Nucleic acid constructs encoding the Trk fragment and their utilization

Abstract

The present invention provides a novel nucleic acid construct encoding a functional fragment of the Trk family and its use. The nucleic acid construct of one embodiment of the present invention encodes a fusion polypeptide comprising the intracellular region and the membrane localization sequence of Trk.

Description

Technical Field

[0001] The present invention relates to a nucleic acid construct encoding a functional fragment of Trk family and its utilization. Background Art

[0002] Known neurotrophic factors that play an important role in the survival of nerve cells, axon growth, and development of the central nervous system include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), Neurotrophin-3 (NT-3), and NT-4 / 5 (Non-Patent Document 1).

[0003] TrkB is known as a high affinity receptor for BDNF, and its extracellular domain usually forms a dimer to accept BDNF. However, TrkB also exists in a truncated form lacking the intracellular tyrosine kinase domain, and its function has not yet been fully elucidated (Non-Patent Document 2).

[0004] The inventors have been conducting research on neurotrophic factors for many years and reported that signal control between glial cells and glial cells and between glial cells and nerve cells is important for neuroprotection (Non-Patent Documents 3 to 5).

[0005] Furthermore, in the process of continuing research on neuroprotection and axon regeneration using TrkB signaling, attention has been paid to the function of Dock3 as a guanine nucleotide exchange factor (GEF) activated by TrkB signaling (Non-Patent Documents 6 to 9).

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent Document 1: International Publication No. WO 2018 / 185468 (International Publication Date: October 11, 2018)

[0009] Non-patent literature

[0010] Non-patent document 1: Park H, Poo MM. Neurotrophin regulation of neural circuit development and function. Nat Rev Neurosci 14(1):7-23, 2013.

[0011] Non-Patent Document 2: Fenner BM. Truncated TrkB: beyond a dominant negative receptor. Cytokine Growth Factor Rev 23(1-2):15-24, 2012.

[0012] Non-Patent Document 3: Harada T, Harada C, Nakayama N, Okuyama S, Yoshida K, Kohsaka S, Matsuda H, Wada K. Modification of glial-neuronal cell interactions prevents photoreceptor apoptosis during light-induced retinal degeneration. Neuron 26(2):533-541, 2000.

[0013] Non-Patent Document 4: Harada T, Harada C, Kohsaka S, Wada E, Yoshida K, Ohno S, Mamada H, Tanaka K, Parada LF, Wada K. Microglia-Muller glia cell interactions control neurotrophic factor production during light-induced retinal degeneration. J Neurosci 22(21):9228-9236, 2002.

[0014] Non-Patent Document 5: Harada C, Guo X, Namekata K, Kimura A, Nakamura K, Tanaka K, Parada LF, Harada T. Glia- and neuron-specific functions of TrkB signalling during retinal degeneration and regeneration. Nat Commun 2:189, 2011.

[0015] Non-patent document 6: Namekata K, Harada C, Taya C, Guo X, Kimura H, Parada LF, Harada T. Dock3 induces axonal outgrowth by stimulating membrane recruitment of the WAVE complex. Proc Natl Acad Sci USA 107(16):7586-7591, 2010.

[0016] Non-patent literature 7: Namekata K, Harada C, Guo X, Kimura A, Kittaka D, Watanabe H, Harada T. Dock3 stimulates axonal outgrowth via GSK-3β-mediated microtubule assembly. J Neurosci 32(1):264-274,2012.

[0017] Non-patent literature 8: Namekata K, Kimura A, Kawamura K, Harada C, Harada T. DockGEFs and their therapeutic potential: neuroprotection and axonregeneration. Prog Retin Eye Res 43: 1-16, 2014.

[0018] Non-patent document 9: Kazuhiko Yukata and Takayuki Harada, Function of Dock3 in axon regeneration, Biochemistry 84(5), 368-373, 2012. Summary of the invention

[0019] Technical problem to be solved by the invention

[0020] As described above, the important role of TrkB in neuroprotection and the like has been elucidated from various viewpoints.

[0021] However, it is difficult to induce high expression of TrkB in a gene vector into which the full-length TrkB gene is inserted. This is because the full-length TrkB gene has a relatively large size, and therefore the size of the promoter that can be used is limited.

[0022] Furthermore, in the treatment of nerve-related diseases, it is believed that not only overexpression of TrkB but also regular administration of BDNF is required. Therefore, gene therapy research that effectively utilizes TrkB has not yet been put into practical use.

[0023] Recently, an expression vector into which a gene encoding a fusion protein of BDNF and TrkB is inserted has been reported (Patent Document 1). However, the molecular size of this fusion protein suggests that the expression level is not that high.

[0024] The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel nucleic acid construct encoding a functional fragment of the Trk family and its use.

[0025] Technical solutions to the problem

[0026] In order to solve the above-mentioned problems, the present invention includes the aspects shown below.

[0027] (i) A nucleic acid construct encoding a fusion polypeptide comprising the intracellular region of Trk and a membrane localization sequence.

[0028] (ii) A method for activating a cell, comprising the step of localizing the intracellular region of Trk to the cell membrane via a membrane localization sequence in the cell.

[0029] (iii) A fusion polypeptide comprising the intracellular region and the membrane localization sequence of Trk.

[0030] Effects of the Invention

[0031] According to one aspect of the present invention, a novel nucleic acid construct encoding a functional fragment of the Trk family and its utilization can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a diagram showing the structure of constitutively activated TrkB (CA-TrkB).

[0033] Figure 2 This is a diagram showing the adeno-associated virus vector prepared in Example 1.

