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WO2007113361A1 - Traitement de maladies causées par des altérations du développement axonal des neurones - Google Patents

Traitement de maladies causées par des altérations du développement axonal des neurones Download PDF

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Publication number
WO2007113361A1
WO2007113361A1 PCT/ES2007/000192 ES2007000192W WO2007113361A1 WO 2007113361 A1 WO2007113361 A1 WO 2007113361A1 ES 2007000192 W ES2007000192 W ES 2007000192W WO 2007113361 A1 WO2007113361 A1 WO 2007113361A1
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Prior art keywords
peptide
nrgl
polynucleotide
seq
fragment
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PCT/ES2007/000192
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English (en)
Spanish (es)
Inventor
Oscar MARÍN PARRA
Beatriz Rico Gonzalo
Guillermina LÓPEZ BENDITO
Nuria Flames Bonilla
Sonia Lorenzo Vidal
Juan Antonio SÁNCHEZ ALCAÑIZ
Original Assignee
Consejo Superior De Investigaciones Científicas
Universidad Miguel Hernández De Elche
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Publication of WO2007113361A1 publication Critical patent/WO2007113361A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1883—Neuregulins, e.g.. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor

Definitions

  • the invention relates, in general, to the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system, particularly in humans, based on the use of neuregulin 1 (NRGl), or a variant or functionally equivalent fragment thereof, or of a polynucleotide encoding said protein, variant or functionally equivalent fragment, or of a compound that enhances the production and / or activity of said NRGl.
  • NRGl neuregulin 1
  • axonal growth is very restricted from the last stages of its development. For this reason, the regeneration of axons after trauma or neurodegenerative diseases is one of the most important challenges of current biomedicine.
  • the restriction of axonal growth in the CNS is mainly due to the existence of factors that slow axonal growth in the adult brain and the absence of factors that promote axon elongation.
  • factors that restrict the growth of axons in recent years much progress has been made in the study of several proteins derived from myelin, such as Nogo, MAG and OMgp.
  • axonal growth The regulation of axonal growth, the formation of collaterals and new connections in the postnatal brain by myelin proteins, is a research area with great therapeutic potential.
  • factors that promote the extension of axons one of the recently used strategies and that constitutes a very active field of research, is the use of the enveloping glia.
  • Other factors that promote the growth of certain axons are Netrin-1 and brain-derived growth factor (BDNF).
  • Axonal regeneration is frequently seen from the perspective of spinal injuries, but there are numerous neuronal pathologies in which the axons of other populations of CNS neurons are affected, such as in schizophrenia, Alzheimer's disease or multiple sclerosis (Principies of Neural Science (2000) Kandel ER, Schwartz JH, Jessell TM Eds. McGraw-Hill, New York).
  • Interneurons are characterized by having short axons, unlike the cortical-spinal or thalamus-cortical neurons that they have with long axons, but in any case, their regeneration could represent a considerable improvement in cortical functioning and therefore represents a target attractive for the development of an appropriate therapy for these diseases.
  • NRGl is one of the members of the protein family called Neuregulins (NRG). To date, this family of proteins has four members called NRGl, NRG2, NRG3 and NRG4, respectively.
  • NRGs contain a domain similar to epidermal growth factor (EGF), hereinafter "EGF domain", through which they activate tyrosine kinase type membrane receptors that are structurally related to the EGF receptor (EGFR or ErbBl).
  • EGF domain epidermal growth factor
  • ErbBl ErbBl
  • NRGl binds and preferentially binds to the ErbB3 and ErbB4 receptors, which (sometimes with the intervention of ErbB2) mediate a very wide range of biological functions.
  • EGF domain variant that contains the protein ( ⁇ or ⁇ )
  • NRGl is essential for the survival of glia cells, as well as for their proliferation and differentiation (Buonanno A, Fischbach GD (2001) Neuregulin and ErbB receptor signaling pathways in the nervous system. Curr Opin Neurobiol 11: 287-296).
  • the functions of NRGl range from proliferation control to very late events in neuronal development, such as the regulation of channel expression (Buonanno A, Fischbach GD (2001) Neuregulin and ErbB receptor signaling pathways in the nervous system Curr Opin Neurobiol 11: 287-296).
  • NRGl is also capable of promoting the migration of Schwann cells and cortical neurons (Mahanthappa NK, Anton ES, Matthew WD (1996) Glial growth factor 2, to soluble neuregulin, directly increases Schwann cell motility and indirectly promotes neurite outgrowth. J Neurosci 16: 4673-4683; Flames N, Long JE, Garratt AN, Fischer TM, Gassmann M, Birchmeier C, Lai C, Rubenstein JL, Mar ⁇ n O (2004) Short- and long-range attraction of cortical GABAergic interneurons by neuregulin- 1.
  • Neuron 44: 251-261 to act as memory modulator (Use of neuregulin-beta as an indicator and / or target, US 20030036101) and to induce differentiation of neural stem cells into glial cells (Methods for differentiating neural stem cells to glial cells using neuregulins, US 6,033,906 ).
  • NRGl is useful in the treatment of schizophrenia (Human schizophrenia gene, US 20050208527).
  • NRGs such as their use to promote cellular myocardial differentiation (Application of growth factor neuregulin and its analogs, CN19990107175), to promote the survival of retinal cells (Methods of treating disorders of the eye) , US 6,750,196) and to treat viral myocarditis and congestive cardiomyopathy (Neuregulin based methods and compositions for treating cardiovascular diseases, US 20060019888).
  • the present invention faces the problem of providing new therapeutic tools for the treatment of diseases caused by an alteration of the axonal development of neurons in the nervous system.
  • the present invention is based on the observation that isoforms of the Nrgl gene that contain the ⁇ variant of the EGF domain (EGFB) of NRGl, are capable of promoting the growth of axons and the development of ramifications in neurons that express receptors for NRGl, such as ErbB4 and ErbB3, both in long-axon neurons (eg, thalamus-cortical neurons) and in short-axon neurons (eg, GABAergic cerebral cortex interneurons) [see the Example that accompanies this description] .
  • EGFB EGF domain
  • NRGl the person responsible for causing this effect appears to be the EGF domain of the NRGl, since a recombinant fragment of that domain is capable of producing the same effects as the complete protein, whether said fragment is part of the diffusible or soluble NRGl protein (Ig-NRGl) such as membrane-anchored NRGl protein (CRD-NRGl). Due to the structural similarity between the ⁇ and ⁇ forms of NRGl, it is expected that the effects obtained for the ⁇ form are extensible to the ⁇ forms of the protein. This suggests that NGR1 or a fragment thereof that comprises its EGF domain, is an important factor for the development or regeneration of axons in different populations of neurons in the nervous system.
  • Ig-NRGl diffusible or soluble NRGl protein
  • CCD-NRGl membrane-anchored NRGl protein
  • said factor can be used to regenerate nerve connections (axons) that depend on NRGl / ErbB4 signaling by therapeutic administration of a compound such as, for example, a polynucleotide comprising the nucleotide sequence encoding the protein. NRGl, or a fragment thereof comprising its EGF domain, or, alternatively, of said NRGl protein, or a fragment thereof comprising its EGF domain.
