRG neurons that die during normal bird development (Hofer and Barde, 1988, Nature 331, 261-2). Our finding of NT-3 effects on chicken peripheral ganglia confirms that individual neurons can respond to a variety of related factors. If it is right, it raises interesting questions. For example, receptor components and / or signal transduction mechanisms for the three related neurotrophic factors may be common. Neurons that respond simultaneously may have multiple receptors specific for a particular neurotrophic factor or a single receptor that can trigger a response to a variety of neurotrophic factors. In vivo, these various factors may simultaneously be present in all reactive neurons. Spatial changes in these receptors over time are more likely to occur (Fig. Davies et al., 1987, Nature, 326, 353-358). It is possible that different factors are located at different locations in the same neuron (for example, the sensory neuron may bind different factors in peripheral and central regions (Kalcheim et al., 1987, Le Douarin, EMBO, 6, 2871-2873). If different factors bind to for some neurons, their effects may be both redundant and complementary.

Demonstration of the individual and potential complementary roles of NGF, BDNF and NT-3 will be based on information that is crucial to understanding the normal development and existence of the nervous system. Animal studies suggest that NGF may be useful in the treatment of neurodegenerative conditions (Snider and Jonson, 1989, Amer. Neurol. 26, 489-506; Ficher et al., 1987, Nature, 329, 65-8; Phelps et al., 1989,

Neurobiol Aging, 10, 205-7). The cloning of a new member of the NGF / BDNF family and its potential interaction with other members of this family initiates new potential therapeutic applications of these proteins in the treatment of neurodegenerative disorders.

An example. Cloning and characterization of the human neurotrophin-3 gene.

8.1. Results.

Preparation of the probe (R1B / 2C) used to identify the rat NT-3 gene was performed as described in Section 7. The probe was obtained by polymerase chain reaction (PCR) from rat genomic DNA using degenerate nucleotide primers corresponding to the two indicated frames for the amino acid residue sequences in the NGF and BDNF genes. After the detection of populations of DNA molecules corresponding to the novel gene with the R1B / 2C probe was initially detected by blotting rat genomic DNA cleaved with EcoRI, this probe detected a new DNA fragment without the expected fragments,

<td>matching</td><td>NGF and BDNF</td><td colspan="2">genes.</td>

<td>When there was</td><td>used</td><td>marked</td><td>32P R1B / 2C probe</td>

<td>in analyzing</td><td>human</td><td>genomic</td><td>DNA cleaved</td>

as with rat DNA, bands corresponding to NGF and BDNF genes, as well as additional bands, hybridized to various restriction enzymes. For example, a new (i.e., non-NGF, non-BDNF) band of about 1.8 kb was observed for HindIII digestion, and a new band of about 15 kb was observed for BamHI; and cleavage with EcoRI - new bands of 8 and 12 kb were observed (two bands may have been due to EcoRI restriction sites in human genomic DNA). These data prove that human DNA is an NT-3 gene that is conservative in rats and humans.

The human NT-3 gene was isolated by genomic library screening as described for the rat NT-3 gene (see, for example, section 7). More specifically, the library formed by partial digestion of genomic DNA from human placenta with the Sau3A restriction enzyme cloned by the bacteriophage vector XEMBL3 / SP / T7 (Clontex Ine.) Was screened with the R1B / 2C probe as well as rat NGF and BDNF probes. The human NT-3 clone was expected to hybridize to the R1B / 2C probe but not to hybridize with the NGF and BDNF probes. One such phage clone was identified from 8x10⁵ check. This clone labeled φΰΝ3 (GI) was found to have a 16 kb human DNA insert. The DNA of this clone was digested with restriction enzymes and the selected restriction fragments were subcloned into pBluescript (Stratagene) for DNA sequence analysis. FIG. Figure 11 shows the sequence of the human NT-3 gene and the deduced amino acid sequence of the product and compared it to the rat NT-3 sequence.