[0034] Figure 3 It is a graph showing the results of Example 1 (expression level of CA-TrkB).

[0035] Figure 4 This is a diagram showing other results of Example 1 (intracellular signaling using CA-TrkB).

[0036] Figure 5 This is a graph showing the results of Example 2 (protection of retinal ganglion cells by CA-TrkB).

[0037] Figure 6 This is a diagram showing the results of Example 2 (optic nerve axon regeneration using CA-TrkB).

[0038] Figure 7This is a graph showing the results of Example 3 (protection of retinal ganglion cells by CA-TrkB using glaucoma model mice).

[0039] Figure 8 It is a diagram showing the results of Example 4 (Verification of signal transduction using CA-TrkB variants).

[0040] Fig. 9 It is a diagram showing the structures of constitutively activated TrkA and TrkC related to Example 5, and the results of Example 5 (intracellular signaling using CA-TrkA and CA-TrkC).

[0041] Fig.10 It is a diagram showing the base sequence of the gene fragment encoding iSH-TrkB in the Example.

[0042] Fig.11 It is a diagram showing the base sequence of the gene fragment encoding CA-TrkA in the Examples.

[0043] Fig.12 It is a diagram showing the base sequence of the gene fragment encoding CA-TrkC in the Example.

[0044] Fig.13 It is a graph showing the results of Example 6 (measurement of visual evoked potential). DETAILED DESCRIPTION

[0045] [Definition of terms, etc.]

[0046] In this specification, "polynucleotide" may also be referred to as "nucleic acid" or "nucleic acid molecule". Unless otherwise specified, "polynucleotide" includes polynucleotides containing known analogs of naturally occurring nucleotides that can function in the same way as naturally occurring nucleotides. In addition, "base sequence" may also be referred to as "nucleic acid sequence" or "nucleotide sequence". Unless otherwise specified, "base sequence" refers to a sequence of deoxyribonucleotides or a sequence of ribonucleotides. In addition, a polynucleotide may be a single-stranded or double-stranded structure, and if single-stranded, it may be a sense strand or an antisense strand.

[0047] In this specification, "gene" refers to a "polynucleotide" encoding a protein.

[0048] In the present specification, the "expression control region" of a gene refers to a "polynucleotide" that controls the expression of a gene. Examples of the "expression control region" include a promoter region, an enhancer region, and the like.

[0049] In this specification, "expression cassette" refers to an expression unit including an expression control region that functions in an expression host and a polynucleotide operably linked to the expression control region. In the expression cassette, the polynucleotide is preferably a gene or a gene fragment. As an example, an expression cassette is formed by genetically linking the expression control region to the polynucleotide. "Operably linked" refers to a state in which the expression of the polynucleotide is controlled by the expression control sequence. The expression cassette can also be in the form of an expression vector.

[0050] In this specification, "polypeptide" may also be referred to as "protein". "Polypeptide" includes a structure in which amino acids are linked by peptide bonds, and may also include structures such as sugar chains or isoprenoid groups. Unless otherwise specified, "polypeptide" includes polypeptides containing known analogs of naturally occurring amino acids that can function in the same manner as naturally occurring amino acids.

[0051] [1. Nucleic acid construct]

[0052] The nucleic acid construct of one embodiment of the present invention is a nucleic acid construct encoding a fusion polypeptide comprising an intracellular region of Trk and a membrane localization sequence. The present invention is based on the following surprising discovery: by expressing the nucleic acid construct in cells, the intracellular signal transduction pathway downstream of Trk can be activated even without administering neurotrophin. That is, the present invention relates to a new method for cell activation, in which the intracellular region of Trk is localized to the cell membrane through the membrane localization sequence possessed by the fusion polypeptide encoded by the nucleic acid construct.

[0053] The nucleic acid construct of the present embodiment exhibits, for example, the following representative effects.

[0054] 1) No need to overexpress or administer the corresponding neurotrophic factors.

[0055] 2) Compared with the full length of Trk, the molecular size required for activation can be greatly reduced. Thus, high expression of the molecule can be achieved. In addition, the design freedom of the nucleic acid construct is greatly increased, so, for example, it is easier to achieve high expression or improvement of gene introduction efficiency.

[0056] (Fusion peptide)

[0057] The fusion polypeptide encoded by the nucleic acid construct of the present embodiment includes the following 1) the intracellular region of Trk, and 2) a membrane localization sequence. Depending on its characteristics, the membrane localization sequence is sometimes preferably configured on the N-terminal side of the fusion polypeptide or the C-terminal side of the fusion polypeptide. In relation to the intracellular region of Trk, the membrane localization sequence can be configured relative to the N-terminal side (the side close to the transmembrane region) of the intracellular region, or it can be configured relative to the C-terminal side. In addition, in the constitutively activated Trk shown in the Examples, the membrane localization sequence is configured on the C-terminal side of the fusion polypeptide, and is configured relative to the C-terminal side of the intracellular region of Trk. Thus, the constitutively activated Trk shown in the Examples causes the intracellular region of Trk to be located at a position opposite to the usual position relative to the cell membrane (see also Figure 1 and Fig. 9 ).

[0058] In one example, the fusion polypeptide may also contain "other sequences" in addition to the "intracellular region and membrane localization sequence of Trk". Examples of "other sequences" include spacer sequences and SH2 sequences of p85. The position of inserting the "other sequence" may be set according to the purpose, for example, between the intracellular region and the membrane localization sequence of Trk, on the N-terminal side of the fusion polypeptide (in this case, the membrane localization sequence is sometimes preferably arranged on the C-terminal side), or on the C-terminal side of the fusion polypeptide (in this case, the membrane localization sequence is sometimes preferably arranged on the N-terminal side).