  • the invention relates to the use of a peptide comprising the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, as well as a polynucleotide encoding said functionally equivalent peptide, variant or fragment. , or of a compound that induces the production and / or activity of said NRGl, in the elaboration of a pharmaceutical composition for the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system.
  • Said polynucleotide may be contained in a gene construct, in a vector or in a host cell, which constitute additional aspects of the present invention.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a peptide comprising the EGF domain of NRG1, or a functionally equivalent variant or fragment thereof, as well as a polynucleotide encoding said functionally equivalent peptide, variant or fragment, or of a compound that induces the production and / or activity of said NRGl, in the elaboration of a pharmaceutical composition for the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system, together with one or more pharmaceutically acceptable carriers or excipients.
  • Said polynucleotide may be contained in a gene construct, in a vector or in a host cell.
  • the target neuron with nerve connections (axons) to regenerate does not endogenously express the ErbB4 receptor, it could promote or induce the expression of said receptor and, thus, give the target neurons the ability to respond to NRGl.
  • This approach which consists in inducing or increasing the expression of a membrane receptor in a target neuron with nerve connections (axons) to regenerate, preferably the ErbB4 receptor or the ErbB3 receptor, receptors to which NRG1 or a fragment binds and activates thereof, which comprises its EGF domain, constitutes an additional aspect of the present invention.
  • FIGURES Figure 1 are images and graphs showing that Ig-NRGl promotes the growth of the thalamus-cortical axons (TCAs).
  • TCAs thalamus-cortical axons
  • A Expression of Ig-Nrgl in the cerebral cortex (NCx) to 3.5.
  • B, C Immunohistochemistry for ⁇ lll-Tubulin, a neuronal marker, showing explants of the dorsal thalamus (dTh) of embryos 3.5 after 96 hours in culture, facing aggregates of control COS cells (B) or transfected with Ig-Nrgl ( C).
  • the boxes show the expression of GFP in the transfected COS cells.
  • Figure 2 are images and graphs that show that Ig-NRGl through its EGF domain promotes the growth of the thalamic axons in collagen and laminin supports.
  • A Experimental paradigm used to test the effect of the EGF domain of Ig-NRGl on the growth of dorsal thalamic axons grown on collagen (B and C) or laminin (E and F).
  • B and C Dorsal thalamus explants grown in collagen with control medium (D) or supplemented with the EGF domain of human NRGl ⁇ .
  • E and F Similar experiments performed on laminin. In all cases the axons are labeled with an antibody against ⁇ lll-Tubulin, a neuronal protein.
  • Figure 3 is an image showing the expression of NRGl and its ErbB4 receptor in the postnatal cerebral cortex.
  • A, A ' Cross sections through the telencephalon of a 30 day postnatal mouse (P30) showing the expression of the CRD-Nrg-1 isoform by in situ hybridization.
  • A Panoramic view of the rare expression of CRD-Nrg-1 in the neocortex (NCx) and hippocampus (HC).
  • a ' Detail of the cortical region bounded by a dashed line in A.
  • FIG. 4 is a graph and images that show that NRGl stimulates the growth of axons and their collaterals in cortical interneurons.
  • A Experimental paradigm used to demonstrate the role of NRGl in the axonal development of GABAergic interneurons.
  • B Image of an interneuron in culture after being treated with control supernatant.
  • the invention is based, in general, on the fact that the inventors have observed that a peptide comprising the EGF domain of NRG1 has promoter or stimulatory activity for the growth of axons of neurons of the nervous system, whereby said peptide, or a functionally equivalent variant or fragment thereof, as well as a polynucleotide encoding said functionally equivalent peptide, variant or fragment, or a compound that induces the production and / or activity of said NRGl, can be used as therapeutic agents, in in particular, as agents promoting or stimulating the growth of axons of neurons of the nervous system, in the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system.
  • Said polynucleotide may be contained in a gene construct, in a vector or in a host cell.
  • the invention relates to the use of a product selected from: (i) a peptide comprising the amino acid sequence of the epidermal growth factor (EGF) domain [EGF domain] of neuregulin 1 [NRGl], or a functionally equivalent variant or fragment of said peptide;
  • a product selected from: (i) a peptide comprising the amino acid sequence of the epidermal growth factor (EGF) domain [EGF domain] of neuregulin 1 [NRGl], or a functionally equivalent variant or fragment of said peptide;
  • the invention relates to the use of a peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment of said peptide, that is, with axon growth stimulating activity of neurons of the nervous system, in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of neurons of the nervous system.
  • peptide refers in general to a compound formed by the union of two or more amino acids by peptide bonds and includes both oligopeptides and polypeptides and proteins. Said peptide can be found in an unmodified form or in a modified form (e.g., by glycosylation, phosphorylation, acetylation, etc.).
  • neuroregulin 1 refers to a protein capable of activating the ErbB2 / ErbB4 or ErbB2 or ErbB3 heterodimer tyrosine kinase membrane receptors, including the various isoforms of said NRGl as well as any NRGl of any organism expressing said protein (eg, human, mouse, etc.).
  • NRGl can be presented either in a diffusible or soluble form, identified as Ig- NRGl or also as NRGl of types I and II, or as a membrane anchored form, identified as CRD-NRGl or also as NRGl of type III (Falls DL (2003) Neuregulins: functions, forms, and signaling strategies. Exp CeIl Res 284: 14-30), or in any of its isoforms, some of which, such as isoforms 1, 2, 3, 4, 6, 7, 8, 9 and 10, produced by alternative splicing, and Its sequences are described in UniProtKB / Swiss-Prot entry Q02297.
  • NRGl contains a domain similar to EGF, called "EGF domain".
  • EGF domain refers to a peptide encoded by the gene encoding the NRGl (Nrgl gene) that binds and activates the ErbB2, ErbB3, ErbB4 receptors, or combinations thereof and modulates them.
  • Biological reactions of NRGl eg, control neuronal proliferation, promote the migration of Schwann cells and cortical neurons, induce differentiation of neuronal stem cells into glial cells, etc.
  • Two variants of said EGF domain have been described, called ⁇ and ⁇ (Falls DL (2003) Neuregulins: functions, forms, and signaling strategies. Exp CeIl Res 284: 14-30).
  • said EGF domain of the NRGl comprises, or is constituted by, the amino acid sequence of the EGF domain of the human NRGl (hNRGl), such as the variant EGF domain ⁇ of the hNRGl, whose amino acid sequence is shown in SEQ ID NO: 1.
  • said EGF domain of the NRGl comprises, or is constituted by, the amino acid sequence of the variant EGF domain ⁇ of the hNRGl, whose amino acid sequence is shown in SEQ ID NO : 2.
  • the peptide comprising said EGF domain of the NRGl comprises, or is constituted by, the amino acid sequence of the diffusible or soluble form of the NRGl, sometimes identified in this description as Ig-NRGl, such as Human Ig-NRGl, which may include the ⁇ or ⁇ variants of the EGF domain, and whose amino acid sequences correspond to SEQ ID NO: 3 (NRGl Alpha, Protein Acc.
