RU2354662C2 - Noggin2-based activin activity blocking method - Google Patents

Abstract

FIELD: chemistry. ^ SUBSTANCE: invention refers to biotechnology, specifically to Noggin2 proteins and can be used in medicine. The signalling cascade activin is blocked by introducing Noggin2 in an organism, tissue or cell in amount effective to inhibit or decrease activin activity. ^ EFFECT: invention allows blocking effectively activity of protein activin. ^ 2 dwg, 8 ex

Description

Field of Invention

This invention relates to proteins that regulate cell differentiation by inhibiting TGF-β superfamily proteins.

State of the art

The superfamily of transforming growth factor P (TGF-P) includes many growth factors that share common structural motifs. These proteins are involved in a wide range of biological processes, such as the processes of cell and tissue differentiation during embryonic development and wound healing, restoration and reconstruction of bone tissue in the adult body. A number of intercellular matrix proteins have been identified that are antagonists of various representatives of the TGF-β superfamily (e.g., bone morphogenesis proteins, BMP) and play an important role in the processes of cell differentiation and development (Balemans and Van Hul, Dev Biol. (2002) 250 (2): 231-250; Avsian-Kretchmer and Hsueh, Mol Endocrinol. (2004) 18 (I): 1-12). BMP antagonists include, for example, chordin, ventroptin, noggin, cerberus and follistatin proteins.

For example, the proteins cerberus and noggin vertebrates induce the formation of head structures in the anterior ectoderm of vertebrate embryos. The noggin protein is also able to change the differentiation of mesoderm primordia from ventral primordia, such as blood and mesenchyme, into dorsal primordia, such as muscle or chorda, or differentiation of epidermal primordia into neural rudiments. The functions of chordin are similar to those of noggin1, reflecting a similar mechanism of action of these proteins as antagonists of BMP.

TGF-β superfamily growth factor antagonists are important pharmaceutical, clinical, and laboratory tools for regulating cell differentiation and therapeutic intervention.

Recently, a new protein noggin homologue, called noggin2, has been described in vertebrates (Fletcher et al., Gene Expr Patterns. 2004, v. 5 (2), pp. 225-230). The noggin2 gene has been shown to have a differential expression pattern during the development of Xenopus. However, the function of noggin2 has not been investigated. The present invention relates to the function of noggin2.

SUMMARY OF THE INVENTION

The authors of the present invention discovered that noggin2 plays a role in the regulation of cell differentiation and is an antagonist of some members of the TGF-P superfamily. Moreover, the spectrum and nature of the action of noggin2 are different from those of noggin1 (formerly “noggin”). In particular, unlike noggin1, noggin2 does not induce the development of muscle tissue from embryonic ventral mesoderm. In addition, it may interfere with the well-known activity of noggin1 in relation to the induction of muscle development. At the same time, like noggin1, noggin2 can induce the development of nervous tissue from the embryonic ectoderm. Thus, noggin2 has unique selective activity in relation to the induction of the development of nervous tissue.

The revealed ability of noggin2 to inhibit differentiation of muscle tissue is explained by its ability to inhibit the general differentiation of embryonic mesoderm due to its interaction with a different (wider) spectrum of factors from the TGF-β superfamily. In addition to inhibiting the epidermal activity of BMP2, BMP4 or BMP7, which is also known to be inhibited by noggin1, noggin2 has the ability to inhibit the activity of an embryonic mesoderm inducer such as activin.

The present invention relates to methods and compositions that use noggin2 or a nucleic acid encoding it.

In a preferred embodiment, the present invention relates to a method for reducing or blocking the activity of specific Noggin2 targets (e.g. BMP2, BMP4, BMP7, activin, or a combination thereof) in an organism, tissue or cell, comprising administering nailgin2 to the specified organism, tissue or cell in an amount effective to inhibit or reduce the activity of specific noggin2 targets by inhibiting the binding of these targets or fragments thereof to specific protein receptors.

In a preferred embodiment, the method and compositions are used to modify cell differentiation, where the method involves bringing the cell or the surrounding medium into contact with the noggin2 protein under conditions where the noggin2 specifically interacts with its target components of the medium and / or cell surface, to modify cell differentiation.

In one preferred embodiment, the exogenous protein noggin2 is used; in another preferred embodiment, an expression construct is used comprising the Noggin2 encoding nucleic acid under the control of a suitable promoter. In this embodiment, a method for altering cell differentiation involves the creation of an expression construct comprising a nucleic acid encoding leggin2 under the control of a suitable promoter; introducing said expression construct into a cell for expressing noggin2 or a functional chimeric protein containing noggin2, where the expression of noggin2 or said chimeric protein leads to a change in cell differentiation.

Brief Description of the Figures

Figure 1 shows the difference between the effects of noggin1 and noggin2 on the differentiation of nervous and muscle tissue in Xenopus laevis embryos and their tissue explants. (VI) a 1 L situ hybridization signal is shown in black.

(A, B). Ventral microinjections of 40 pg of synthetic noggin1 gene mRNA in X. laevis embryos at the early stages of development lead to the formation of an additional body axis (black arrow indicates the main body axis, gray arrow indicates the additional body axis), while microinjections of the same amount of synthetic mRNA noggin2 leads to the formation of a mushroom-like phenotype (A: 80%, 68 out of 85 embryos in total; B: 90%, 68 out of 75 embryos in total).

(VJ). In situ hybridization of control embryos (to the left of each of their photographs) and embryos microinjected with noggin2 mRNA with mRNA samples of the given genetic markers shows an increase in neural (B, D) and inhibition of epidermal (D) and mesoderm (E, G) differentiations. The signal is shown in black.

(H). Explants of the ventral marginal zone (VMZ) of embryos micro-injected with ventral RNA of noggin1 are extended towards the end of neurulation due to differentiation of skeletal muscles.

(AND). The explants of the ventral marginal zone (VMZ) of embryos microinjected ventrally with Noggin2 RNA retain a rounded shape at the end of neurulation.

(TO). Scheme of experiments with ectoderm explants of the animal region of the embryo (animal caps - AS) and explants of the ventral marginal zone of the embryo (VMZ).

(L). Reverse transcription - PCR analysis of total RNA samples of ectoderm explants of the animal region of the embryos and explants of the ventral marginal zone of the embryos, microinjected mRNAs of noggin1 and noggin2 with primers for neuroectodermal (NCAM, Xanf-1), epidermal (keratin-arterial) muscle mesoderm (brachyury) and endo mesoderm (cerjberus) molecular markers and Ef-1α as a control for the amount of mRNA. Noggin1 and noggin2 activate the expression of neural markers (NCAM, Xanf-1) and inhibit the expression of epidermal (keratin), while only moggin1 mRNA displays the ability to activate the expression of the muscle molecular marker α-actin in explants of the ventral marginal zone.