8.2. Discussion.

Analyzing the sequences revealed that the nucleotide and amino acid residue sequences of human and rat NT-3 are highly conserved with each other. Inside the region encoding the mature residue, rats have a homology of 92% of the amino acid DNA sequence protein (119 human genes).

But there is no difference in the rat and human gene nucleotide sequence that would replace the encoded amino acid residues presented in mature rat and human NT-3 (and mature mouse NT-3, see Section 6, supra). the amino acid residue sequences of the protein are identical. This is reminiscent of the high conservatism of BDNF, the sequences of which are completely identical to those of rat, mouse, human and porcine. In contrast, the amino acid residues of mature human NGF and rodent (mouse or rat) protein differ by about 10%.

In addition, the amino acid residue sequences of human and rat NT-3 probaltites also show little difference (different regions are underlined in Figure 11). Above the putative proteinase cleavage site, Arg-Arg-Lys-Arg polypeptide, the human protein does not contain a single codon (upstream of Pro) in the rat gene. One human amino acid residue sequence block from four is different from the rat preprotein NT-3, and the 6 amino acid residues are altered between these blocks and the putative proteinase cleavage region.

9th Human NT-3 biological activity

Since the derived amino acid residue sequence of mature human NT-3 protein is identical to that of mature rat NT-3, it can be reliably predicted that human and rat NT-3 proteins will have the same identical biological properties. The neurotrophic activity of human NT-3 was confirmed by inserting a cloned human gene into the plasmid expression vector pCDM8 by transfecting the resulting plasmid pC8-hN3 (PI) in COSM5 cells (as described by Okayama, 1987, Mol. Cell. Biol., 7, 1745-52) and subsequently assaying for neurotrophic activity in the medium of transfected cells. The human NT-3 gene was amplified by PCR from the bacteriophage &lt;RTIgt; (I) &lt;/RTI&gt; hN3 (GI) and inserted into the plasmid expression vector pCM8 in the same manner as the rat NT-3 gene (Example 7); the resulting plasmid was labeled with pC8hN3 (PI). The complete nucleotide sequence of the NT-3 gene insert was determined and compared to the genomic sequence as described above to confirm that no mutations were introduced during PCR and cloning. The transfection and assay methods were identical to those used to evaluate rat NT-3 activity.

As expected, human NT-3 was found to exhibit neurotrophic activity in the dorsal basal ganglia and nodosa ganglia of exploratory explants of 9-day-old chick embryos (see Fig. 8, Tab. IV) 24 hours after exposure to mock-transfected COS-M5 cells, conditioned in supernatant, transfected with plasmids (all from expression vector pCDM8) encoding recombinant human BDNF or recombinant rat NT-3 (rNT-3; plasmid pC8-rN). (Pi), or recombinant human NT-3 (hNT-3; plasmid pC8-hN3 (PI)). The BDNF plasmid was chosen as a positive control because BDNF exhibits neurotropic activity in both nodular ganglia and dorsal basal ganglia. As seen in Tab. IV, and rat and human recombinant NT-3 at moderate doses (cf. Table IV) exhibit the same activity as BDNF in the dorsal basal ganglia and significantly higher activity than BDNF in the nasal ganglia. No differences were observed between human and rat NT-3 activity.

Table VI.

Recombinant human NT-3 protein produced in COS cells exhibits the same activity as recombinant rat NT-3 in nasal ganglia and in dorsal basal ganglia of chick embryo explants.