  • VAR_SEQ or VSP sequence variants
  • VSP_003426, VSP_003426, VSP_003426, VSP_003426, VSP_003426, VSP_003426 VSP_003430 or as the mouse Ig-NRGl, whose amino acid sequence is shown in SEQ ID NO: 4 (Protein Acc. # Q6DR99).
  • the peptide comprising said EGF domain of NRGl comprises, or is constituted by, the amino acid sequence of the membrane-anchored form of NRGl, sometimes identified in this description as CRD-NRGl, such as Human CRD-NGRl, which may include the ⁇ or ⁇ variants of the EGF domain, whose amino acid sequence is shown in SEQ ID NO: 5 (Protein Acc. # Q 15491) or as the mouse CRD-NRGl, whose sequence of Amino acids are shown in SEQ ID NO: 6 (Protein Acc # AAT68240).
  • variant refers to a peptide substantially homologous and functionally equivalent to the peptide comprising the amino acid sequence of the EGF domain of NRGl.
  • a variant includes conservative amino acid substitutions that do not substantially alter its promoter or stimulatory activity of axon growth of the nervous system neurons of the EGF domain of NRGl.
  • a peptide is "substantially homologous" to said peptide comprising the amino acid sequence of the EGF domain of the NRGl when it has a good alignment with the amino acid sequence of said EGF domain of the NRGl, that is, when its amino acid sequence has a degree of identity with respect to the amino acid sequence of said EGF domain of the NRGl, of at least 30%, typically of at least 50%, advantageously of at least 60 %, preferably of at least 70%, more preferably of at least 85%, and even more preferably of at least 95%.
  • sequences homologous to the sequence of the EGF domain of the NRGl can be easily identified by a person skilled in the art with the help of an appropriate computer program to compare sequences, for example, the BLAST program (Altschul et al. 1997. Nucleic Acids Res .25: 3389).
  • the term “functionally equivalent”, as used herein, means that the peptide in question maintains, at least, the function related to the promoter or stimulatory activity of axon growth of the nervous system neurons of the EGF domain of the NRGl. Said activity promoting or stimulating the growth of axons of neurons of the nervous system can be determine by conventional methods such as the tests described in Examples 1 and 2 that accompany this description.
  • said variant is a mutant form of the peptide comprising the amino acid sequence of the EGF domain of NRGl that maintains the promoter or stimulating activity of axon growth of neurons in the nervous system.
  • Said mutant form may have insertions, deletions or modifications of one or more amino acids with respect to the peptide comprising the amino acid sequence of the EGF domain of the NRGl, for example, any of the amino acid sequences shown in SEQ ID NO: 1-6, with the proviso that it retains said promoter or stimulatory activity of axon growth of nerve system neurons.
  • a variant of a peptide according to the present invention will include conservative amino acid substitutions that do not substantially alter its axon growth promoter or stimulator activity of the nervous system neurons of the EGF domain of NRGl.
  • the person skilled in the art knows the appropriate conservative amino acid substitutions that can be made without significantly altering the biological activity of the resulting molecule.
  • substitutions that affect a single amino acid in non-essential regions of a peptide do not substantially alter its activity (Wa ⁇ son el al. Molecular Biology of ⁇ he Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub. Co., P. 224).
  • fragment refers to a peptide comprising a portion of said peptide comprising the amino acid sequence of the EGF domain of NRGl, that is, a contiguous amino acid sequence comprised within of said sequence.
  • said fragment comprises a portion of any of said peptides that comprise, or are consisted of, of any of the amino acid sequences shown in SEQ ID NO: 1-6.
  • said fragment must be functionally equivalent to said peptide comprising the amino acid sequence of the EGF domain of NRGl, that is, it must have promoter or stimulating activity of the growth of axons of neurons of the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ID NO: 1, or a functionally equivalent variant or fragment of said peptide, in the preparation of a composition pharmaceutical for the treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ED NO: 2, or a functionally equivalent variant or fragment of said peptide, in the preparation of a pharmaceutical composition for treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ID NO: 3, or a functionally equivalent variant or fragment of said peptide, in the preparation of a pharmaceutical composition for treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ID NO: 4, or a functionally equivalent variant or fragment of said peptide, in the preparation of a pharmaceutical composition for treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ED NO: 5, or a functionally equivalent variant or fragment of said peptide, in the preparation of a pharmaceutical composition for treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the invention relates to the use of a peptide comprising, or is constituted by, SEQ ID NO: 6, or a functionally equivalent variant or fragment of said peptide, in the preparation of a pharmaceutical composition for treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • said peptide comprising the amino acid sequence of the EGF domain of NRGl, or a variant or fragment of the same functionally equivalent, it has promoter or stimulating activity of the growth of axons of the neurons of the nervous system, so it can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations of the axonal development of the neurons of the system nervous.
  • Said peptide comprising the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment of said peptide can be obtained by conventional methods known to those skilled in the art, for example, from an organism producing said peptide, variant or fragment, either natively or recombinantly, by a method comprising culturing said organism under appropriate conditions for the expression of said peptide, variant or fragment, and recovering it from the medium.
  • the EGF domain, ⁇ variant of hNRG1 can be purchased commercially from Peprotech (Cat # 100-03).
  • said peptide, variant or functionally equivalent fragment thereof can be obtained by conventional methods of chemical peptide synthesis known to those skilled in the art, eg, by solid phase chemical synthesis techniques (Merrifield RB. J Am Chem Soc 1963; 85: 2149-2154) using the Atherton Fmoc variant (Atherton, E., Logan, JC and Sheppard, RC 1989. Peptide synthesis II. Procedures for solid phase synthesis using N-fluorenyl methoxycarbonyl aminoacids on polyamide supports. substance P and of acyl carrier protein 65-74 decapeptide J. Chem. Soc. Perkin Trans.
  • the peptides obtained can be purified by conventional methods, eg, by high performance liquid chromatography (HPLC) or reverse phase HPLC, and, if desired, can be analyzed by conventional techniques, for example, by sequencing and mass spectrometry (MS), amino acid analysis, nuclear magnetic resonance, etc.
  • HPLC high performance liquid chromatography
  • MS mass spectrometry
  • said peptide comprising the amino acid sequence of the EGF domain of ⁇ RG1, variant or functionally equivalent fragment thereof can be part of a fusion protein.
  • said fusion protein may contain a region A consisting of a first polypeptide comprising the peptide comprising the amino acid sequence of the EGF domain of ⁇ RG1, variant or fragment functionally equivalent thereof, linked to a B region comprising a second polypeptide.
  • Said second polypeptide can be any suitable peptide, for example, a peptide with axon growth promoter or stimulating activity of neurons of the nervous system.
  • said second polypeptide may also be a peptide comprising the amino acid sequence of the EGF domain of NRG1, a variant or an equivalent functional fragment thereof.