Figure 2 shows the difference between the effects of noggin1 and noggin2 on the expression of the jbrachyury, BF-1, and Pax-6 genes and analyzes the ability of the noggin2 protein to bind to BMP-4 and activin. (AE) - results of in situ hybridization of total preparations of Xenopus laevis embryos with probes for the corresponding genes (the in situ hybridization signal is shown in black); (A'-E ') - the same embryos are shown under ultraviolet light, which allows you to visualize microinjected cells containing in addition to the mRNA tag - fluorescein-lysine-dextran (FLD).

(A, A '). Suppression of expression of a mesoderm molecular marker (brachyury) in embryos microinjected with noggin2 mRNA. A '- green fluorescent dye shows the distribution of microinjected material.

(B, B '). Expression of a mesoderm molecular marker (brachyury) in the embryos microinjected with noggin1 mRNA does not show changes compared to the control embryo (arrow).

(B, B ', D, D'). The formation of the additional axis of the body in embryos microinjected with 2-5 ng noggin2 mRNA into one of the ventral blastomeres. The presence of the expression of molecular markers BF-1 and Pax-6 indicates the presence of anterior-head and ocular structures in the additional axes.

(G, G ', E, E'). Additional body axes formed upon microinjection of 40 ng moggin1 mRNA into one of the embryo ventral blastomeres of the embryo do not contain forehead and ocular structures.

(G). Noggin2 protein exhibits the ability to bind bone morphogenetic protein (BMP) molecules and activin molecules. A detailed description of the experiments is given in the text of examples 4 and 5.

(H). The noggin2 protein shows the ability to inhibit the phosphorylation of intracellular BMP activin signal transmitters, the Smad1 and Smad2 proteins, respectively. A detailed description of the experiments is given in the text of examples 4 and 5.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term "ortholog" refers to a polypeptide or protein derived from one species that is a functional copy of a polypeptide or protein of another species. Differences in ortholog sequences are the result of speciation.

As used herein, the term “homologue” or “homology” is a term used in the art to describe the affinity of a nucleotide or protein sequence of another nucleotide or protein sequence, determined by the degree of identity and / or similarity between said compared sequences.

As summarized above, methods for changing the activity of certain members of the TGF-J3 superfamily are disclosed, as well as compositions containing a noggin2 protein, a nucleic acid encoding it, or anti-noggin2 antibodies for use in them. These methods and compositions for their practical use can be used in a number of research, diagnostic or therapeutic applications, including, but not limited to, inhibition or reduction of inflammation; regulation of bone reconstruction in the adult skeleton; the prevention and treatment of BMP and activin-related disorders in animals, in particular humans; research and treatment of heart diseases and neurological disorders.

The present invention relates to the use of the specific function of noggin2. Using the example of the noggin2 protein from Xenopus laevis with the amino acid sequence given in SEQ ID NO: 2, the authors discovered that nuggin2 plays a role in regulating cell differentiation and is different from noggin1 and other known growth factor antagonists belonging to superfamily TGF-P. Unlike noggin1, noggin2 suppresses the general differentiation of the embryonic mesoderm, inhibiting the action of the mesoderm inducer, activin.

At the same time, like noggin1, noggin2 induces the development of nervous tissue from the embryonic ectoderm. Thus, noggin2 has unique selective activity in relation to the induction of the development of nervous tissue. The revealed ability of noggin2 to inhibit dorsalizing activity of noggin1 is the result of its ability to bind to some members of the TGF-β superfamily other than BMP2, BMP4 or BMP7. In particular, activin and its related proteins, necessary for muscle induction from embryonic mesoderm, are additional TGF-β factors that bind to noggin2.

The term “noggin2” as used herein means a protein having unique and selective activity for inducing the development of neural tissue, binding to noggin2-specific binding targets selected from the group consisting of BMP2, BMP4, BMP7 and activin, or a combination thereof, and a homologue and an ortholog of protein noggin2 selected from the group consisting of Xenopus laevis noggin2; Noggin2 Xenopus tropicalis; Noggin2 Fugu rubripes; Noggin2 Gallus gallus (SEQ ID NO: 2, 4, 6, 8). Mammal noggin2, including human noggin2, is of particular interest.

Noggin2-specific activity or function can be determined by known cell biology approaches in vitro or in vivo systems, e.g. in vitro binding assays, cell culture assays, in animals (e.g. immune response, gene therapy, transgenic animals, etc.) .) etc. Binding assays include any assay that assesses the specific molecular interaction of noggin2 with a binding target. The target for binding may be a natural target for binding or non-natural target for binding, such as a specific immune protein, such as an antibody. Typically, binding specificity is assessed by biological analysis (for example, the ability to induce nerve tissue from an injected embryonic ectoderm) or by determining the binding constant of noggin2 to its targets from the TGF-β superfamily, etc.

By protein homologue is meant a protein whose amino acid sequence is at least about 68%, typically at least about 75%, and most often at least about 85%, is identical to the known amino acid sequences of noggin2 given in SEQ ID NO: 2 , 4, 6, 8, as determined using the MegAlign algorithm, DNAstar clustal algorithm, as described in DG Higgins and P.M. Sharp, "Fast and Sensitive multiple Sequence Alignments on a Microcomputer", CABIOS, 5 pp. 151-3 (1989) (using multiplicity 1 parameters, pass penalty 3, windows 5 and saved diagonals 5). In many implementations, the homologues of interest have a much greater sequence identity, e.g., 90% (e.g., 92%, 93%, 94%) or higher, e.g., 95%, 96%, 97%, 98%, 99%, 99, 5%, especially for the amino acid sequence that forms the functional regions of the protein.

Also of interest are proteins that are substantially identical to noggin2 proteins with the amino acid sequences of SEQ ID Nos. 2, 4, 6, 8, where significant identity means that the amino acid sequence of the protein is at least about 68% identical to that of the protein typically at least about 75% and most often at least about 80%, where in some cases the identity may be more than, for example, 85%, 90%, 95% or more.

Proteins of interest include naturally occurring proteins and recombinant proteins containing the amino acid sequence of noggin2 or its functional protein domains having definable analysis of the specific activity of noggin2. Thus, proteins can be deletion mutants of natural noggin2 proteins, and they can be obtained in the form of hybrid products containing noggin2 polypeptides or its functional protein domains. The fragments of interest are polypeptides, typically at least about 30 amino acids long, typically at least about 50 amino acids long, preferably at least about 75 to 100 amino acids long, and can be up to 300 amino acids or more in length, but, as a rule, do not exceed 250 amino acids in length, where the fragment contains a portion of the amino acid sequence identical to the protein in question, at least about 25 amino acids in length, and, as a rule, at least 45 amino acids, and in many embodiments, at least 50 amino acids. In some embodiments, the subject polypeptides are approximately 25 amino acids, approximately 50, approximately 75, approximately 100, approximately 125, approximately 150, approximately 200, or approximately 250 amino acids in length, up to a full length protein. The fragments of interest retain all or substantially all of the specific functions of the wild-type protein.