Table VI ·

<td></td><td colspan="7">Evaluation of growth regrowth</td>

<td></td><td colspan="5">DRG</td><td colspan="2">NG</td>

<td>Control</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td> 0.5</td><td> 0</td><td> 0</td>

<td>BDNF: 20 μϊ COS supernatant</td><td> 3</td><td> 3</td><td> 3</td><td> 3</td><td> 3</td><td> 1</td><td> 3</td>

<td>Imitation: 20 μϊ COS supernatant</td><td> 0</td><td> 0.5</td><td> 0</td><td> 0</td><td> 0</td><td> 0</td><td></td>

<td>Human NT-3: 20 μΐ COS supernatant</td><td> 3</td><td> 2</td><td> 4</td><td> 3</td><td></td><td> 3</td><td> 3</td>

<td>Rat NT-3: 20 μΐ COS supernatant</td><td> 3</td><td> 4</td><td> 4</td><td> 3</td><td> 4</td><td> 3</td><td> 4</td>

Growth rating is given from 0 to 5 according to 1-5 individual ganglia after 24 hours of culture. An evaluation of each ganglion is given.

10th Identification of human NT-3 gene product by metabolic labeling.

[ ³⁵S] methionine and [ ³⁵S] was also collected by electrophoresis

The predicted size of the mature NT-3 polypeptide (rat and human) is 119 amino acid residues with a molecular weight of 13.6 kD. To determine the size of the experimentally mature human NT-3 polypeptide, cells were transfected with human NT-3 expression plasmid and metabolically labeled, and then the coded medium was screened for the desired polypeptide. In the experiment whose results are shown in Figs. 12, COS-M5 cells were transfected with plasmid pC-hN3 (PI) (described above), cells were labeled with cysteine ​​mixture, growth medium was fractionated on SDS PAGE 15% acrylamide gel, and protein was transferred onto membrane (as described by Fowbin et al. , 1979, Proc Natl Acad Sci.

USA, 76, 4350-4354), labeled polypeptides were detected by autoradiography. Simulating transfection. cells were used as a control (lane marked Mosck). As shown in Figs. 12, the expression plasmid pC8-hN3 (PI) expressed a single polypeptide with an estimated size of about 14 kD (indicated in Figure NT-3), which was not in control. By the error of this method, it was in good agreement with the presumed size of the mature NT-3 protein.

11th Neurotrophin-3 maintains viability of rat ventral mesencephalic cultures of dopamineergic neurons

The ventral midbrain culture of rat embryo E14 was prepared according to US patent no. No. 07 / 400,591, issued August 30, 1989, which is incorporated by reference in its entirety. The culture was inoculated at a cell density of 100,000. cells 1 cm or 50 thousand. cells per cm 2 (see Figures 13 and 14), and were grown in the absence of neurotrophic factor (control) or with increasing amounts of COS cell supernatant containing recombinant human neurotrophin-3. After 8 days in culture, cells were fixed and stained with monoclonal antibody against tyrosine-hydroxylase (TH) with a dopaminergic neuron marker. As we can see in Figs. 13 and 14 increase in the number of TH-positive cells surviving after 8 days with increasing NT-3 levels, with a maximum of 2.5-fold higher than in the control diluted 1:25 in NT-3 COS cell supernatant. Purified neuronal growth factor was found to be unaffected, and the effects of NT-3 were analogous to those of BDNF.

12th An example. NT-3, BDNF and NGF in the developing rat nervous system: parallel and receptor expression distributions

12.1. Methods

12.1.1. Materials and Dissections

Spraque-Dawley rats from Harlan Spraque-Dawley Ine. were used for all dissections. Dissections of the brain of mature animals were performed under standard microscopic anatomical markings. The cortex samples included the neocortex and the dorsal areas of the olfactory cortex. Intermediate brain samples were taken using cerebellar sections (stria medullaria) and crossing the optic nerves as dorsal and ventral marks, respectively. The midbrain specimens were from colliculi superior and colliculi inferior, dorsally, and continued toward the ventral surface of the brain to the most protruding edge of the pituitary gland. The posterior brain samples did not contain the cerebellum but contained the pituitary gland and the oblong brain. It should be noted that only the major areas of strictness were used, so that caudal pole dissection was performed on the tubercle tissue to avoid contamination. The hippocampus from the fimbria / fornix region to the neonatal brain is performed using analogous markings except for stria medullaria. Artificially fertilized rats were used to obtain embryonic tissues, indicating the date of fertilization marked with EI, and the day of neonatal appearance is indicated as PO. Adult rats weighed 150 to 275 grams (6-8 weeks).