  • Said region B may be attached to the ammo-terminal region of said region A, or alternatively, said region B may be linked to the carboxyl-terminal region of said region A. Both regions A and B may be linked directly or through of a spacer peptide (linker) between said regions A and B.
  • the fusion protein can be obtained by conventional methods known to those skilled in the art, for example, by gene expression of the nucleotide sequence encoding said fusion protein in appropriate host cells.
  • the invention relates to the use of a polynucleotide encoding said peptide that comprises, or is constituted by, the amino acid sequence of the EGF domain of NRG1, or a variant or functionally equivalent fragment of said peptide, is that is to say, with promoter or stimulating activity of the growth of axons of the neurons of the nervous system, in the elaboration of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of the neurons of the nervous system.
  • polynucleotide refers in general to a nucleotide polymer including both deoxyribonucleotides and ribonucleotides that give rise to deoxyribonucleic acids (DNA), eg, genomic DNA (gDNA) or DNA copy ( cDNA), etc., and ribonucleic (RNA), eg, messenger RNA (rnRNA), etc., respectively.
  • DNA deoxyribonucleic acids
  • gDNA genomic DNA
  • cDNA DNA copy
  • RNA ribonucleic
  • rnRNA messenger RNA
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence encoding the EGF domain of NRGl, such as the EGF domain of hNRGl.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 7, which contains the nucleotide sequence encoding the ⁇ variant of the EGF domain of the hNRGl.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 8, which contains the nucleotide sequence encoding the ⁇ variant of the EGF domain of the hNRG1.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence encoding the amino acid sequence of the diffusible or soluble form of NRGl (Ig-NRGl), such as the diffusible or soluble form of human NRGl. or murine.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 9 (Nucleotide Acc # M94165), which contains the nucleotide sequence encoding the diffusible or soluble form of the hNRGl ( Human Ig-NRGl).
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 10 (Nucleotide Acc # AY648976), which contains the nucleotide sequence encoding the diffusible or soluble form of the NRG1 of mouse (mouse Ig-NRGl).
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence encoding the amino acid sequence of the membrane-anchored form of NRGl (CRD-NRGl), such as the membrane-anchored form of human NRGl. or murine.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 11 (Nucleotide Acc # BC064587), which contains the nucleotide sequence encoding the membrane-anchored form of the hNRGl ( Human CRD-NRGl).
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence shown in SEQ ID NO: 12 (Nucleotide Acc # AY648975), which contains the nucleotide sequence encoding the membrane-anchored shape of the NRG1 of mouse (mouse CRD-NRGl).
  • the invention relates to the use of a polynucleotide comprising, or is constituted by, SEQ ID NO: 7 in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the Axonal development of nervous system neurons.
  • the invention relates to the use of a polynucleotide that comprises, or is constituted by, SEQ ID NO: 8 in the elaboration of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of Nervous system neurons.
  • the invention relates to the use of a polynucleotide that comprises, or is constituted by, SEQ ID NO: 9 in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of Nervous system neurons.
  • the invention relates to the use of a polynucleotide that comprises, or is constituted by, SEQ ID NO: 10 in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of Nervous system neurons.
  • the invention relates to the use of a polynucleotide that comprises, or is constituted by, SEQ ID NO: 11 in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of Nervous system neurons.
  • the invention relates to the use of a polynucleotide which comprises, or is constituted by, SEQ ID NO: 12 in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of Nervous system neurons.
  • said polynucleotide may have variations in its sequence with respect to the nucleotide sequences shown in SEQ ID NO: 7-
  • polynucleotide substantially homologous and functionally equivalent to said polynucleotides shown in SEQ ID NO: 7- 12.
  • a polynucleotide is
  • substantially homologous to another polynucleotide when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of the other polynucleotide of at least 30%, typically of at least 50%, advantageously of at least 60%, preferably of at least 70%, more preferably of at least 85%, and, even more preferably, at least 95%.
  • Homologous sequences can be easily identified by a person skilled in the art with the help of an appropriate computer program to compare sequences.
  • a polynucleotide substantially homologous to another polynucleotide can be isolated from an organism producing a peptide comprising the amino acid sequence of the EGF domain of the NRGl, a variant or fragment thereof, based on the information contained in said SEQ ID NO: 7-12 or is constructed based on the sequence of the polynucleotides shown in SEQ ID NO: 7-12 by, for example , the introduction of conservative or non-conservative substitutions.
  • Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides at any end of the sequence, or the deletion of one or more nucleotides at either end or inside the sequence. sequence.
  • polynucleotide eg, the polynucleotide defined in any of SEQ ID NO: 7-12
  • “functionally equivalent fragment” applied to a given polynucleotide refers, but is not limited to, a fragment of the polynucleotide whose Nucleotide sequence is shown in SEQ ID NO: 7-12 comprising the nucleotide sequence encoding a peptide comprising the amino acid sequence of the EGF domain of NRGl, a variant or fragment thereof, which maintains at least the function related to the activity promoting or stimulating the growth of axons of neurons of the nervous system of the EGF domain of NRGl.
  • said polynucleotide encoding said peptide comprising the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof possesses promoter or stimulatory activity of axon growth of neurons of the nervous system , so it can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations in the axonal development of neurons in the nervous system.
  • Said polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, can be obtained by conventional methods known to those skilled in the art, by example, from an organism producing said peptide, variant or fragment, either natively or recombinantly.
  • said polynucleotide comprises, or is constituted by, the nucleotide sequence encoding said fusion protein comprising said regions A and B.
  • the invention relates to the use of a gene construct, hereinafter "gene construct of the invention", comprising said polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment of said peptide, that is, with promoter or stimulating activity of axon growth of neurons of the nervous system, in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of neurons of the nervous system.
  • gene construct of the invention comprising said polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment of said peptide, that is, with promoter or stimulating activity of axon growth of neurons of the nervous system, in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of neurons of the nervous system.
  • the gene construct of the invention can incorporate, operably linked, a sequence regulating the expression of said polynucleotide, thereby constituting an expression cassette.
  • operably linked means that said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment thereof, encoded by said polynucleotide, is expressed in the correct reading frame under the control of control or regulatory expression sequences.
  • Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment of the It also includes promoter sequences that direct its transcription (eg, pCMV, pT7, plac, ptrc, ptac, pBAD, laugh, etc.), transcription initiation and termination signals (eg, tlt2, etc.), polyadenylation signal , ribosome binding sequences (RBS), coding sequences of transcriptional regulators (enhancers), transcriptional silencers (silencers), repressors, etc.
  • promoter sequences that direct its transcription (eg, pCMV, pT7, plac, ptrc, ptac, pBAD, laugh, etc.), transcription initiation and termination signals (eg, tlt2, etc.), polyadenylation signal , rib
  • said expression control sequence is functional in prokaryotic cells and organisms, for example, bacteria, etc.
  • said expression control sequence is functional in eukaryotic cells and organisms, for example. , mammalian cells, mammalian tumor cell lines, etc.
  • the construction of the invention further comprises a marker or gene encoding a motif or a phenotype that allows the selection of the host cell transformed with said construction.