Also of interest are proteins that are derivatives or mutants of naturally occurring proteins with specific noggin2 activity. Mutants and derivatives may retain the biological properties of wild-type proteins (eg, those found in nature) or may possess biological properties different from those of wild-type proteins. The term “biological property” of the proteins of the present invention refers to biochemical properties, such as in vivo and / or in vitro stability (eg, half-life); ripening speed, ability to aggregate or to form oligomers and other such properties, but is not limited to them. Mutations include substitutions of individual amino acids, deletions or insertions of one or more amino acids, shortening or lengthening at the N-terminus, shortening or lengthening at the C-terminus, and the like.

Also of interest is the nucleic acid encoding leggin2 comprising a nucleic acid selected from the group consisting of SEQ ID NO: I, 3, 5, 7; nucleic acids, substantially identical to these nucleic acids and their mutants, derivatives and homologs. Nucleic acids encoding mammalian noggin2 proteins (e.g., human noggin2) are of particular interest.

By a nucleic acid homologue is meant a nucleic acid identical to the corresponding nucleotide sequence by at least about 30%, and preferably by about 40%, 50%, 55%, 60%, 65%, 70% or higher, including 75%, 80%, 85%, 90% and 95% or higher. The control sequence is typically at least about 30 nucleotides long, more often at least about 60 nucleotides in length, and can reach the length of the complete sequence to be compared. Sequence similarity is calculated based on the control sequence. Algorithms for sequence analysis such as BLAST described in Altschul et al., J. Mol. Are known in the art. Biol., 215, pp. 403-10 (1990) (for example, using default values, i.e., parameters w = 4 and T = 17).

The source of homologous nucleic acids and protein can be any type of plant or animal, or the sequence can be fully or partially synthesized, including nucleic acid analogs.

Mutants or derivatives of noggin2 proteins and nucleic acids can be formed using standard molecular biology methods to a matrix nucleic acid encoding a noggin2 protein by modifying, deleting, or adding one or more nucleotides of a template sequence or combination thereof to form a template nucleic acid variant. Substitutions, additions or deletions can be made by any method known in the art (see, for example, Gustin et al., Biotechniques (1993) 14: 22; Barany, Gene (1985) 37: 111-123 and Colicelli et al., Mol Gen. Genet. (1985) 199: 537-539, Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108), which is a PCR with errors, permutation, oligonucleotide- directed mutagenesis, assembly PCR, mutagenesis through sexual PCR, in vivo mutagenesis, cassette mutagenesis, recursive group mutagenesis, exponential group mutagenesis, site-directed mutagenesis, random mutagenesis, gene rearrangement in, saturation mutagenesis of gene portions (GSSM), synthetic ligation permutation (SLR) or a combination thereof.

Substitutions, additions or deletions can also be introduced by a method involving recombination, recursive recombination of sequences, mutagenesis using a modified DNA phosphothioate, mutagenesis using a uracil matrix, mutagenesis using a duplex gap, mutagenesis using the recovery of point mismatches, mutagenesis using a host strain with a deficiency in the recovery system, chemical mutagenesis, radiogenic mutagenesis, deletion mutagenesis, mutagenesis has been applied restriction-selection, mutagenesis using restriction-purification, synthesis of artificial genes, group mutagenesis, the creation of multimers of chimeric nucleic acids and their combination.

In addition, degenerate variants of the protein-encoding noggin2 nucleic acids can be obtained. Degenerate nucleic acid variants contain substitutions of nucleic acid codons for other codons encoding the same amino acids. Specifically, degenerate nucleic acid variants are prepared to enhance their expression in the host cell. In this embodiment, nucleic acid codons that are not preferred or less preferred in the host cell genes are replaced by the codons most represented in the host cell coding sequences, wherein said substituted codons encode the same amino acid.

Derivatives can also be obtained using standard methods, including RNA modification, chemical modifications, post-translational and post-transcriptional modifications, etc. For example, derivatives can be obtained using methods such as changing the profile of phosphorylation or glycosylation or acetylation or lipidization, or using various types of cleavage of mature forms and the like.

Proteins and fragments of interest are isolated, i.e. exist outside the natural environment. The considered proteins and protein domains can be synthesized or obtained by recombinant technologies or isolated from their natural environment. There are many molecular and biochemical methods for the biochemical synthesis, molecular expression, and purification of these compositions, see, e.g., Molecular Cloning, A Laboratory Manual (Sambrook, et al., Cold Spring Harbor Laboratory), Current Protocols in Molecular Biology (Eds. Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, NY).

In preferred embodiments, the proteins of the present invention are recombinant proteins obtained using standard approaches using a Noggin2-encoding nucleic acid, for example, a nucleic acid selected from the group consisting of SEQ ID NO: I, 3, 5, or 7.

The nucleic acid molecule referred to herein is a DNA molecule, such as a genomic DNA molecule or a cDNA molecule, or an RNA molecule, such as an mRNA molecule. Specifically, said nucleic acid molecules are open reading frame cDNA molecules encoding the noggin2 protein of the present invention or a functional fragment thereof and are capable of being expressed under suitable conditions as the active noggin2 or functional domain of the noggin2 of the present invention.

The genomic sequence of interest may contain a nucleic acid located between the initiating codon and the stop codon, as defined in the listed sequences, including all introns normally present in the original chromosome. The genomic sequence of interest may include 5'- and 3'-untranslated regions located in mature mRNA, as well as specific transcriptional and translational regulatory sequences, such as promoters, enhancers, etc., including about 1 t. bp, but possibly more, flanking genomic DNA at either the 5 ′ or 3 ′ end of the transcribed region.

The nucleic acids of interest can be isolated and obtained in substantially purified form. The term "substantially purified form" means that the nucleic acids are at least about 50% pure, typically at least 90% pure and, as a rule, are "recombinant", i.e. flanked by one or more nucleotides with which they are normally not associated in the natural chromosome in their natural host organism.

Nucleic acids of the present invention, corresponding cDNAs, full-length genes and constructs can be artificially produced using various protocols known to those skilled in the art. Appropriate nucleic acid constructs are purified using standard recombinant DNA methods, for example, as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, and rules described, for example, in United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.

Also of interest are chimeric proteins containing noggin2 or fragments thereof, fused, for example, with a degradation sequence, a signal protein, or any protein or polypeptide of interest. Fusion proteins may contain, for example, the noggin2 protein and the second polypeptide (“fusion partner”), combined into a single reading frame at the N-terminus and / or C-terminus of the noggin2 polypeptide. Fusion partners include, but are not limited to, polypeptides capable of binding antibodies specific to the fusion partner (e.g., epitope tags), antibodies or their binding domains, polypeptides providing catalytic function or induction of a cellular response, ligands or receptors, or mimetics thereof etc. In such fusion proteins, the fusion partner is generally associated with a portion of the fusion protein, which is the noggin2 protein, a non-natural method and, as a rule, is not the noggin2 protein of the present invention or its derivative / fragment. Nucleic acids encoding the fusion proteins described above are also of interest.