[0059] In a preferred example, the number of amino acids in the fusion polypeptide is within the range of 300 to 650, and sometimes more preferably within the range of 330 to 630. In addition, in a preferred example, the number of amino acids in the "intracellular region and membrane localization sequence of Trk" in the fusion polypeptide is 300 to 450, and sometimes more preferably within the range of 330 to 440, or 350 to 440, or 360 to 440 (or 370 to 440), or 360 to 410 (or 370 to 410). If the size of the fusion polypeptide is small, it has the following advantages, for example: 1) the design freedom of other structures in the nucleic acid construct (i.e., structures other than the region encoding the fusion polypeptide) is increased, 2) the expression efficiency of the fusion polypeptide is improved, etc. In particular, the options of promoters, etc. in the nucleic acid construct are increased, which greatly contributes to the improvement of the expression efficiency of the fusion polypeptide.

[0060] In one example, the fusion polypeptide may also include a portion of the extracellular region of Trk and / or a portion of the transmembrane region. In a preferred example, the fusion polypeptide does not include the extracellular region and / or the transmembrane region of Trk. In a more preferred example, the fusion polypeptide does not include both the extracellular region and the transmembrane region of Trk.

[0061] (Intracellular domain of Trk)

[0062] In this embodiment, the intracellular region of Trk refers to a polypeptide corresponding to the intracellular region of the high affinity receptor for neurotrophic factor (Trk) which is a single transmembrane protein, and a polypeptide functionally equivalent to the intracellular region. The intracellular region is also called a cytoplasmic domain.

[0063] As a family of Trk, three types of TrkA, TrkB and TrkC have been reported so far. TrkA, TrkB and TrkC are all domains that receive neurotrophic factors in their extracellular regions, and their intracellular regions have tyrosine kinase activity. In the cells of the nervous system, TrkA, TrkB and TrkC all activate their downstream intracellular signal transduction pathways by receiving the corresponding neurotrophic factors. In addition, TrkA is a receptor for NGF, TrkB is a receptor for BDNF and NT-4 / 5, and TrkC is a receptor for NT-3.

[0064] The intracellular region of Trk can be any one as long as it 1) has tyrosine kinase activity and / or 2) activates the downstream intracellular signal transduction pathway of Trk when localized in the cell membrane. The activation of the downstream intracellular signal transduction pathway of Trk can be understood as a known method, such as increasing the expression of at least one selected from Ras, ERK1 / 2, PI3K, Akt and phospholipase C-γ.

[0065] The intracellular region of Trk is not particularly limited, and for example, the following polypeptides are preferred.

[0066] 1) A polypeptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 2, 6, 9, 12, or 14. In addition, SEQ ID NO: 2 is an example of an amino acid sequence of the intracellular region of TrkB derived from human, SEQ ID NO: 6 is an example of an amino acid sequence of the intracellular region of TrkA derived from mouse, SEQ ID NO: 9 is an example of an amino acid sequence of the intracellular region of TrkC derived from mouse, SEQ ID NO: 12 is an example of an amino acid sequence of the intracellular region of TrkA derived from human, and SEQ ID NO: 14 is an example of an amino acid sequence of the intracellular region of TrkC derived from human.

[0067] 2) A polypeptide that is functionally equivalent to a polypeptide represented by an amino acid sequence as described in any one of SEQ ID NOs. 2, 6, 9, 12, or 14. Examples of functionally equivalent polypeptides include polypeptides represented by an amino acid sequence that exhibits 90% or more sequence identity to an amino acid sequence as described in any one of SEQ ID NOs. 2, 6, or 9. In addition, the sequence identity is more preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0068] In addition, the 104th amino acid (Lys) in the amino acid sequence shown in SEQ ID NO: 6 is an amino acid essential for tyrosine kinase activity or activation of an intracellular signal transduction pathway downstream of TrkA, and therefore, it is preferably free of mutation.

[0069] Similarly, the 134th amino acid (Lys) in the amino acid sequence shown in SEQ ID NO: 2 is an amino acid essential for tyrosine kinase activity or activation of an intracellular signal transduction pathway downstream of TrkB, and therefore, preferably has no mutation.

[0070] Similarly, the 118th amino acid (Lys) in the amino acid sequence shown in SEQ ID NO: 9 is an amino acid essential for tyrosine kinase activity or activation of an intracellular signal transduction pathway downstream of TrkC, and therefore, preferably has no mutation.

[0071] Similarly, the 104th amino acid (Lys) in the amino acid sequence shown in SEQ ID NO: 12 is an amino acid essential for tyrosine kinase activity or activation of an intracellular signal transduction pathway downstream of TrkA, and therefore, preferably has no mutation.

[0072] Similarly, the 118th amino acid (Lys) in the amino acid sequence shown in SEQ ID NO: 14 is an amino acid essential for tyrosine kinase activity or activation of an intracellular signal transduction pathway downstream of TrkC, and therefore, preferably has no mutation.

[0073] (Membrane localization sequence)

[0074] The membrane localization sequence is a peptide sequence for localizing the above-mentioned fusion polypeptide to the cell membrane. The membrane localization sequence is preferably a sequence that induces lipid modification of amino acids. The type of lipid modification of amino acids is not particularly limited, and examples thereof include 1) fatty acylation, 2) prenylation, 3) glycosylphosphatidylinositolization, or 4) cholesterolation, and more specifically, myristoylation, palmitoylation, farnesylation, or geranylgeranylation, and among these, farnesylation or geranylgeranylation is preferred.