  • the gene construct of the invention may also comprise, if necessary and to allow a better isolation, detection or secretion outside the peptide cell that comprises, or is constituted by, the amino acid sequence of the EGF domain of the NRG1, or a functionally equivalent variant or fragment of said peptide (active peptide), a nucleotide sequence encoding a peptide capable of being used for the purpose of isolation, detection or secretion of said peptide (control peptide).
  • the gene construct of the invention comprises, in addition to said polynucleotide encoding said active peptide, a nucleotide sequence encoding said control peptide; by way of illustration, not limitation, said control peptide comprises, or is constituted by, a polyhistidine sequence (eg, 6xHis), a peptide sequence recognizable by a monoclonal antibody for identification, or any other that serves to purify the fusion protein resulting by immunoaffinity chromatography (eg, tag peptides such as c-myc, HA, E-tag) (Using antibodies: a laboratory manual. Ed. Harlow and David La ⁇ e (1999). CoId Spring Harbor Laboratory Press. New York.
  • a polyhistidine sequence eg, 6xHis
  • a peptide sequence recognizable by a monoclonal antibody for identification or any other that serves to purify the fusion protein resulting by immunoaffinity chromatography (eg, tag peptides such as c-myc,
  • said gene construct of the invention comprising the polynucleotide encoding said peptide comprising the amino acid sequence of the EGF domain of NRG1, or a functionally equivalent variant or fragment thereof, possesses growth promoting or stimulating activity. of axons of the neurons of the nervous system, so it can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system.
  • the gene construct of the invention can be obtained by using techniques widely known in the state of the art [Sambrook et to the., "Molecular Cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989 VoI 1-3] and, if desired, can be inserted into an appropriate vector, such as a gene expression vector that allows its expression to be regulated under suitable conditions inside the cells.
  • the invention relates to the use of a vector, hereinafter "vector of the invention", comprising said polynucleotide encoding said peptide comprising, or consisting of, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment of said peptide, that is, with promoter or stimulatory activity of axon growth of neurons of the nervous system, or said gene construct of the invention, in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • vector of the invention comprising said polynucleotide encoding said peptide comprising, or consisting of, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment of said peptide, that is, with promoter or stimulatory activity of axon growth of neurons of the nervous system, or said gene construct of the invention, in the preparation of a pharmaceutical composition for the treatment of a disease caused by an alteration
  • an expression vector comprises, in addition to the polynucleotide encoding said peptide that comprises, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment thereof, or said gene construct of the invention, a promoter that directs its transcription (eg, pCMV, pT7, plac, ptrc, ptac, pBAD, ret, etc.), to which it is operatively linked, and other necessary or appropriate sequences that control and regulate said transcription and, where applicable, product translation of interest, for example, transcription initiation and termination signals (eg, tlt2, etc.), polyadenylation signal, origin of replication, ribosome binding sequences (RBS) 5 coding sequences of transcriptional regulators (enhancers), transcriptional silencers (silencers), repressors, etc.
  • a promoter that directs its transcription eg, pCMV, pT7, plac,
  • Appropriate expression vectors can be selected according to the conditions and needs of each specific case among expression plasmids, viral vectors (DNA or RNA), cosmids, artificial chromosomes, etc. which may also contain markers that can be used to select cells transfected or transformed with the gene or genes of interest.
  • the choice of the vector will depend on the host cell into which it will be subsequently introduced.
  • the vector of the invention is an expression vector and the polynucleotide of the invention is under the control of an appropriate promoter, such as the early cytomegalovirus (CMV) promoter.
  • said vector into which said polynucleotide or gene construct of the invention is introduced is a plasmid which, when introduced into a host cell, is integrated or not into the genome of said cell.
  • Illustrative, non-limiting examples of vectors into which said polynucleotide or gene construct of the invention can be inserted include the vector pCMV2 (Invitrogen).
  • the invention provides the vector identified as pCMV2NRGl-SMDF [containing the polynucleotide encoding the mouse CRD-NRGl isoform (Mus musculus)], while, in another particular embodiment, the invention provides the vector identified as pCMV2NRGl-Igbeta [containing the polynucleotide encoding the mouse Ig-NRGl isoform].
  • the vector of the invention is a viral vector.
  • said vector of the invention comprising said polynucleotide encoding said peptide comprising the amino acid sequence of the EGF domain of NRG1, or a functionally equivalent variant or fragment thereof, or said gene construct of the invention, it allows obtaining said peptide, variant or fragment with axon growth promoter or stimulating activity of neurons of the nervous system, so it can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system.
  • said vector may be performed by methods conventional methods known to those skilled in the art [Sambrook et to the., "Molecular Cloning, a Laboratory Manual", 2nd ed., Cold Spring Harbor Laboratory Press, NY, 1989 VoI 1 -3].
  • said vector is a vector useful for transforming prokaryotic cells, while, in another particular embodiment, said vector is a vector useful for transforming prokaryotic cells.
  • the vector of the invention can be used to transform, transfect or infect cells capable of being transformed, transfected or infected by said vector by conventional methods known to those skilled in the art (eg , by chemical transformation, electroporation, microinjection, etc.
  • a strategy includes the use of lentiviruses to infect the target cells [Ralph GS, Binley K, Wong LF, Azzouz M, Mazarakis ND ( 2006) Gene therapy for neurodegenerative and ocular diseases using lentiviral vectors.Clin Sci (Lond) 110: 37-46]
  • Such cells can be prokaryotic or eukaryotic.
  • the vector of the invention can be used to transform eukaryotic cells or prokaryotes
  • the invention relates to the use of a host cell, hereinafter "host cell of the invention", comprising said polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or an equivalent functional variant or fragment of said peptide, that is, with promoter or stimulatory activity of axon growth of neurons of the nervous system, or said gene construct of the invention or said vector of the invention, in the development of a pharmaceutical composition for the treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • host cell of the invention comprising said polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or an equivalent functional variant or fragment of said peptide, that is, with promoter or stimulatory activity of axon growth of neurons of the nervous system, or said gene construct of the invention or said vector of the invention, in the development of a pharmaceutical composition
  • Said host cell of the invention is capable of expressing the protein of the invention and can be obtained by transformation, transfection or infection with a vector of the invention, as previously mentioned, by conventional methods known to those skilled in the art [ Sambrok et al., 1989, cited supra].
  • the host cell of the invention may be a prokaryotic or eukaryotic cell, preferably a human cell and, more preferably, a genetically modified neuron as previously indicated.
  • the host cell of the invention is capable of adequately expressing the peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment of said peptide, which may be, if desired, isolated, purified and applied.
  • the host cell of the invention can be used for numerous applications, eg, (i) to produce peptides with axon growth promoter or stimulating activity of neurons of the nervous system that can serve as the basis for a pharmaceutical composition, (ii) to recombinantly amplify the polynucleotide encoding said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment thereof, contained in said host cell of the invention, (iii) can be used per se in gene therapy, etc.