Also disclosed are a vector and other nucleic acid constructs comprising a Nuggin2 encoding nucleic acid. Applicable vectors include viral and non-viral vectors, plasmids, cosmids, phages, etc., preferably plasmids, and are used for cloning, amplification, expression, transfer, etc. the nucleic acid sequences of the present invention in a suitable host. The selection of a suitable vector is well known to the person skilled in the art, and many such vectors are commercially available. To obtain constructs, a partial or full-length nucleic acid is inserted into a vector, usually by attaching a DNA ligase to a restriction enzyme digested region in a vector. Alternatively, the desired nucleotide sequence can be inserted by homologous in vivo recombination, typically by attaching homology regions to the vector on the sides of the desired nucleotide sequence. For example, homology regions are added by ligation of nucleotides or by polymerase chain reaction using primers containing a homology region and part of a desired nucleotide sequence.

Expressing cassettes or systems are also disclosed, inter alia, used to produce the noggin2 protein or its fusion protein, or to replicate nucleic acid molecules of the present invention. The expression cassette can exist as an extrachromosomal element, or it can be integrated into the genome of a cell as a result of introducing said cassette into the cell. For expression, the product of the gene encoded by the nucleic acid of the invention is expressed in any suitable expression system, including, for example, bacterial, yeast, insect, amphibian or mammalian systems. In the expression vector, the nucleic acid of the invention is operably linked to a regulatory sequence, which may contain promoters, enhancers, terminators, operators, repressors and inducers. Methods for preparing expression cassettes or systems capable of expressing a desired product are well known to those skilled in the art.

Cell lines stably expressing noggin2 can be selected by methods well known in the art (e.g., co-transfection with a selectable marker, such as dhfr, gpt, neomycin, hygromycin, which allows identification and isolation of transfected cells containing a gene integrated into the genome).

The expression systems described above can be used in prokaryotic or eukaryotic hosts. To obtain the protein, such host cells as E. coli, B. subtilis, S. cerevisiae, insect cells in combination with baculovirus vectors, or cells of higher organisms such as vertebrates, for example, COS7, HEK293, CHO cells, Xenopus oocytes, etc. can be used. .d.

When any of the above host cells or other applicable host cells or host organisms are used to replicate and / or express the nucleic acids of the invention, the resulting replicated nucleic acid, expressed protein or polypeptide fall within the scope of the invention as a host cell product or the host organism. The product can be isolated by any suitable methods known in the art.

Also disclosed are antibodies specifically binding to noggin2 of the present invention. Applicable antibodies can be prepared using methods known in the art. For example, polyclonal antibodies can be prepared as described in Harlow and Lane Antibodies: A Laboratory Manual, (1988) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, and monoclonal antibodies can be prepared as described in Coding Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology; 3rd edition, (1996) Academic Press. Hybrid antibodies are also of interest, including humanized antibodies, as well as single chain antibodies and antibody fragments such as Fv, F (ab ') ₂ and Fab.

Noggin2, as well as the other components of the present invention described above, are used in many different applications, including the use of immunogens, targets in screening assays, biologically active substances for modulating cell growth, differentiation and / or functioning, etc.

In a preferred embodiment, the present invention relates to a method for reducing or inhibiting the activity of specific noggin2 targets (e.g. BMP2, BMP4, BMP7, activin, nodal, or a combination thereof), comprising administering the noggin2 into the specified organism, tissue or cell in an amount sufficient to inhibit or reduce activity of noggin2-specific targets by inhibiting the binding of these targets or fragments thereof to specific protein receptors.

In a preferred embodiment, the methods and compositions provided are used to modify cell differentiation. In particular, a method for modifying cell differentiation involves bringing the cell or the environment surrounding the cell into contact with the noggin2 protein under conditions where the noggin2 specifically interacts with its targets, the components of the medium and / or cell surface, to effect a change in cell differentiation.

In one preferred embodiment, the exogenous protein noggin2 is used; in another preferred embodiment, an expression construct is used comprising the Noggin2 encoding nucleic acid under the control of a suitable promoter. Noggin2 protein can be added to in vitro culture media and body fluids such as blood, synovial fluid, etc. The protein can also be introduced or expressed in specific cell populations by any suitable method, such as microinjection, promoter-dependent expression of the recombinant enzyme, targeted delivery of lipid particles, etc. These methods can be used to reduce unwanted osteogenesis, inhibit cell growth requiring morphogenetic protein (e.g. BMP-dependent neuroblastomas and gliomas), regulate cartilage formation and growth, change morphogen-dependent cell development / differentiation directions in culture, such as cells for transplantation or infusion, etc. Since noggin2 causes activity in relation to the induction of nerve tissue, it can be used to treat peripheral neuropathies in the growth of the spinal cord and processes of sensitive neurons, for example, it can be included in therapeutic treatment for the regeneration of processes of neurons after strokes, brain damage caused by head injuries , and paralysis caused by spinal cord injuries. The use may also be in the treatment of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and multiple sclerosis, by stimulating the processes of neurons. Additional applications may include restoration of dissected axons, which often occurs in foci of damage in multiple sclerosis.

As an antagonist of a number of growth factors (for example, BMP2, BMP4, BMP7, activin, or a combination thereof), leggin2 or a fragment thereof is used as a suppressor of the activity of these factors, for example, morphogenetic bone protein (BMP), activin, or their orthologs. Preferably, the invention relates to a method for counteracting the functioning of said specific target, comprising bringing said target into contact with the noggin2 protein or fragment thereof. The method according to the invention is carried out under conditions when noggin2 or a fragment thereof binds to a specific target. As an antagonist of BMP, noggin2 may be useful for the prophylaxis or treatment of BMP-related disorders in animals, in particular humans. For example, noggin2 can be used to treat diseases or disorders that include, but are not limited to, progressive ossifying fibrodysplasia (FOP), and also to treat abnormal bone growth, such as abnormal bone growth after hip replacement surgery, trauma, burns, or spinal cord injuries. In addition, noggin2 may be useful in treating or preventing the undesirable effects of BMP associated with abnormal bone growth observed in association with metastatic prostate cancer or osteosarcoma. Since it is known that the content of the BMP2 and BMP4 ligands increases significantly with asthma (Rosendahl et al., Am J Respir Cell Mol Biol. 2002 Aug; 27 (2): 160-9) and with some other inflammatory diseases, in particular with rheumatoid arthritis (Lories et al., Arthritis Rheum. 2003 Oct; 48 (10): 2807-18.), then noggin2 can also be used to bind BMP and, thus, to treat inflammatory syndromes.