[0075] Specific examples of the amino acid sequence motif constituting the membrane localization sequence include the following sequences: X refers to any amino acid, A refers to an aliphatic amino acid (eg, isoleucine, leucine, valine, alanine, methionine), and C refers to cysteine.

[0076] 1) Met-Gly-XXX-Ser-XXX (N-terminal lipid modification motif (N-myristoylation)), 2) CAAX motif, CC motif, CXC motif, CCXX motif (C-terminal lipid modification motif).

[0077] (Nucleic Acid Construct Form)

[0078] One form of the nucleic acid construct of the present embodiment is a gene encoding the above-mentioned fusion polypeptide comprising the intracellular region and membrane localization sequence of Trk. One example is a gene having any of the base sequences shown in SEQ ID NOs. 4, 5, 8 or 11, or a gene having a base sequence showing 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with any of the base sequences shown in SEQ ID NOs. 4, 5, 8 or 11. In addition, an example of a gene encoding the intracellular region of Trk constituting the above-mentioned fusion polypeptide is a gene having any of the base sequences shown in SEQ ID NOs: 3, 7, 10, 13 or 15, or a gene having a base sequence showing 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with any of the base sequences shown in SEQ ID NOs: 3, 7, 10, 13 or 15.

[0079] Another form of the nucleic acid construct of the present embodiment is an expression cassette for expressing the above-mentioned gene. The expression cassette is preferably an expression vector, more preferably a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, an adeno-associated viral vector, a herpes virus vector, a vaccinia virus vector, or other viral vectors. Among them, from the perspective of gene therapy for neurological diseases, adeno-associated viral vectors are preferred, wherein, from the perspective of gene therapy for neurological diseases, serotype 1 (AAV1) showing tissue tropism for the central nervous system, serotype 2 (AAV2) with a wider tissue tropism, and serotype 5 (AAV5) showing tissue tropism for the central nervous system or retina are sometimes preferred.

[0080] The expression cassette includes any expression control region that functions in the expression host. The type of promoter used as the expression control region is not particularly limited. Specifically, for example, CAG promoter, CMV promoter, SYN (synaptophysin) I and other promoters that cause high expression in mammals (especially humans) or selectively function in specific tissues are sometimes preferred. As a promoter, CAG promoter is sometimes more preferred. In the expression cassette, the promoter is configured on the upstream side of the gene.

[0081] The above expression cassette may also include a functional sequence as needed, which has the effect of improving the stability of mRNA produced by transcription, and plays a role in increasing the expression of the gene, etc. As an example of a functional sequence, a WPRE (woodchuck hepatitis virus posttranscriptional regulatory element) sequence can be cited. In the expression cassette, the WPRE sequence is arranged on the downstream side of the gene.

[0082] Another example of a functional sequence that may be included in the expression cassette is a signal sequence for adding a poly A tail. An example of a signal sequence for adding a poly A tail includes a sequence corresponding to simian virus 40, a sequence corresponding to human growth hormone, etc., and a sequence corresponding to human growth hormone is sometimes more preferred. In the expression cassette, the WPRE sequence is arranged on the downstream side of the gene.

[0083] The expression cassette may further include ITRs (inverted terminal repeat sequences) etc. ITRs are arranged in the expression cassette so as to sandwich the entire transcription unit (from the promoter to the signal sequence for adding the poly A tail).

[0084] [2. Pharmaceutical composition]

[0085] The pharmaceutical composition of one embodiment of the present invention is a gene therapy drug comprising the above-mentioned nucleic acid construct. In one example, the pharmaceutical composition is a drug for a nerve-related disease, and in a more specific example, the pharmaceutical composition is used for neuroprotection or nerve regeneration. The pharmaceutical composition can exert a strong neuroprotective effect and axon regeneration effect, and is used for the treatment and / or prevention of nerve-related diseases. Within the scope of the present invention, a method for treating and / or preventing nerve-related diseases by administering an effective amount of the pharmaceutical composition to a subject is included.

[0086] (Target of administration)

[0087] The human or non-human animal as the subject of administration is preferably any one selected from the group consisting of mammals including humans. The type of mammal as the subject of administration is not particularly limited, and examples thereof include experimental animals such as mice, rats, rabbits, guinea pigs, primates other than humans, pets such as dogs and cats, livestock such as cattle and horses, and humans, and humans are particularly preferred. The human or non-human animal as the subject of administration has, for example, the following neurological diseases.

[0088] (Neurological diseases)

[0089] The types of nerve-related diseases to be treated and / or prevented are not particularly limited, and refer to diseases in which the functions of nervous system cells are impaired due to degeneration and / or cell death of nervous system cells. The causes of degeneration or cell death of nervous system cells are not particularly limited, and include damage to the nervous system caused by accidents, etc.

[0090] As the nerve-related diseases of the visual nervous system, neurodegenerative diseases such as glaucoma, retinitis pigmentosa, age-related macular degeneration, diabetic retinopathy, and damage to the visual nervous system caused by accidents, etc. can be listed, wherein, diseases with degeneration of retinal ganglion cells represented by glaucoma, and damage to the visual nervous system (optic nerve trauma) can sometimes be more preferably used as the target disease of the present invention. As the nerve-related diseases of the auditory nervous system, sensory nerve degeneration diseases such as neurological deafness, and damage to the auditory nervous system caused by accidents, etc. can be listed.

[0091] (Dosage method, dosage, frequency of administration, etc.)