  • a host cell of the invention resulting from the genetic transformation of a cell to express said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally variant or fragment equivalent thereof, it can allow the secretion of said peptide to the extracellular medium where it exerts its action, or, alternatively, it can present said peptide in the membrane of the host cell of the invention itself.
  • Gene expression systems may or may not allow the integration of the new genetic material into the genome of the host cell.
  • both the polynucleotide and the gene construct or vector provided by this invention can be used as a medicament to protect human cells, preferably human neurons affected from an axonal alteration, in a procedure of treatment and prophylaxis of gene therapy of a human being affected by a disease that involves neuronal alterations.
  • the host cells of the invention can be used as a medicament for the regeneration or implantation of tissues or cells in humans.
  • Biopharmaceutical tools and gene therapy procedures are sufficiently known to a person skilled in the art in such a way that with the information described in the present invention they can be developed without undue effort.
  • the invention relates to the use of a host cell of the invention, such as a human cell transformed with the polynucleotide, gene construct or vector provided by this invention, of virtually any cell line, preferably of the central nervous system , and, more preferably, a neuron, as a regenerating cell of human tissues, and, more preferably, of the central nervous system.
  • a host cell of the invention such as a human cell transformed with the polynucleotide, gene construct or vector provided by this invention, of virtually any cell line, preferably of the central nervous system , and, more preferably, a neuron, as a regenerating cell of human tissues, and, more preferably, of the central nervous system.
  • the aforementioned peptides, provided by this invention, as well as the cells themselves can be converted into biopharmaceuticals.
  • said host cell of the invention comprising said vector of the invention or said gene construct of the invention can be used to express said polynucleotide encoding said peptide comprising the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, which possesses promoter or stimulatory activity of the growth of axons of neurons of the nervous system, so it can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations of the Axonal development of nervous system neurons.
  • the invention relates to the use of a compound that induces the production and / or activity of NRGl, that is, with activity stimulating the growth of axons of neurons of the nervous system, in the preparation of a composition Pharmaceutical for the treatment of a disease caused by an alteration of the axonal development of neurons in the nervous system.
  • the term "compound that induces the production and / or activity of NRGl” refers, in general, to a compound that mimics and / or increases the intensity and / or prolongs the duration of the promoter activity or axon growth stimulator of nervous system neurons, of the
  • a “NRGl production inducing compound” refers, in general, to a compound that increases the transcription and / or translation of the Nrgl gene, or a compound that increases the post-translational modification and / or cell traffic of a precursor of the NRGl, or a substance that prolongs the half-life of the NRGl.
  • a "compound that induces the activity of NRGl” refers, in general, to a compound that increases the potency of the promoter or stimulatory activity of the growth of axons of neurons of the nervous system of the NRGl, or that decreases the potency of an antagonist of
  • NRGl activity inducing compound is neither said peptide comprising, or is constituted by, the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, or said polynucleotide encoding said peptide , variant or functionally equivalent fragment.
  • said compound that induces the production and / or activity of NRGl in particular, of the promoter or stimulating activity of the growth of axons of neurons of the nervous system, can be used as a therapeutic agent, in particular, in the treatment of diseases caused by alterations of the axonal development of neurons of the nervous system.
  • peptides that comprise the EGF domain of NRGl that have axon growth promoter or stimulator activity of neurons of the nervous system can be used in the treatment of diseases caused by alterations in axonal development of Nervous system neurons
  • disease caused by an alteration of the axonal development of neurons of the nervous system includes any disease, disorder or pathology in which there has been an alteration of the axonal development of one or more neurons of the nervous system.
  • diseases include:
  • neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc .
  • neuropsychiatric diseases such as bipolar disorder, autism, learning disorders, etc .
  • Y neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc .
  • neuropsychiatric diseases such as bipolar disorder, autism, learning disorders, etc .
  • Y neurodegenerative diseases, such as multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc .
  • neuropsychiatric diseases such as bipolar disorder, autism, learning disorders, etc .
  • Y Y
  • neuron includes all types of neurons, e.g., (i) neurons that are characterized by having long axons, as well as (ii) neurons that are characterized by having short axons.
  • Illustrative, non-limiting examples of neurons characterized by having long axons include thalammo-cortical neurons that travel long distances to reach their target in the cerebral cortex.
  • illustrative, non-limiting examples of neurons characterized by having short axons include cerebral cortex inhibitor interneurons, whose axons form local circuits in a restricted area of the cortex.
  • the invention relates to a pharmaceutical composition, hereinafter "pharmaceutical composition of the invention", which comprises a product selected from: (i) a peptide comprising the amino acid sequence of the domain
  • NRF1 EGF or a functionally equivalent variant or fragment of said peptide
  • the pharmaceutical composition of the invention can be used in the treatment of diseases, disorders or pathologies caused by an alteration of the axonal development of neurons in the nervous system of a subject.
  • subject refers to a member of a mammalian species, and includes, but is not limited to, pets, primates and humans; preferably, the subject is a human being, male or female, of any age or race.
  • the pharmaceutical composition of the invention comprises one or more pharmaceutically acceptable carriers or excipients, in addition, of said product (active ingredient) selected from: (i) a peptide comprising the amino acid sequence of the EGF domain of IaNRG1, or a variant or fragment functionally equivalent thereof, (ii) a polynucleotide encoding said functionally equivalent peptide, variant or fragment thereof, (iii) a gene construct of the invention, (iv) a vector of the invention, (v) a host cell of the invention, and (vi) a compound that induces the production and / or activity of said NRGl, for administration even subject to the need for treatment.
  • active ingredient selected from: (i) a peptide comprising the amino acid sequence of the EGF domain of IaNRG1, or a variant or fragment functionally equivalent thereof, (ii) a polynucleotide encoding said functionally equivalent peptide, variant or fragment thereof, (iii) a gene construct of the invention, (iv
  • the pharmaceutical composition of the invention comprises a peptide comprising the amino acid sequence of the EGF domain of NRG1, or a functionally equivalent variant or fragment thereof, such as a peptide selected from peptides whose amino acid sequences are shown in SEQ ID NO: 1-6.
  • the pharmaceutical composition of the invention comprises a polynucleotide encoding said peptide comprising the amino acid sequence of the EGF domain of the NRGl, or a functionally equivalent variant or fragment thereof, such as a polynucleotide selected from the polynucleotides whose Nucleotide sequences are shown in SEQ ID NO: 7-12.
  • the pharmaceutical composition of the invention comprises a gene construct of the invention. In another particular embodiment, the pharmaceutical composition of the invention comprises a vector of the invention.
  • the pharmaceutical composition of the invention comprises a host cell of the invention, preferably human, and more preferably a neuron, transformed by the polynucleotide, construct or vector provided by this invention.
  • the pharmaceutical composition of the invention will be presented in a pharmaceutical form suitable for administration to a subject by any appropriate route of administration.
  • the pharmaceutical composition of the invention will include the pharmaceutically acceptable carriers and excipients necessary for the preparation of the pharmaceutical form of administration chosen.