Since noggin2 can bind activin, it also finds use in the prevention and treatment of activin-related diseases, for example, inhibition of the progression of chronic renal failure, acute renal failure, pancreatitis and neonatal bronchopulmonary dysplasia, etc. It can also be used for eye injuries, such as trauma to the cornea due to burns or contact with a foreign body or inflammatory eye diseases such as uveitis.

In addition, the nucleic acids, proteins, and fragments of noggin2 of the invention can be used to alter the activity of specific noggin2 targets in animals, in particular humans.

Compositions of interest may include a noggin2 protein, for example, a recombinant purified protein, or a chimeric protein containing noggin2, or a functional fragment of noggin2. These compositions may also include nucleic acid encoding a noggin2 protein capable of expressing noggin2 protein in cells, tissues, organs or organisms of interest. These compositions may also contain a suitable pharmaceutical carrier for systemic or local administration in vivo by any suitable method including, but not limited to, injection (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, endoneural, perineural, intraspinal, intraventricular, intravitreal, intrathecal and etc.) by absorption through the epithelial or mucocutaneous lining (e.g., through the mucosa of the oral cavity, rectal or intestinal mucosa, etc. e.) or delayed release implants, including cellular or tissue implants. Depending on the route of administration, the composition may contain a liquid carrier, such as saline, be enclosed in liposomes, microcapsules, polymer or paraffin-based controlled-release formulations, or be formulated in the form of a tablet, pill or capsule. The concentration of the active ingredient (s) used depends on the type of disease or signs that are affected, the method of administration used, and also the characteristics of the biological system. Effective doses can be obtained by extrapolating dose-response curves obtained in in vitro test systems or in animal models.

As used herein, the term "effective dose" refers to the amount of protein required to achieve a biological effect, i.e. suppressing the function of the above representatives of the TGF-beta superfamily. The biological effect can be expressed in reducing signs, symptoms or eliminating the causes of the disease or any other changes in the biological system. Typically, an effective dose is in the range of 0.1-100 mg / kg of the subject.

Administration may result in the distribution of noggin2 in the body or in a localized area. For example, in many conditions involving the distal regions of the nervous system, intravenous or intrathecal administration of noggin2 can be used. Alternatively, and not by way of limitation, when localized regions of the nervous system are involved, local administration may be used. In such situations, an implant containing a nuggin2 can be placed in or near a specific area. Suitable implants include, but are not limited to, a gelatin sponge, wax, or microparticle based implants.

The following examples are provided for illustration and not limitation.

Example 1

Noggin2 cDNA cloning

Noggin2 cDNA was cloned during a search for Xenopus laevis genes involved in the regulatory network of the homeobox-containing Xanf-1 gene (Zaraisky et al. Developmental Biology 1992, v.152, pp.373-382; Ermakova et al., Development 1999, v. 126, pp. 4513-4523). To identify Xanf-1 genetic targets, the authors carried out subtractive hybridization of cDNA samples obtained from anterior ectoderm explants of two groups of Xenopus embryos: in one group, translation of Xanf-1 and its pseudo-allele in Xenopus laevis, Xanf-2 was reduced using microinjections of 1 mM morpholine oligon Xanf-1 (5'-ATCCTTTCTGAAGAGCAGGAGACAT and 5'-ATCCTTTCTGGAGACCAGTAGACAT, Gene Tools); in another group, control morpholine oligonucleotides (MOs) provided by Gene Tools Co. were introduced into the embryos by microinjection In the embryos of Xenopus laevis, at the stage of two blastomeres, microinjections of MOs against Xanf or control MOs mixed with vital dye fluorescein lysine dextran (FLD) were performed at the animal poles of both blastomeres. For each of the two types of embryos in the middle neurula stage, as described in Zaraisky et al. Developmental Biology 1992, v. 152, pp. 373-382, excised approximately 30 anterior neuroectoderm explants, taken together with the underlying enthemesoderm. For the operation, only those embryos were selected that possessed a uniform FLD signal in the anterior region. Subtractive hybridization and differential screening was performed using the PCR Select kit (BD Clontech). The cDNA inserts of all differentially distributed clones (approximately 50 total) were sequenced and analyzed online using BLAST programs using the GeneBank and JGI databases. As a result, a clone was identified containing the complete coding region of the noggin2 protein. cDNA containing the complete coding region of noggin2 was reamplified in PCR using primers 5′-ATTACCGGTGGGAGAACCTTGTTCTTCATT-3 ′ and 5′-ATTCTCGAGTTAGCATGAACACTTACACTCTG-3 ′ and subcloned into the p35T vector. The nucleotide and amino acid sequences of noggin2 are shown in SEQ ID NO: 1 and 2.

Example 2

The effect of noggin2 on the direction of cell development

The p35T vector containing the complete coding part of the noggin2 cDNA (p35T-noggin2 vector) was obtained as described in Example 1.

The complete coding sequence of noggin1 was obtained by PCR amplification from a Xenopus laevis cDNA sample using noggin1-specific primers 5'-TATCCATGGCAAGAAATCGGGAGCA-3 'and 5'-ATTCTCGAGTCTCAGCATGAGCATTTGCA-3' and cloned into vector p35 (vector).

Synthetic mRNAs of noggin2 and noggin1 were obtained using the mMESSAGE MASHINE Kit (Ambion) reagent kit from p35T vectors - noggin2 and p35T vector - noggin1, respectively, linearized with EcoRI restriction endonuclease (Fermentas). The effects of noggin2 mRNA microinjections on the development of Xenopus laevis were compared with the previously described effects of noggin1 mRNA injections (Smith and Harland, Cell 1992, v.70, pp. 829-840; Lamb et al., Science 1993, v.262, pp.713- 718; Smith et al. Nature 1993, v. 361, pp. 547-549). During these experiments, the mRNA of noggin1 and noggin2 were injected into the following blastomeres using an Eppendorf microinjector.

Ventral injections of 40 pg of noggin1 mRNA into early embryos induced the secondary axis of the body (Fig. 1A, black arrow indicates the primary axis; gray arrow indicates the secondary axis, the effect was observed in 68 of 85 embryos subjected to microinjection), whereas similar injections of noggin2 mRNA (40 pkg) ) led to the development of mushroom-shaped embryos (Fig. 1B, the effect was observed in 68 of 75 embryos). Thus, unlike noggin1 (Dale and Slack, Development 1987, v. 100, pp. 279-295; Cell 1992, v. 70, pp. 829-840; Smith et al., Nature 1993, v. 361, pp.547-549), noggin2, despite its alleged ability to bind BMP, did not induce the secondary axis of the body when its mRNA was injected into both ventral blastomeres of 8-cell embryos. Instead, abnormal mushroom-shaped embryos arose.

In situ hybridization on a total preparation of embryos with injection of noggin2 mRNA with probes for mRNA of neural (NCAM, Xanf-1), epidermal (keratin gene), muscle (α-actin gene) genetic markers and genetic markers of late mesoderm (brachyury) revealed giant neuralization ectoderm and a decrease in muscle and epidermal differentiation (Fig. 1B-G). Taken together, these results indicate that noggin2 can neutralize the embryonic ectoderm, but inhibits the differentiation of the dorsal mesoderm and, thus, can inhibit the dorsalizing effect of endogenous noggin1.