[0092] The method of administration of the pharmaceutical composition is not particularly limited, and the composition may be administered locally by injection (using a syringe or an infusion pump, etc.), eye drops, transdermal administration, sublingual administration, etc., or systemically by oral administration, intravenous or intraarterial intravascular administration, enteral administration, etc. In a preferred administration form, the composition is administered locally near the nervous system to be treated, etc.

[0093] The dosage (effective amount) of the pharmaceutical composition can be appropriately set according to the age, sex, symptoms, administration route, administration frequency, etc. of the above-mentioned human or animal as the administration subject. In addition, if necessary, the above-mentioned dosage can be determined without excessive experiments by conducting in vivo measurements using the pharmaceutical composition in advance.

[0094] The frequency of administration of the pharmaceutical composition is not particularly limited as long as the effect can be obtained, and can be appropriately set according to, for example, the above-mentioned dosage, administration route, symptoms, age or sex of humans or animals.

[0095] (Components other than nucleic acid constructs)

[0096] The pharmaceutical composition of one embodiment of the present invention may also be constituted to include at least the above-mentioned nucleic acid construct and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is not particularly limited, and preferably has the following properties: it does not substantially inhibit the function of the above-mentioned nucleic acid construct, and does not bring substantial adverse effects to humans or non-human animals as the subject of administration. In addition, the pharmaceutical composition is sometimes preferably a liquid carrier including water, and in one example, it may also be a liposome preparation.

[0097] Further examples of the components constituting the pharmaceutical composition include lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, buffers, colorants, antioxidants, and viscosity modifiers, but are not limited thereto.

[0098] [3. Methods of cell activation, etc.]

[0099] The cell activation method of one embodiment of the present invention comprises the following steps (localization step): the intracellular region of Trk is localized to the cell membrane via a membrane localization sequence in the cell. Typically, the localization step can be performed by expressing any one of the nucleic acid constructs described in the column [1. Nucleic acid construct] and the pharmaceutical composition described in the column [2. Pharmaceutical composition] in the nervous system cells. Here, the types of nervous system cells are not particularly limited, and include cells of the central nervous system, cells of the peripheral nervous system, progenitor cells of these cells, etc. Nervous system cells can be isolated or exist in the body of a human or non-human animal.

[0100] According to the above cell activation method, even without administering neurotrophic factors, the intracellular signaling pathway downstream of Trk can be activated. Furthermore, as a result of cell activation, a strong neuroprotective effect and axonal regeneration effect are exerted, which can be used for the treatment and / or prevention of nerve-related diseases.

[0101] One embodiment of the present invention also provides a nervous system cell into which the nucleic acid construct described in the column [1. Nucleic acid construct] is expressibly introduced, or a non-human animal having the nervous system cell.

[0102] [4. Conclusion]

[0103] As described above, the present invention includes the following aspects.

[0104] (1) A nucleic acid construct encoding a fusion polypeptide comprising the intracellular region and membrane localization sequence of Trk.

[0105] (2) The nucleic acid construct according to (1), wherein the intracellular region of Trk is selected from any one of the following: 1) a polypeptide represented by the amino acid sequence set forth in any one of SEQ ID NOs: 2, 6, 9, 12, or 14;

[0106] 2) A polypeptide represented by an amino acid sequence that is 90% or more identical to the amino acid sequence described in any one of SEQ ID NOs: 2, 6, 9, 12, or 14.

[0107] (3) The nucleic acid construct according to (1) or (2), wherein the membrane localization sequence induces lipid modification of amino acids.

[0108] (4) The nucleic acid construct according to (3), wherein the membrane-localizing sequence is a farnesylation sequence.

[0109] (5) The nucleic acid construct according to any one of (1) to (4), wherein the nucleic acid construct is a vector.

[0110] (6) The nucleic acid construct according to (5), wherein the nucleic acid construct is an adeno-associated virus vector.

[0111] (7) A pharmaceutical composition comprising the nucleic acid construct according to any one of (1) to (6) above.

[0112] (8) The pharmaceutical composition according to (7), wherein the pharmaceutical composition is a drug for a neurological disease.

[0113] (9) The pharmaceutical composition according to (8), wherein the pharmaceutical composition is used for neuroprotection or neuroregeneration.

[0114] (10) A nervous system cell into which the nucleic acid construct described in (1) above has been expressibly introduced.

[0115] (11) A method for activating a cell, comprising the step of localizing the intracellular region of Trk to the cell membrane via a membrane localization sequence in the cell.

[0116] (12) The method according to (11), wherein the above step is performed by expressing any one of the nucleic acid constructs selected from any one of (1) to (6) and the pharmaceutical composition selected from any one of (7) to (9) in nervous system cells.

[0117] (13) A fusion polypeptide comprising the intracellular region and the membrane localization sequence of Trk. The fusion polypeptide may also be encoded by the nucleic acid construct described in any one of (1) to (4) above.

[0118] The following examples are provided to explain the embodiments of the present invention in more detail. Of course, the present invention is not limited to the following examples, and the details may have various forms. In addition, the present invention is not limited to the above-mentioned embodiments, and various changes may be made within the scope shown in the claims, and the embodiments obtained by appropriately combining the technical means disclosed respectively are also included in the technical scope of the present invention. In addition, all the documents recorded in this specification are cited as references.