12.1.2. RNA isolation and Nothern blotting.

Selected tissues were excised from rats and freeze frozen in liquid nitrogen. iRNA was isolated by homogenization on 3M LiCl / 6M in urea as described (Bothwell et al., 1990, Methods for Eukaryotic Gene Detection and Analysis, RNA (10 µg) Assay in Four

Jons and Bartlett, Boston, MA). was fractionated by electrophoresis on 1% stitched agarose gels (Bothwell et al.) and transferred onto nylon membranes (Magna Graph, Micron Separations) using 10x standard citrate saline, pH 7.0. These RNAs were UV ligated (Stratalinker®, Stratagene Ine.) And hybridized at 68 ° C with radioactive probes in 0.5 M sodium phosphate buffer (pH 7.0) with 1% bovine serum albumin (Fraction V, Sigma), 7% SDS, 1 mM EDTA (Mahmondi and Liss, 1989, Biotechniques, 7, 31-33) and

100 pg / ml of digested salmon sperm DNA. Filters were washed at 68 ° C with 2xSSC, 0.1% SDS, and exposed for 1 day to two weeks with a reinforcing screen (Cronex®, Du Pont) using X-ray tape (XAR-5, Kodak) at 70 ° C. The development of four gels with ethidium bromide revealed that the same amounts of total RNA were found in various tissues (Mainsonpierre et al., 1990, Science, 247, confirmed by hybridization with several specific 28SrRNAs).

1446-1451), it was probes blots,

12.1.3. Preparation of NT-3, BDNF, NGF and NGFR probes

NT-3, BDNF Expression Proc. Natl described above).

and cloning of NGF coding regions into and Secel, 8573-8577),

1987, was viewed.

vector pCMM8 (Aruffo Acad. Sci. USA, 84, supra (Maisanpiere

All of the Xhol inserts in these plasmids were 800 base pairs (bp) isolated on an acrylamide gel and electroeluted (Botthwell et al., 1990), followed by labeling with the 32 P random sixes (Bothwell et al.); by hybridizing with each probe synthetic NT-3, BDNF, and NGF transcripts (see below), it was found that the probe specific for one neurotrophin did not hybridize with the related neurotrophin transcripts. The rat NGFR probe was a 1.6 kb Ncol cDNA fragment extending the coding region of the rat NGFR protein (Radeke et al., 1987, Nature 325, 593597).

12.1.4. Obtaining and Quantifying a Synthetic Transcript.

The phage T7 promoter contained in the expression constructs pCDM8 / neurotropin described for the production of synthetic RNA regions, originally identified above, contained transcripts corresponding to the sense orientation of the encoding NT-3, BDNF and NGF. the amount of these synthetic transcripts was spectrophotometrically. Subsequently, the transcripts were examined by labeling its end [Bothwell et al. ) With a 30 nucleotide probe hybridized to the common 5 'end (immediately after the T7 promoter) for all three Densitometric Scanning Densitometers Series 300, Molecular Dynamics, Ine.) Synthetic transcripts for dot- and Northern blots. (The computerized hybridization (temperature hybridization, above) was performed with an oligonucleotide probe performed at 55 C as described and the washes were other operations, confirming that the amounts of synthetic transcripts can be used as accurate standards (data shown in Figure 15A).