The authors also used explants obtained from the ventral marginal zone (VMZ) of embryos at the stage of early gastrula to analyze the effects of noggin2 on the differentiation of the mesoderm. Microinjection of noggin1 mRNA causes a strong elongation of these explants, transforming the nature of their ventral mesoderm into muscle mesoderm (dorsalization) (Dale and Slack, Development 1987, v. 100, pp. 279-295; Cell 1992, v. 70, pp. 829-840 ; Smith et al., Nature 1993, v. 361, pp. 547-549; Fig. 1H). In contrast, like VMZ non-injection explants, expressing noggin2 or both noggin1 and noggin2 explants remain rounded (Fig. 1I).

For independent testing of the effects of noggin2 on the differentiation of ectodermal cells, the authors used reverse transcription-PCR (RT-PCR) and compared the expression of various genetic markers in explants of the "naive" ectoderm (animal caps - AS) extracted from embryos at the stage of early gastrula, into which microinjections introduced mRNA noggin1 or noggin2 (Fig. 1K, L). The authors also used explants extracted from the ventral marginal zone (VMZ) of the embryos at the stage of early gastrula to analyze the effects of noggin2 on the differentiation of the mesoderm (Fig. 1K, L).

AS explants were removed from the embryos subjected to injection using a micro-knife and a glass rod, as previously described in Zaraisky et al. Developmental Biology 1992, v. 152, pp. 373-382. All explants were incubated overnight in a 0.5 × MMR solution. Of the ten explants of each type, total RNA was isolated and RT-PCR was performed as described in Zaraisky et al. Developmental Biology 1992, v. 152, pp. 373-382. The following primers were used: α-actin gene: 5'-GCTGACAGAATGCAGAAG and 5'-TTGCTTGGAGGAGTGTGT; brachyury: 5'-GCTGGAAGTATGTGAATGGAG and 5'-TTAAGTGCTGTAATCTCTTCA; Cerberus: 5'-GCTTTGGTAAATGCATCTCTCTCC and 5'-GGTGACCAGTAAATCATACCTGCT; keratin gene XK81: 5'-CTGAATAACATGAGAGCTG and 5'-TGGTTCTAGTTGTGGATGT; NCAM: 5'-CCTGCTTATGTGTATCGC and 5'-TTCACAGTACTGGGCTGTGCTT; Xanf-1: described in Groppe et al., 2002.

RT-PCR based on total RNA from AS and VMZ explants of embryos injected with moggin1 or noggin2 mRNA was performed with primers for markers of neuroectoderm (NCAM, Xanf-1), epidermis (keratin), muscle (α-actin), late mesoderm ( brachyury) and entomesoderm (cerberus) and Ef-1-α as a control (Fig. 1L). Noggin1 and noggin2 have been shown to activate the expression of neural markers (NCAM, Xanf-1) and inhibit the epidermal marker (keratin), while only noggin1 activates the expression of muscle marker (α-actin) in VMZ explants. Thus, in this analysis, noggin2 showed strong neuralizing activity similar to that of noggin1 (Smith and Harland, Cell 1992, v.70, pp. 829-840; Lamb et al., Science 1993, v.262, pp.713-718 ), inducing the expression of markers of the anterior part of the nervous system, but inhibiting the epidermal marker. The ability of noggin2 to neutralize the ectoderm, together with its ability to suppress the dorsalizing activity of noggin1, looks very unusual and means that noggin2, in addition to BMP, can bind some other factors necessary for muscle differentiation.

Example 3

Inhibition of differentiation of embryonic mesoderm using noggin2

The effect of microinjections of noggin2 mRNA on the differentiation of the embryonic mesoderm was evaluated by analyzing the expression of the universal marker of the early stages of the development of the mesoderm - the brachyury gene - at the stage of the early gastrula Xenopus laevis. In the course of these experiments, moggin1 or noggin2 mRNA obtained as described in example 2 was injected into the equatorial region of one of the ventral blastomeres at the stage of 8 blastomeres in an amount of 40 pg using an Eppendorf microinjector.

The experimental results are shown in FIG. 2. As can be seen from FIG. 2A and A ', microinjections of noggin2 mRNA cause inhibition of the expression of the brachyury gene in the area of microinjection, which indicates the suppression of differentiation of embryonic mesoderm at this point. At the same time, similar microinjections of noggin1 mRNA do not cause inhibition of brachyury expression and, accordingly, inhibition of the development of mesoderm (Fig.2B and B ').

The results of these experiments clearly indicate that, unlike noggin1, noggin2 has a unique ability to suppress the differentiation of embryonic mesoderm.

Example 4

Inhibition of the BMP signaling cascade using noggin2

The ability of noggin2 to inhibit the BMP signaling cascade was assessed by the ability of noggin2 to suppress the expression of the main genetic target of this cascade - the Vent2 homeobox gene - in explants of embryonic ectoderm embryos (AS explants) microinjected with a mixture of noggin2 mRNA and BMP2, BMP4, or BMP4 mRNA. Considering the literature data that different BMPs are able to exhibit increased activity by forming heterodimers, in a special series of experiments, moggin2 mRNA was mixed for microinjections with mRNA of all of the above BMPs simultaneously. As a control, similar experiments were performed with mRNA and noggin1.

Vent2 gene expression was analyzed by RT-PCR based on total RNA from AS explants as described in Example 2. The following Vent2-specific primers were used: 5'-ACTGAACACAAGGACTAATACA and 5'-AGAGGCCAGAGACTGCCCAA.

It has been found that noggin2 is able to suppress activation of Vent2 expression induced by any of these BMPs, as well as mixtures thereof. Moreover, Vent2 inhibition was observed even if the concentration of microinjected BMP mRNA exceeded that of noggin2 by 30 times. In contrast, inhibition of BMP cascade of noggin1 was observed only if the concentration of BMP mRNA did not exceed the concentration of noggin1 mRNA. These data indicate that noggin2 is a much more effective inhibitor of the BMP cascade than noggin1.

To directly study the ability of the noggin2 protein to bind BMP molecules in X.laevis embryos, the active forms of noggin2 and BMP4 were expressed in the expression vectors pCS2-3Myc-noggin2 and pCS2-3Flag-BMP4, respectively.