  • the pharmaceutical form of administration of the active ingredient (product) may vary within a wide range of possibilities known to those skilled in the art, depending on the nature of said active ingredient (peptide, polynucleotide, or cell).
  • the pharmaceutical composition of the invention will be formulated in a pharmaceutical form of solid administration (eg, tablets, capsules, dragees, granules, suppositories, etc.) or liquid (eg, solutions, suspensions, emulsions, etc.) for administration by any appropriate route of administration, for example, orally, parenterally (eg, intramuscularly, subcutaneously, intravenously, etc.), etc.
  • This pharmaceutical form of administration of the active ingredient is especially suitable when the active ingredient is said peptide comprising the amino acid sequence of the EGF domain of NRG1, or a variant or functionally equivalent fragment thereof.
  • the pharmaceutically acceptable carriers and excipients appropriate for the pharmaceutical form of administration and route of administration chosen will be chosen.
  • the pharmaceutical composition of the invention can be formulated in the form of a composition intended for use in gene therapy; by way of illustration, not limitation, in this case, the pharmaceutical composition of the invention may contain a vector, viral or non-viral, comprising a polynucleotide of the invention or a gene construct of the invention.
  • said vectors may be viral vectors, for example, based on retroviruses, adenoviruses, etc., or non-viral such as DNA-liposome, DNA-polymer, DNA-polymer-liposome complexes, etc.
  • Such vectors which contain a polynucleotide or a gene construct of the invention can be administered directly to the human or animal body by conventional methods.
  • said vectors can be used to transform, transfect or infect cells, for example, mammalian cells, including man, ex vivo, and then implant them in the human or animal body to obtain the desired therapeutic effect.
  • said cells will be formulated in a suitable medium that does not adversely affect the viability of said cells.
  • the pharmaceutical composition of the invention comprises at least one product (active ingredient) selected from: (i) a peptide comprising the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, (ii ) a polynucleotide encoding said peptide, variant or functionally equivalent fragment thereof, (iii) a gene construct of the invention, (iv) a vector of the invention, (v) a host cell of the invention, and (vi) a compound that induces the production and / or activity of said NRGl, in a therapeutically efficient amount.
  • active ingredient selected from: (i) a peptide comprising the amino acid sequence of the EGF domain of NRGl, or a functionally equivalent variant or fragment thereof, (ii ) a polynucleotide encoding said peptide, variant or functionally equivalent fragment thereof, (iii) a gene construct of the invention, (iv) a vector of the invention, (v) a host cell of the
  • the term "therapeutically efficient amount” refers to the amount of product (active ingredient) calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of protein and the therapeutic effect to be achieved.
  • the pharmaceutical composition of the invention may contain only one of said products (active ingredients), or, if desired, a combination of two or more of said different products.
  • the dose of active ingredient to be administered to a subject will be a therapeutically efficient amount and may vary within a wide range.
  • the pharmaceutical composition of the invention can be administered one or more times a day for preventive or therapeutic purposes.
  • the dose of active ingredient to be administered will depend on numerous factors, including the characteristics of the product to be administered, such as, for example, its activity and biological half-life, the concentration of the product in the pharmaceutical composition, the clinical situation of the subject, the severity of the pathology, the pharmaceutical form of administration chosen, etc. For this reason, the doses mentioned in this invention should be considered only as guidelines for the person skilled in the art, and he must adjust the doses according to the variables mentioned above.
  • the pharmaceutical composition of the invention is used as a promoter or stimulator composition for the growth of axons of neurons of the nervous system, so it can be used in the treatment of diseases caused by alterations of axonal development of neurons of the nervous system.
  • diseases include chronic or acute spinal injuries; traumatic brain injuries; neurodegenerative diseases, e.g., multiple sclerosis, Alzheimer's disease, Parkinson's disease, etc .; neuropsychiatric diseases, e.g., autism, bipolar disorder, learning disorders, etc .; and other diseases of the nervous system, e.g., epilepsy.
  • the pharmaceutical composition of the invention can be used with other drugs, for example, drugs with promoter or stimulatory activity of the axon growth of additional nervous system neurons, in order to increase the efficiency of the pharmaceutical composition of the invention, thereby generating a combination therapy.
  • additional drugs may be part of the same pharmaceutical composition or, alternatively, may be provided as a separate pharmaceutical composition for administration at the same time (simultaneous administration) as the pharmaceutical composition of the invention or at different times (sequential administration) with respect to the administration of the pharmaceutical composition of the invention.
  • the invention relates to a method for the treatment of a disease caused by an alteration of the axonal development of neurons of the nervous system, which comprises administering to a subject in need of treatment, a therapeutically efficient amount of a selected product. between:
  • Virtually any disease caused by an alteration of the axonal development of neurons of the nervous system can be treated according to said method, such as traumatic spinal and brain injuries, neurodegenerative diseases, neuropsychiatric diseases, neurological diseases related to GABAergic function, etc.
  • diseases include chronic or acute spinal injuries; traumatic brain injuries; neurodegenerative diseases, such as sclerosis multiple, Alzheimer's disease, Parkinson's disease, etc .; neuropsychiatric diseases, such as autism, bipolar disorder, learning disorders, etc .; and other diseases of the nervous system, such as epilepsy.
  • said product for administration to said subject, said product (active ingredient) will be formulated in the form of a pharmaceutical composition, such as the pharmaceutical composition of the invention previously described, and will be dosed in a suitable dose that allows recovery of the normal development of axons in neurons.
  • a pharmaceutical composition such as the pharmaceutical composition of the invention previously described
  • Organotypic sliced cultures were performed as described in detail previously (Anderson SA, Eisenstat DD, Shi L, Rubenstein JLR (1997) Interneuron migration from basal forebrain to neocortex: dependence on DIx genes. Science 278: 474- 476).
  • the slices were grown on polycarbonate membranes for culture (8 ⁇ m pore size; Corning Costar) in culture plates with 1 ml of medium (Neurobasal / B-27) (Life Technologies) supplemented with glutamine, 5 horse serum % and penicillin / streptomycin. In these trials, the TCAs begin to grow after 36 hours of growth; The slices were grown for 72-96 hours.
  • the COS7 cell aggregates (commercially available from Sigma-Aldrich) were prepared by diluting the transfected cells with matrigel as described previously (Flames N, Long JE, Garratt AN 5 Fischer TM, Gassmann M, Birchmeier C, Lai C, Rubenstein JL, Mar ⁇ n O (2004) Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1. Neuron 44: 251-261).
  • the COS7 cell line was developed from the standardized African green monkey kidney line, after transforming these cells with a mutant form of the SV40 virus containing the wild form of the large tumor antigen. They are cells with a typical fibroblast morphology, widely used in DNA transfection experiments.
  • DMEM fetal calf serum
  • glutamax 2 mM
  • penicillin / streptomycin 100 U / ml penicillin and 100 ⁇ g / ml streptomycin
  • the dorsal thalamus explants of wild mice were dissected at 3.5 and we cultured them in collagen, laminin or matrigel for 96 hours.