[0119] Example

[0120] [Example 1] Preparation of constitutively active TrkB (hereinafter referred to as CA-TrkB) vector

[0121] (method)

[0122] An AAV vector expressing the intracellular region of the TrkB molecule (referred to as AAV-CA-TrkB) was prepared as follows. The AAV vector into which the gene fragment encoding CA-TrkB was inserted was pAAV-CAG-shuttle-WPRE manufactured by Applied Viromics. A DNA fragment encoding the following protein was prepared as the gene fragment encoding CA-TrkB: a myc-tag was added to the N-terminus of the intracellular region of TrkB, and a farnesylation signal sequence was added to the C-terminus of the region (see also Figure 1 ). That is, CA-TrkB does not contain the extracellular region and transmembrane region of TrkB, but uses only the intracellular region of TrkB, and a myc-tag and a farnesylation signal sequence are added to the intracellular region.

[0123] In addition, the farnesylation signal sequence (the base sequence of which is shown in sequence number 1) is a signal sequence for localizing the protein to the membrane. In addition, the amino acid sequence of the intracellular region of TrkB is shown as sequence number 2, and the base sequence of the gene (DNA) encoding the intracellular region of TrkB is shown as sequence number 3. And, the base sequence of the gene fragment encoding CA-TrkB (the full length of the gene fragment inserted into the AAV vector) is shown as sequence number 4.

[0124] AAV-CA-TrkB uses the CAG promoter as an expression control system for the gene fragment encoding CA-TrkB, and further achieves high expression by fusing the WPRE sequence with a poly A sequence derived from human growth hormone (refer to Figure 2 ). The WPRE sequence and the poly A sequence derived from human growth hormone are placed downstream of the farnesylation signal sequence in the AAV vector. In addition, the CAG promoter, the WPRE sequence, and the poly A sequence derived from human growth hormone are inserted into the original AAV vector from the beginning.

[0125] Next, the obtained AAV-CA-TrkB was transfected into Cos7 cells, and its expression level or activity was evaluated.

[0126] (result)

[0127] Figure 3 and Figure 4 The results of transfection of AAV-CA-TrkB into Cos7 cells are shown in FIG. Figure 3The following results are shown: AAV-CA-TrkB was transfected into Cos7 cells, and the cells were lysed after culturing for 24 hours, and protein blotting was performed using an anti-myc antibody (c-Myc antibody (9E10) manufactured by Santa Cruz). It was confirmed that the expression level of AAV-CA-TrkB was significantly increased compared to the full-length TrkB vector subjected to the same experiment. In addition, the full-length TrkB vector refers to an adeno-associated virus vector in which a gene fragment encoding full-length TrkB is inserted instead of a gene fragment encoding CA-TrkB compared to the structure of AAV-CA-TrkB.

[0128] Figure 4 The following results are shown: AAV-CA-TrkB was transfected into Cos7 cells, and the cells were lysed after culturing for 24 hours, and protein blotting was performed using anti-pERK antibody (Phospho-p44 / 42 (Erk1 / 2) (Thr202 / Thr204) manufactured by Cell Signaling), anti-pAKT antibody (Phospho-Akt (Ser473) (587F11) manufactured by Cell Signaling), anti-ERK antibody (p44 / 42 MAPK (Erk1 / 2) (137F5) manufactured by Cell Signaling), and anti-AKT antibody (Akt antibody manufactured by Cell Signaling). It was found that in Cos7 cells expressing AAV-CA-TrkB, the production of pERK and pAKT increased, and both signals were activated. Among them, the total amount of ERK and AKT did not change. In addition, pERK and pAKT are both intracellular signals located downstream of TrkB. In addition, Figure 4 The control in the figure is the result of the same experiment using an empty vector (CA-TrkB sequence removed from the AAV-CA-TrkB vector), in which almost no production of pERK and pAKT was found.

[0129] [Example 2] Pathological analysis of optic nerve crush model

[0130] (method)

[0131] =Method for evaluating neuroprotective effect=

[0132] For mice (system name C57BL / 6: purchased from Charles River), 2 μL of AAV-DsRed (control group) or AAV-CA-TrkB was administered into the eye 14 days before optic nerve crush. In addition, AAV-DsRed refers to an adeno-associated virus vector in which a gene fragment encoding the fluorescent protein DsRed is inserted instead of a gene fragment encoding CA-TrkB, compared to the structure of AAV-CA-TrkB.

[0133] Next, the optic nerve of the above-mentioned mouse was exposed, and the optic nerve was physically crushed at a position of about 1 mm behind the eyeball (the details of the method are also referred to non-patent documents 6 and 7). Then, a retinal expansion specimen was prepared 14 days after the optic nerve was crushed, and the number of residual retinal ganglion cells was quantitatively counted. In addition, 10 days before the optic nerve was collected (the same as 10 days before the preparation of the retinal expansion specimen), the dye Fluoro-Gold (trade name made by Fluorocrome) was injected from the superior colliculus of the brain to retrogradely mark the retinal ganglion cells.

[0134] =Evaluation Method of Optic Nerve Regeneration=

[0135] For mice (system name C57BL / 6: purchased from Charles River Corporation), 2 μL of AAV-DsRed (control group) or AAV-CA-TrkB was administered into the eye 14 days before optic nerve crush.

[0136] The optic nerve was crushed in the same manner as in the above-mentioned "Evaluation Method for Neuroprotective Effect". Then, 14 days after the optic nerve was crushed, the mice were perfused and fixed, and the pathological specimens of the optic nerve were prepared, and the amount of axon regeneration from the crushed part was quantified. In addition, 3 days before the optic nerve was collected (which means the same as 3 days before the pathological specimens of the optic nerve were prepared), the fluorescent dye CTB was injected from the eyeball to make the regenerated axon fibers color.