To obtain the pCS2-3Myc-noggin2 vector, the nucleotide sequence encoding the three amino acid sequences of the Myc protein epitope (EQKLISEEDLEQKLISEEDLEQKLISEEDL) was inserted by PCR into the noggin2 cDNA directly at the 3 'end of the sequence encoding the noggin2 signal peptide. For this purpose the first cloning step were obtained, respectively 5 'and 3' overlapping part noggin2 cDNA by PCR with the primers 5'-AATTGGATCCGCCACCATGAAGAGGATAAATCTGC-3 'and 5'-GAGGTCTTCCTCCGATATCAGCTTCTGTTCCAGATCCTCTTCAGAGATGAGTTTCTGCTCATAAGGCTGACAGCACCCCTGA-3', 5'-GAACAGAAGCTGATATCGGAGGAAGACCTC GAGCAGAAACTCATCTCTGAAGAGGATCTGCTCAGGCTTAGACCCTCT-3 'and 5'- ATTCTCGAGTTAGCATGAACACTTACACTCTG-3 ', respectively. In the second stage, these cDNA fragments were purified from unincorporated primers, mixed with each other, denatured by heating, annealed and subjected to a second round of PCR with end primers 5'-AATTGGATCCGCCACCATGAAGAGGATAAATCTGC-3 'and 5'-ATTCTCGTT-3. The resulting full-length noggin2 cDNA containing a sequence encoding the three amino acid sequences of Myc was cloned into the pCS2 vector at the restriction saigas NcoI (dull) / AgeI (dull) and XhoI / XhoI. To obtain the pCS2-3Flag-BMP4 vector, the nucleotide sequence encoding the Flag three amino acid sequences (DYKDDDDKDYKDDDDKDYKDDDDK) was inserted by PCR into BMP4 cDNA directly at the 3 'end of the sequence encoding the proregion of Xenopus laev BMP4 protein. In the first step were prepared by cloning the 5 'and 3' overlapping part BMP4 cDNA by PCR with the primers 5'-AATTGGATCCGCCACCATGATTCCTGGTAACCGAATG-3 'and 5'-TTTGTCATCATCGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCCTGTTTTGGACT TCTTTTTGAC-3', 5'-GACTACAAAGACGATGATGACAAAGATTACAAGGATGACGACGATA AGCAGAGACCCCGTAAAAAAAAC-3 'and 5'-AATCTCGAGTCAACGGCACCCACACCCTTCCA- 3 ', respectively. In the second stage, these cDNA fragments were purified from unincorporated primers, mixed with each other, denatured by heating, annealed and subjected to a second round of PCR with end primers 5'-AATTGGATCCGCCACCATGATTCCTGGTAACCGAATG-3 'and 5'-AATCGTAC-3A. The resulting full-length BMP4 cDNA containing a sequence encoding the three amino acid sequences of the Flag epitope was cloned into the pCS2 vector at the restriction sites NcoI (dull) / AgeI (dull) and XhoI / XhoI.

600 pg of synthetic 3Myc-noggin2 mRNA and 600 pg of synthetic 3Flag-BMP4 mRNA were microinjected into developing embryos of X. laevis at the stage of 2 blastomeres. Subsequently, these embryos were incubated in a 0.1 MMR solution until the early gastrula stage.

The expression of expressed proteins was carried out by lysing the embryos at the stage of early gastrula according to the method described in Tanegashima et al., Int.J.Dev. Biol (2004) 48: 275-283.

After isolation, the expressed 3Flag-BMP4 protein was absorbed on Flag-Agarose resin (Sigma) according to the manufacturer's protocol. After washing the resin in accordance with the manufacturer's protocol, the presence of 3Myc-noggin2 protein bound on this resin was analyzed by protein blotting using Myc-specific monoclonal antibodies (Sigma).

This experiment revealed the ability of the noggin2 protein molecules to bind BMP4 protein molecules (Fig. 2G).

To study the ability of the noggin2 protein to inhibit BMP-induced BMP signaling in embryonic cells, the effect of noggin2 on the phosphorylation of the intracellular transmitter of this signaling pathway, Smadl, was analyzed. For this, embryos of the Spur frog were injected at the stage of 2-4 cells with either BMP4 mRNA (100 pg / embryo) or a mixture of BMP4 mRNA (10 pg / embryo) and noggin2 (1 pg / embryo). The analysis of Smadl phosphorylation in the injected embryos was carried out at the stage of the beginning of gastrulation (stage 10) by Western blotting using commercial monoclonal antibodies to Phospho-Smadl (Cell Signaling Technology). As a result, it was shown that noggin2 is able to block the increase in the concentration of Phospho-Smadl caused by BMP4 (Fig. 2, C).

Example 5

Inhibition of the activin / nodal signaling cascade with noggin2

The ability of noggin2 to inhibit the activin / nodal signaling cascade was assessed by the ability of noggin2 to suppress the expression of the main genetic target of this cascade, the brachyury gene, in explants of embryonic ectoderm of embryos (AS explants) microinjected with a mixture of noggin2 mRNA and mammalian Xnr1, Xnr2, or nnr2, mice ) As a control, similar experiments were performed with mRNA and noggin1.

Brachyury gene expression was analyzed by RT-PCR for total RNA from AS explants as described in Example 2.

Noggin2 has been found to be able to suppress activation of brachyury expression induced by any of these Xnr. Moreover, inhibition of brachyury was observed only if the concentration of microinjected Xnr mRNA did not exceed that of noggin2. In contrast, noggin1 is not able to inhibit the activation of the brachyury gene in similar tests. These data demonstrate that, unlike noggin1, noggin2 is able to inhibit not only the BMP signaling cascade, but also the cascade activated by members of the other large superfamily of TGF-β factors - activin / nodal.

To study the ability of the noggin2 protein to bind activin protein molecules in X.laevis embryos, the active forms of noggin2 and activin-B were expressed in the expression vectors pCS2-3Myc-noggin2 and pCS2-3Flag-activin-B, respectively.

Obtaining pCS2-3Myc-noggin2 vector was carried out as described in Example 4.

To obtain the pSP64T-3Flag-activin-B vector, the nucleotide sequence encoding the Flag three amino acid sequences (DYKDDDDKDYKDDDDKDYKDDDDK) was inserted by PCR into the activin-B cDNA immediately behind the sequence encoding the proregion of the Xenopus laevis activin-B protein. In the first step of cloning were obtained 5 'and 3' overlapping part of activin-B cDNA by PCR with the primers 5'-AATTGGATCCGCCACCATGGCTCTCCTGTTACTGCCTCTG-3 'and 5'-TTTGTCATCATCGTCTTTGTAGTCCTTATCGTCGTCATCCTTGTAATCCTCGAGGCCTCTCTTACGGA-3', 5'-GACTACAAAGACGATGATGACAAAGATTACAAGGATGACGACGATAAG TGCGATGGACACACAAATT-3 'and 5'-AATGAATTCATGCACAGCCGCACTCGTCCA -3 'respectively. In the second stage, these cDNA fragments were purified from unincorporated primers, mixed with each other, denatured by heating, annealed, and subjected to a second round of PCR with end primers 5'-AATTGGATCCGCCACCATGGCTCTCCTGTTACTGCCTCTG-3 'and 5'-ACG-ATGATCATGATCATGATCATGCCTG-3AT. The resulting full-length activin-B cDNA containing a sequence encoding the three amino acid sequences of the Flag epitope was cloned into the pSP64T vector at the restriction sites HindIII (dull) / Agel (dull) and EcoRI / EcoRI.