12.1.5. Quantification of neurotrophic transcripts in densimetry.

Concentrations of transcripts in the various samples were normalized to a standard sample from a mature rat brain (see above) in the following manner. Equivalent aliquots of mature brain RNA samples were plated on each blot. Densitometric scanning of multiple exposure autoradiographs was performed to determine the signal intensity of each sample. The signal intensity of each sample was determined for each scan exposure. For each scan exposure, the signal intensity of each sample was divided by the signal intensity of the mature brain sample at the same exposure, thereby normalizing all settings to standardized mature brain samples. FIG. The level of transcripts presented in Fig. 18 was normalized to mature brain level in various samples, and mature brain level was accepted at 1.0. The detection of fg neurotrophic factor in a total brain sample of 1 mg of mature brain sample was successful in determining true transcription levels (fg / hg) in samples normalized to the mature brain sample.

12.2. RESULTS

12.2.1. Quantification of NT-3, BDNF, and NGF iRNAs and comparison of their levels in adult rat brainstem.

for comparison

Quantitatively transcript

Northern blotting was used to quantify and express NT-3, BDNF, and NGF transcripts in various tissue samples, each of the neurotrophic factors in various samples being determined by precise synthetic standards. Synthetic NT-3,

RNAs of BDNF and NGF transcripts, the concentration of which was accurately determined (see Fig. 15A and explanatory notes), were introduced during blotting as well as 10? G of total RNA isolated from mature rat brain. They were hybridized with radioactive probes and autoradiographed (Fig. 15B). Scanning densimetry was used for mature. for comparison of hybridization signals of brain sample and synthetic standards. In these studies, all samples had approximately iRNA levels (40 fg NT-3 transcripts, 45 fg BDNF transcripts and 30 fg 1 ng NGF transcripts) in the mature rat brain of all neurotrophins. In all other blots, an aliquot of a standard mature rat brain sample was added, thereby quantifying expression levels of neurotrophic factor transcripts (see below). To facilitate visual assessment of the expression levels of all three neurotrophin transcripts in the various samples, exposures were selected so that the signal intensity from a standard mature rat brain sample is the same for all neurotrophins by normalizing signals in other tissue samples to the appropriate standard.

12.2.2. Gene expression of NT-3, BDNF, and NGF exhibits common and individual features of development.

In the study of the expression of a neurotrophic gene in rat embryos, all three neurotrophins were found to be abruptly expressed between day 11 and day 12 of embryo development. FIG. 16A: All three neurotrophin transcripts are prominent in Eli and E12 embryos. The time course of a sharp increase in neurotrophin gene expression coincides with the time when neurogenesis is actually occurring (both peripheral and central) and coincides with the onset of axon formation by these neurons (see, e.g., Altman and Bayer, 1982, Adv. (Anat. Embryol. Cell. Biol. Vol. 74; Altman and bayer, 1984, ibid., Vol. 85).

Despite this coordination of neurotrophic gene expression during embryogenesis, NT-3 iRNA expression was found to be higher in early embryos (180 fg / ng total RNA) when compared to the standard sample of mature brain (5). -10 fg / ng total iRNA) and NGF iRNA at intermediate level (30 fg / ng total iRNA) (Figure 16A). There is a reciprocal relationship between NT-3 and BDNF expression in the developing brain (Figs. 16B) or in a strongly innervated heart (Fig. 16C), initially high NT-3 expression decreases, while low baseline BDNF expression increases until the adult animal finally converges; NT3 expression remains constant. Interestingly, expression of NT-3 increases with the development of liver and thymus, organs that are weakly innervated and do not express BDNF iRNA expression (Fig. 16C).

Embryonic expression of the NGFR transcript appears to precede the increase in neurotropic gene expression and is unexpectedly high in the early spinal cord and decreases during prenatal development of the brain and postnatal development of the heart (Fig. 16A, B and C).