  • the explants were: (i) confronted with aggregates of COS7 cells transfected with Gjp or co-transfected with Ig-Nrgl and GJp; or (2) cultured with medium supplemented with a peptide constituted by the EGF domain, variant ⁇ , of human NRG1 (0.1 ⁇ M Heregulinal ⁇ , Peprotech). After fixing the dorsal thalamus explants, they were divided into four sectors, and the length of the 15 longest axons in each explant was measured using Sigma Sean Pro Software.
  • the cells were transfected at 60-70% confluence using Lipofectamine® or Fugene® following the manufacturers instructions. Transfections were carried out with the following plasmids: pCMV2NRGl-SMDF (which allows the expression of the CRD-Nrgl isoform of Mus musculus), pCMV2NRGl-Igbeta (which allows the expression of the Ig-Nrgl isoform of M. musculus), or pCMV2 as a control. All these plasmids were generously donated by C.
  • NRGl The role of NRGl in the development of axons of neurons of the nervous system
  • the role of NRGl in the development of axons of neurons of the nervous system has been fundamentally investigated in two different neuronal types, (i) thalamus-cortical neurons, which are characterized for having long axons that travel long distances to reach their target in the cerebral cortex, and (ii) the cerebral cortex inhibitor interneurons, which have short axons that form local circuits in a restricted area of the cortex.
  • the thalamus-cortical axons grow into the cerebral cortex after 72 hours of culture in the vast majority of cases, even when the area that expresses NRG1 ⁇ has been cut but has remained in the same place ( Figures IF and IH).
  • the TCAs grow very little and in virtually no case (2/21) reach the cortex ( Figures IG and IH).
  • Nrgl is a potent stimulator of the growth of long axons, as is the case of TCAs 5, it was proposed to investigate the role that this molecule could play in the development of short axons, as is the case of interneuron axons cortical
  • the distribution of Nrgl in the postnatal cortex seems to indicate that it is found mainly in projection neurons, also called pyramidal cells.
  • NRGl was able to stimulate the development of the axons of the GABAergic interneurons of the cerebral cortex.
  • COS7 cells were transfected with a control plasmid or with a plasmid encoding NRGl (mouse CRD-NRGl form), or with the EGF motif of human NRGl ⁇ , and its supernatant was collected after 48 hours in culture.
  • These supernatants were added to 7-day in vitro (div) cultures from neurons dissociated from the medial ganglionic eminence (MGE, the main source of cortical interneurons) of Gad65: GFP mice, and after 2 div the cultures were fixed and analyzed (Figure 4A).
  • MGE medial ganglionic eminence

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Abstract

L'invention concerne l'utilisation d'un peptide qui comprend la séquence d'acides aminés du domaines semblable au facteur de croissance épidermique (EGF) [domaine EGF] de la neuréguline 1 [NRG1], ou un variant ou un fragment fonctionnellement équivalent à ce peptide, ou d'un polynucléotide codant pour ledit peptide, variant ou fragment fonctionnellement équivalent, ou d'un composé induisant la production et/ou l'activité de ladite NRG1, dans l'élaboration d'une composition pharmaceutique destinée au traitement de maladies causées par des altérations du développement axonal des neurones du système nerveux.
PCT/ES2007/000192 2006-04-05 2007-04-03 Traitement de maladies causées par des altérations du développement axonal des neurones WO2007113361A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148023A3 (fr) * 2007-05-23 2009-01-22 Med College Georgia Res Inst Compositions et procédés pour traiter des troubles neurologiques
WO2009062750A3 (fr) * 2007-11-16 2009-09-24 Proteosys Ag Isoformes solubles actives de neuréguline modifiées de façon post-traductionnelle
WO2011021916A3 (fr) * 2009-08-21 2011-07-07 경희대학교 산학협력단 Utilisation du peptide à domaine de type egf de l'héréguline bêta 1

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11426447B2 (en) * 2016-04-19 2022-08-30 Leibniz-Institut Fur Alternsforschung—Fritz-Lipmann-Institut E.V. (Fli) Neuregulin for the treatment of tumors of the nervous system
CN108277272A (zh) * 2018-04-09 2018-07-13 重庆医科大学附属第医院 一种辅助癫痫诊断的标志物及其检测试剂盒

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001026607A2 (fr) * 1999-10-08 2001-04-19 Uab Research Foundation Isoformes de variant d'epissage de neureguline smdf et ggf et utilisations de celles-ci
US20030129688A1 (en) * 1997-02-10 2003-07-10 Genentech, Inc. Heregulin variants
US20050208527A1 (en) * 2000-02-28 2005-09-22 Decode Genetics Ehf. Human schizophrenia gene

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030129688A1 (en) * 1997-02-10 2003-07-10 Genentech, Inc. Heregulin variants
WO2001026607A2 (fr) * 1999-10-08 2001-04-19 Uab Research Foundation Isoformes de variant d'epissage de neureguline smdf et ggf et utilisations de celles-ci
US20050208527A1 (en) * 2000-02-28 2005-09-22 Decode Genetics Ehf. Human schizophrenia gene

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ESPER R.M. ET AL.: "Neuregulins: versatile growth and differentiation factors in nervous system development and human disease", BRAIN RES. REV., vol. 51, no. 2, August 2006 (2006-08-01), pages 161 - 175 *
FLAMES N. ET AL.: "Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1", NEURON., vol. 44, no. 2, 2004, pages 251 - 261 *
HO W.-H. ET AL.: "Sensory and motor neuron-derived factor. A novel heregulin variant highly expressed in sensory and motor neurons", J. BIOL. CHEM., vol. 270, no. 24, 1995, pages 14523 - 14532, XP002003223 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008148023A3 (fr) * 2007-05-23 2009-01-22 Med College Georgia Res Inst Compositions et procédés pour traiter des troubles neurologiques
WO2009062750A3 (fr) * 2007-11-16 2009-09-24 Proteosys Ag Isoformes solubles actives de neuréguline modifiées de façon post-traductionnelle
AU2008323169B2 (en) * 2007-11-16 2013-08-29 Mind-Nrg Sa. Active soluble post-translationally modified neuregulin isoforms
CN105561299A (zh) * 2007-11-16 2016-05-11 意识-Nrg公司 经翻译后修饰的有活性的可溶性神经调节蛋白同种型
US10350269B2 (en) 2007-11-16 2019-07-16 Mind-Nrg Sarl Method of effecting neuroprotection using soluble Neuregulin isoforms
US10813978B2 (en) 2007-11-16 2020-10-27 Mind-NRG Sàrl Method of effecting neuroprotection using soluble neuregulin isoforms
US12251423B2 (en) 2007-11-16 2025-03-18 Mind-NRG Sàrl Method of effecting neuroprotection using soluble neuregulin isoforms
WO2011021916A3 (fr) * 2009-08-21 2011-07-07 경희대학교 산학협력단 Utilisation du peptide à domaine de type egf de l'héréguline bêta 1
KR101186218B1 (ko) 2009-08-21 2012-10-08 경희대학교 산학협력단 히레귤린 베타1의 ｅｇｆ-성 도메인 펩타이드의 용도

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