Analysis of the ability of the expressed 3Myc-noggin2 protein to bind 3Flag-activin-B protein molecules was carried out using specific antibodies according to the procedure described in Example 4.

This experiment revealed the ability of the noggin2 protein molecules to bind activin-B protein molecules (Fig. 2 G).

To study the ability of the noggin2 protein to inhibit activin-induced activin / nodal signaling in embryonic cells, the effect of noggin2 on the phosphorylation of the intracellular transmitter of this signaling pathway, Smad2 protein, was analyzed. For this purpose, the frog embryos were injected at the stage of 2-4 cells with either activinB mRNA (100 pg / embryo) or a mixture of activinB mRNA (10 pg / embryo) and Noggin2 (1 ng / embryo). The analysis of Smad2 phosphorylation in the injected embryos was carried out at the stage of the beginning of gastrulation (stage 10) by Western blotting using commercial monoclonal antibodies to Phospho-Smad2 (Cell Signaling Technology). As a result, it was shown that noggin2 is able to block the increase in the concentration of Phospho-Smad2 caused by activinB (Fig. 2, C).

Example 6

Simultaneous inhibition of BMP and activin / nodal signaling cascades and induction of head structures using noggin2

As is known, the differentiation of the structures of the main axis of the vertebral body is inhibited on the ventral side of the embryo as a result of the permanent activation of two main TGF-β cascades: BMP and activin / nodal. Moreover, selective inhibition of the BMP cascade leads by default to the development of structures characteristic of the trunk and posterior part of the body axis, in particular, the development of the spinal cord and muscles. At the same time, the simultaneous inhibition of both BMP and activin / nodal cascades induces the anterior-head structures, including the eye and forebrain.

In normal development, the suppression of the BMP cascade on the dorsal side of the embryo is carried out by specific protein factors secreted by the cells of the Shperman organizer and able to bind BMP. The most famous of them is noggin1. Microinjections of noggin1 mRNA into the ventral vegetative blastomeres of the Xenopus embryo can cause the development of additional trunk structures on the ventral side of the embryo body. Moreover, the second body axes induced by exogenous noggin1 never contain anterior-headed structures, the development of which requires simultaneous inhibition of both BMP and activin / nodal cascades. In normal development, inhibition of the activin / nodal cascade, as well as the closely related positive feedback of the Wnt signaling cascade, is carried out by other factors secreted by the cells of the head part of the Shperman organizer. The most famous of these are Dikkopf and Cerberus. Moreover, the last protein is able to bind three factors at once: BMP, nodal and Wnt. As a result, in the rostral part of the embryo, the ban on activation of the anterior-head differentiation program is lifted and the anterior-head structures develop.

The ability of noggin2 to simultaneously inhibit both the BMP cascade and the activin / nodal cascade indicates a potential ability of this factor to induce anterior-headed structures. However, in the experiments described in Example 2, a similar effect could be masked by the strong neuralizing effect of noggin2, caused by relatively high concentrations of microinjectable mRNA.

In this regard, the ability of low concentrations of noggin2 to induce the development of additional anterior-headed structures in whole embryos was studied. In this series of experiments, synthetic noggin2 mRNA, also obtained as described in Example 2, was microinjected into one of the ventral vegetative blastomeres of Xenopus laevis embryos at the stage of 8-16 blastomeres in the amount of 2-5 pg. As a result, in 25% of embryos, the development of an additional anterior-head section was observed. As shown by the in situ hybridization method with specific probes for mRNA of the forehead markers BF-1 and Pax-6, such additional brain structures included the end brain and eyes (Figure 2, B and B '; D and D'). Similar structures were absent in the additional body axes induced by microinjections of noggin1 mRNA (Fig. 2, D and G '; E and E'). These data demonstrate a qualitative difference between the effects of noggin2 and the effects of noggin1, associated with the ability of noggin2 to inhibit both BMP and the activin / nodal signaling cascade.

Example 7

Obtaining recombinant protein noggin2

To obtain the active homodimer noggin2, expression of noggin2 was carried out in a culture of green monkey monkey CV-1 epithelial cells as part of the pcDNA3-6His-noggin2 expression vector. To obtain the pcDNA3-6His-noggin2 vector, a nucleotide sequence encoding 6 amino acid residues of histidine was inserted by PCR into the noggin2 cDNA immediately behind the sequence encoding the noggin2 signal peptide. The first cloning step were obtained, respectively, 5 'and 3' overlapping part noggin2 cDNA by PCR with the primers 5'-ATTACCGGTGGGAGAACCTTGTTCTTCATT-3 'and 5'-GTGATGGTGATGGTGATGCCCGGGATAAGGCTGACAGCACCCCT-3' and 5'-CATCACCATCACCATCACGGGCCCTATCTCAGGCTTAGACCCTC-3 'and 5'-ATTCTCGAGTTAGCATGAACACTTACACTCTG -3 '. In the second stage, these cDNA fragments were purified from not included primers, mixed with each other, denatured by heating, annealed and subjected to a second round of PCR with primers 5'-ATTACCGGTGGGAGAACCTTGTTCTTCATT-3 'and 5'-ATTCTCTCAGTTACT. The resulting full-length noggin2 cDNA containing a sequence encoding 6 histidines was cloned into the pcDNA3 vector by the restriction saigas NcoI (blunt) and XhoI.

Green monkey epithelial cells (CV-1) were cultured in DMEM medium (Sigma) with 10% serum (Biolot), 100 U / ml penicillin and 100 mg / ml streptomycin at 37 ° C and 1 atm. To obtain a constant cell line producing 6His-noggin2, these cells were transfected with pcDNA3-6His-noggin2 vector. Subsequent selection was performed in a medium containing 1 μg / ml geneticin (Gibco BRL). Secreted recombinant 6His-Noggin2 was obtained from the culture medium by affinity chromatography on Ni-NTA agarose (Qiagen). Protein yield was estimated as 50 μg / million cells per day. Protein activity was confirmed by its ability to activate neurogenesis in explants of embryonic ectoderm.

Example 8

Obtaining polyclonal antibodies against noggin2 cDNA containing the complete coding region of noggin2 was reamplified as described in Example 1, subcloned into the pQE30 vector and used to transform Escherichia coli. A recombinant protein containing six histidine residues was purified using Talon Resin affinity chromatography (Clontech Laboratories, Inc.) under denaturing conditions and used to immunize rabbits using standard technology. Polyclonal serum was tested against recombinant protein using ELISA and Western blot methods.

Claims (1)

A method for blocking the activin signaling cascade by binding of noggin2 to an activin protein, comprising administering noggin2 into an organism, tissue or cell in an amount effective to inhibit or reduce activin activity.

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