12.2.3. Comparison of NT-3, BDNF, NGF, and NGFR Expression in Adult and Neonatal Nervous Systems.

To determine the spatial distribution of neurotrophic gene expression in the rat nervous system and to understand how distinctly developed brain profiles interact between distinct areas of the brain, neurotrophic gene expression in neonatal brain and adult rat brain was investigated. The expression of all three factors was discrete in space and time of development (Fig. 17). Quantification of expression levels of transcripts, including in peripheral tissues, is presented graphically in Figs. 4. The main similarity between all three factors is the high level of expression in the adult hippocampus. The differences are in peripheral tissues of adults where NT-3 and NGF expression is similar (Maisonpierre et al., Supra). There is a clear parallel between NT-3 and BDNF expression in the adult brain (Figs. 17B, 18B); interestingly, both factors are absent in the striatum. But NT-3 and BDNF exhibit interesting and apparently interrelated differences when comparing expression in neonatal and adult brains (Figs. 17A, B and Fig. 18A, B). NT-3 expression is highest in neonates, and is significantly higher than in adults in less mature areas of the brain (ie, cerebellum, hippocampus (ammonia rage) and neocortex). BDNF expression in both of these areas is lowest and highest, similar to that in adults, in the more caudal areas of the brain than in areas that mature earlier (ie, hindbrain, midbrain, and midbrain). As in the mature brain, NT-3 and BDNF transcripts are not detected in the neonatal striatum.

Comparing NT-3 and BDNF expression levels with NGF iRNA expression levels, we found that NGF expression levels in neonatal and adult animal brain tissues are less variable (Fig. 17, 18). NGF expression levels in olfactory cavities are higher in neonates when NGF expression is levels in the hippocampus and neocortex are higher in adult animals. NGFR iRNA expression levels were higher in neonates than in adult brains, with exceptionally high levels in neonatal cerebellum and hindbrain (Fig. 17A, B).

12.2.4. Examination of NT-3, NGF, and DBNF expression during development of discrete central nervous system areas.

To confirm that NT-3 is more overexpressed in the early stages of development of the central nervous system, whereas BDNF is mainly expressed in the later stages of the development of the same regions, neurotrophic factor gene expression was investigated in three central nervous system maturations. . Neurogenesis followed by natural cell death begins very early in the spinal cord (E12E13) and ends a few days before birth (Altman and Bayer, see above). In contrast, most neurons in the cerebellum and hippocampus (ammonium rage) (estimated by granulocyte populations) occur at birth (e.g., Altman, 1996, J. Comp. Neur., 128, 431474; Schlessinger et al., 1975, J. Comp. Neur. ., 159, 149-176). In the later stages of cerebellar development, intensive neurogenesis, neuroblast migration, and neuronal differentiation occur during the first three weeks after birth (Large et al., 1986, Science 234, 352355); this increase occurs much later in the perinatal process, characterized by granulocellular proliferation (e.g., Altman, 1986, see above) and by the interference of cholinergic neurons from the basal precursor brainstem (Koh and Ioy, 1989, J. Neurosvi, 9, 2999-3018), but coincides with further cholinergic differentiation of these neurons (Xarge et al., Supra).

Our evolving spinal cord studies (Fig. (Fig.5A, E)) showed high levels of NT-3 expression in E12-E13 (150-280 fg / ng total RNA), which are lower at birth and almost undetectable in mature animals. BDNF iRNA is barely detectable in E12-E13, reaches its peak at birth (10-20 fg / ng total RNA) and then decreases. The highest expression of NGF iRNA is observed in the E12-E13 spinal cord (15-25 fg / ng total RNA) but 10-fold lower than the expression of NT-3 iRNA at the same stage. Interestingly, NGFR expression is higher in early spinal cord; this expression was previously correlated with the natural period of cell death in the early spinal cord of new forming neurons (Ernfors et al., 1989, Neuron, 2, 1605-1613).

The late developmental cerebellum (Fig. 19B, G) expresses quite high levels of NT-3 iRNA (500-820 fg / ng total RNA) during the first three weeks after birth, and BDNF expression begins to increase only towards the end of this period, in the early stages of cerebellar development. only very low levels of NGF iRNA were detected. NDFR is expressed in large amounts in the early stages of cerebellar development and then decreases before NT-3 iRNA levels begin to decline.