[0137] (result)

[0138] =Neuroprotective Effect=

[0139] The number of residual retinal ganglion cells was quantified 14 days after optic nerve crush. 2 ) group (664±14 cells / mm 2 ) compared with the residual retinal ganglion cells ( Figure 5 ). p<0.01 (one-tailed Student's t test).

[0140] =Optic nerve regeneration=

[0141] The number of regenerated axons labeled with CTB was measured 14 days after optic nerve crush, and the results showed that the AAV-CA-TrkB group had significant improvements in both the number of regenerated axons and the elongation distance compared with the AAV-DsRed group ( Figure 6 ).in addition, Figure 6The X-axis of the graph shown is the distance from the optic nerve crush site (μm), and the Y-axis is the number of axons. p<0.05 (one-tailed Student's t test).

[0142] [Example 3] Protective effect of CA-TrkB on retinal ganglion cells (glaucoma model mice)

[0143] (method)

[0144] GLAST knockout mice (reference paper: Harada T, et al. The potential role of glutamate transporters in the pathogenesis of normal tension glaucoma. The Journal of Clinical Investigation 117: 1763-1770, 2007.) as glaucoma model mice disappear during the 3-5 weeks of age, causing a decline in visual function. For 10-day-old GLAST knockout mice, 1 μL of AAV-DsRed (control group) or AAV-CA-TrkB was administered into the eyeball (see also Example 2). Next, the retina of these knockout mice reaching 12 weeks of age was expanded and the number of remaining retinal ganglion cells was counted. Immunostaining using RBPMS antibody (anti-RBPMS antibody manufactured by Merck Millipore) was used in the detection of retinal ganglion cells. RBPMS is a marker for retinal ganglion cells.

[0145] (result)

[0146] like Figure 7 As shown, in the AAV-CA-TrkB-administered group, the number of residual retinal ganglion cells was significantly increased compared with the control group (P<0.05 (one-tailed Student's t test)).

[0147] [Example 4]

[0148] (method)

[0149] Next, the following variants expressing CA-TrkB were used (see also Figure 8 ) vector to study the activity of signal transduction.

[0150] "Cyto-TrkB" vector: a DNA fragment encoding a protein with a myc tag added to the N-terminus of the intracellular region of TrkB was inserted into the AAV vector of Example 1. Compared with AAV-CA-TrkB of Example 1, the only difference is that no farnesylation signal sequence is added to the C-terminus of the intracellular region of TrkB.

[0151] "KD-TrkB" vector: Compared with AAV-CA-TrkB in Example 1, the only difference is that the encoded protein has a single amino acid mutation in the intracellular region of TrkB that abolishes its kinase activity (the 134th amino acid mutates from lysine to asparagine).

[0152] "iSH-TrkB" vector: Compared with AAV-CA-TrkB in Example 1, the only difference is that the encoded protein also has the SH2 sequence of p85 inserted between the N-terminus of the intracellular region of TrkB and the myc-tag. In addition, the SH2 sequence is a sequence required for the formation of p110, a component factor of PI3K, and the complex. The base sequence of the gene fragment encoding iSH-TrkB (the full length of the gene fragment inserted into the AAV vector) is represented by SEQ ID NOs. 5 and Fig.10 .

[0153] Next, in the same manner as in Example 1, these vectors were transfected into Cos7 cells, and the cells were lysed after culturing for 24 hours, and the effects on the activation of ERK and AKT were examined by Western blotting.

[0154] (result)

[0155] like Figure 8 As shown, it is clear that when the "Cyto-TrkB" vector and the "KD-TrkB" vector were transfected, there was no increase in pERK and pAKT, and no activation of the two signals was found. On the other hand, when the "iSH-TrkB" vector was transfected, compared with the transfection of AAV-CA-TrkB (indicated as CA-TrkB in the figure), a significant increase in AKT activity was found. In addition, Figure 8 The control in is the same as that in Example 1.

[0156] [Example 5] Preparation of constitutively activated TrkA vector and constitutively activated TrkC vector

[0157] (method)

[0158] An AAV vector expressing the intracellular region of the TrkA molecule (referred to as AAV-CA-TrkA) was prepared according to the method of Example 1. That is, an AAV-CA-TrkA (also referred to as AAV-CA-TrkA) was prepared according to the method of Example 1, except that the intracellular region of TrkA was substituted for the intracellular region of TrkB, and a fragment having the same structure as the gene fragment encoding CA-TrkB (see Example 1) was used. Fig. 9 ).

[0159] The amino acid sequence of the intracellular region of TrkA is represented by SEQ ID NO: 6, and the base sequence of the gene (DNA) encoding the intracellular region of TrkA is represented by SEQ ID NO: 7. In addition, the base sequence of the gene fragment encoding CA-TrkA (the full length of the gene fragment inserted into the AAV vector) is represented by SEQ ID NO: 8 and 9. Fig.11 .

[0160] Similarly, an AAV vector expressing the intracellular region of the TrkC molecule (referred to as AAV-CA-TrkC) was prepared according to the method of Example 1. That is, an AAV-CA-TrkC (also referred to as AAV-CA-TrkC) was prepared according to the method of Example 1, except that the intracellular region of TrkC was substituted for the intracellular region of TrkB, and a fragment having the same structure as the gene fragment encoding CA-TrkB (see Example 1) was used. Fig. 9 ).

[0161] The amino acid sequence of the intracellular region of TrkC is represented by SEQ ID NO: 9, and the base sequence of the gene (DNA) encoding the intracellular region of TrkC is represented by SEQ ID NO: 10. In addition, the base sequence of the gene fragment encoding CA-TrkC (the full length of the gene fragment inserted into the AAV vector) is represented by SEQ ID NO: 11 and Fig.12 .