Ammonium horn (Fig. 19C, G) as well as BDNF and NGF iRNA levels increase from small amounts in E17 to medium at birth and reach peak levels in adult animals. Although all three neurotrophin transcripts are expressed in similar amounts in adult animal ammonia rage, NT-3 expression is significantly higher than in BDNF and NGF E17 and neonatal ammonia rage; The expression level of NT-3 in ammonia horn is high as in perinatal cerebellum (820 fg / ng total RNA). Unlike neurotrophins, NGFR expression is decreased during development of the ammon horn.

In all three CNS domains tested, expression of NT-3 is higher with development of these domains and then decreased to the level of mature animals, whereas small amounts of BDNF iRNA initially increase to mature levels, similar to NT-3. Unlike the interconnected expression profiles of NT-3 and BDNF, NGF expression does not exhibit any sequential sequence; NGF is more expressed (albeit in very small amounts) in the early cerebellum and cerebellum than in the late ammonium rage.

12.3. Discussion.

Expression similarities and differences in the temporal and expression domains of the three neurotrophin transcripts were identified in this analysis. The amount of NT-3, BDNF, and NGF transcripts increases simultaneously between day 11 and day 12 of rat embryogenesis. These expressions coincide with intense neurogenesis (Fig.,

Bayer, 1982, cf. above); (Fig., Altman and Bayer, 1984, see above). This relationship confirms that neurotrophins are very important to the nerves can be distinguished in a period when all neurotrophins become necessary for the viability of post-mitotic neurons. However, their expression moments may indicate other roles for neurotrophins in the developing nervous system (see below).

increase in Altman and all three for development,

Although the peak of expression intensity occurs simultaneously for all three neurotrophins, the levels of expression they reach in early embryos differ markedly. NT-3 iRNA levels are highest in embryos; and BDNF iRNA is expressed in trace amounts. This contrast between NT-3 and BDNF expression persists at almost every instant examined. The development of NT-3 expression is most pronounced in CNS regions involved in proliferation, migration, and neuronal and proneuronal differentiation; and usually abrupt decrease in mature CNS areas. In contrast, BDNF expression is highest in areas of the CNS that have already undergone neurogenesis and are usually increased as these CNS areas mature. Surprisingly, the amounts of NT-3 and BDNF transcripts present in the CNS regions of mature animals are the same. The interplay between NT-3 and BDNF expression in the CNS, complemented by their similar expression profiles in the adult animal CNS, suggests that NT-3 and BDNF may in some cases interact with the same neuronal populations in the CNS. If true, our findings suggest that NT-3 is critical for the development of these neurons (apparently during target innervation), whereas BDNF acts on neurons later (ie, as a factor in maturation and viability). NGF expression changes locally with development, but is different from NT-3 and BDNF. The levels of NGFR iRNA expressed have no specific association with the expression of any of these three neurotrophin genes, which may be attributed to the fact that NGFR is a common component of individual neurotrophic receptors (Radriguez-Tebar et al., 1990, Neuron, 4, 487-492). .

NGFR expression tends to be higher in the early stages of CNS development; our studies have identified interesting, developmentally regulated, changes in NGFR expression that are the subject of further research.

The distribution of NT-3 and BDNF in the mature CNS is similar and the distribution is different in the peripheral tissues of adult animals; the broader peripheral distribution of NT-3 transcripts may reflect its activity on a wider spectrum of peripheral cells (nerve cells and non-neural connections), unlike BDNF (Maisonpierre et al., supra).

Although there is important evidence that NGF and BDNF are important in the early stages of nervous system development, our analysis found a more consistent and pronounced correlation between high NT-3 expression and early nervous system development. The levels of NT-3 iRNAs in the developing cerebellum and in the neonatal amniotic fluid are several times higher than those of any neurotrophin in any tissue or brain region and more than several times that of any neurotrophin in the brain of adult animals. The finding that NT-3, a novel cognate NGF protein, can support the viability of at least some BDNF and NGF-dependent neurons (Maisonpierre et al., Supra), and that its expression over time clearly coincides with critical periods of development of the nervous system, that NT-3 is a physiological agent responsible for some important developmental functions that were previously attributed to BDNF and NGF. New studies on the role of members of this gene family are very important because antibodies against these factors can affect their function (Whitlmore, senger, 1987, Brain Res. Rev., 12, 439-464).

Although we have previously demonstrated that NT-3 may act as a classical neuronal viability-maintaining compound (Maisonpierre et al., Supra), it is appreciated that NT-3 acts as a target-derived factor limiting expression of neuronal selection and selection. in no way eliminates other important functions of NT-3 (as with other neurotrophins). The distribution of NT-3 expression in the developing nervous system is similar to that of nestin (a novel filamentous intermediate whose expression is specific for CNS domains during neurogenesis Lendahl et al., 1990) and SNAAPA, an early neurite antigenic marker (Jamamoto et al., 1986, J. Neurosci, 6, 3576-3594). Unlike NGF (Cleoo et al., 1989, Devel. Biol., 134, 30-37), high expression of NT-3 is observed before sympathetic growth reaches the heart. In this way, NT-3 may be associated with developmental processes that do not involve maintenance of neuronal viability, including proliferation (proneuron differentiation) and / or management of cell or axon migration; a further assumption about such a potential physiological role for NT-3 is based on new studies - one neurotrophin (ie, NGF) may influence proneuron proliferation in vitro. In contrast, BDNF, although transcribed in the early stages of development, may have a more general role much later in the initial neuronal death and selection period.

The expression profiles of all three neurotrophins in adult animals are surprisingly similar - all three neurotrophins are expressed in relatively high amounts in adult ammonia. Discontinuation of basal forebrain ammonium rage in cholinergic neuronal outgrowth contributes to atrophy and decreases neuronal synthesis of mediators (reviewed in Smider and Jonson, 1989, Am. Neurol., 26, 489-506). Similar atrophy has been associated with poor performance in memory tests in old rats, and in humans with Alzheimer's disease. Atrophy of cholinergic neuronal sprouting in the basal forebrain can be reversible in rat models by the introduction of NGF. The data presented here do not exclude the possibility that the mature ammonium horn normally supplies all three neurotrophins to the basal forebrain, given that NGF and BDNF are complementary to cholinergic neurons in culture, reflecting the true physiological roles of these compounds. But NT-3 expression is very high in the early stages of ammon horn development and then decreases to adult size, which is similar to NGF and BDNF. This high level of early expression of NT-3 suggests that NT-3 may have a unique role in the control or generation of early derivatives from the basal forebrain or other hippocampal afferents, or the proliferation of starved progranulocytes; the relatively low levels of NGF expression in the early stages of ammonium horn development suggest that NGF does not play such a role (Large, supra).

13th Deposition of microorganisms

The microorganism strains shown below were deposited in 1990. February. March 28 In the American Type Culture Collection, 12301 Parkland Drive, Rock Ville, Maryland, 20852:

<td></td><td>Strain</td><td>ATCC</td>

<td>Bacteriophage DNA</td><td>&lt;To) hN3 (GI)</td><td>collection number 40763</td>

<td>Bacteriophage DNA</td><td>&lt;j) rN3 (GI)</td><td> 40764</td>

<td>Plasmid</td><td>pC8-rN3 (PI)</td><td> 40766</td>

<td>Plasmid</td><td>pC8-hN3 (PI)</td><td> 40765</td>

The present invention is not limited to the following exemplary embodiments of the invention. In fact, various modifications of the invention are possible, in addition to the description which will be apparent to those skilled in the art from the description and accompanying drawings. Such modifications include the definition of the present invention. The description cites various sources used as references.