[0162] Next, in the same manner as in Example 1, these vectors were transfected into Cos7 cells, and after culturing for 24 hours, the cells were lysed, and the effects of TrkB homologs TrkA and TrkC on the activation of ERK and AKT were examined by Western blotting.

[0163] (result)

[0164] like Fig. 9 As shown, when AAV-CA-TrkA and AAV-CA-TrkC were transfected, pERK and pAKT were found to increase, and both signals were activated. Fig. 9 The control in is the same as that in Example 1.

[0165] [Example 6] Effect of CA-TrkB on the recovery of visual function (optic nerve crush model mice)

[0166] (method)

[0167] For mature mice, an AAV-CA-TrkB administration group was obtained according to the method of Example 2. The optic nerve was crushed 14 days after administration, and an electrode port was surgically installed in the superior colliculus of the brain 14 days and 28 days after crushing to measure the visual evoked potential (F-VEP: Flash-Visual Evoked Potential) caused by flash stimulation.

[0168] (result)

[0169] like Fig.13 As shown, the visual evoked potential response disappeared 2 weeks after the crush injury, but visual evoked potential was found 4 weeks after the crush injury, and it was confirmed that some visual function was restored.

[0170] Industrial Applicability

[0171] The present invention can be used for medical purposes targeting, for example, nerve-related diseases and the like. Sequence Listing <110> Tokyo Metropolitan Institute of Medicine <120> Nucleic acid constructs encoding Trk fragments and their use <130> AJ20648PT2102 <150> JP 2019-038503 <151> 2019-03-04 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 63 <212> DNA <213> Artificial sequence <220> <223> signal sequence <400> 1 gccatgagca aagatggtaa aaagaagaaa aagaagtcaa agacaaagtg tgtaattatg 60 taa 63 <210> 2 <211> 384 <212> PRT <213> Homo sapiens <400> 2 Lys Leu Ala Arg His Ser Lys Phe Gly Met Lys Asp Phe Ser Trp Phe 1 5 10 15 Gly Phe Gly Lys Val Lys Ser Arg Gln Gly Val Gly Pro Ala Ser Val 20 25 30 Ile Ser Asn Asp Asp Asp Ser Ala Ser Pro Leu His His Ile Ser Asn 35 40 45 Gly Ser Asn Thr Pro Ser Ser Ser Glu Gly Gly Pro Asp Ala Val Ile 50 55 60 Ile Gly Met Thr Lys Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Gly 65 70 75 80 Ile Thr Asn Ser Gln Leu Lys Pro Asp Thr Phe Val Gln His Ile Lys 85 90 95 Arg His Asn Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly 100 105 110 Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Cys Pro Glu Gln Asp Lys 115 120 125 Ile Leu Val Ala Val Lys Thr Leu Lys Asp Ala Ser Asp Asn Ala Arg 130 135 140 Lys Asp Phe His Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu 145 150 155 160 His Ile Val Lys Phe Tyr Gly Val Cys Val Glu Gly Asp Pro Leu Ile 165 170 175 Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg 180 185 190 Ala His Gly Pro Asp Ala Val Leu Met Ala Glu Gly Asn Pro Pro Thr 195 200 205 Glu Leu Thr Gln Ser Gln Met Leu His Ile Ala Gln Gln Ile Ala Ala 210 215 220 Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg Asp Leu Ala 225 230 235 240 Thr Arg Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys Ile Gly Asp 245 250 255 Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly 260 265 270 Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Met 275 280 285 Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Leu Gly Val Val 290 295 300 Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Tyr Gln Leu Ser 305 310 315 320 Asn Asn Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val Leu Gln Arg 325 330 335 Pro Arg Thr Cys Pro Gln Glu Val Tyr Glu Leu Met Leu Gly Cys Trp 340 345 350 Gln Arg Glu Pro His Met Arg Lys Asn Ile Lys Gly Ile His Thr Leu 355 360 365 Leu Gln Asn Leu Ala Lys Ala Ser Pro Val Tyr Leu Asp Ile Leu Gly 370 375 380 <210> 3 <211> 1152 <212> DNA <213> Homo sapiens <400> 3 aagttggcaa gacactccaa gtttggcatg aaagatttct catggtttgg atttgggaaa 60 gtaaaatcaa gacaaggtgt tggcccagcc tccgttatca gcaatgatga tgactctgcc 120 agcccactcc atcacatctc caatgggagt aacactccat cttcttcgga aggtggccca 180 gatgctgtca ttattggaat gaccaagatc cctgtcattg aaaatcccca gtactttggc 240 atcaccaaca gtcagctcaa gccagacaca tttgttcagc acatcaagcg acataacatt 300 gttctgaaaa gggagctagg cgaaggagcc tttggaaaag tgttcctagc tgaatgctat 360 aacctctgtc ctgagcagga caagatcttg gtggcagtga agaccctgaa ggatgccagt 420 gacaatgcac gcaaggactt ccaccgtgag gccgagctcc tgaccaacct ccagcatgag 480 cacatcgtca agttctatgg cgtctgcgtg gagggcgacc ccctcatcat ggtctttgag 540 tacatgaagc atggggacct caacaagttc ctcagggcac acggccctga tgccgtgctg 600 atggctgagg gcaacccgcc cacggaactg acgcagtcgc agatgctgca tatagcccag 660 cagatcgccg cgggcatggt ctacctggcg tcccagcact tcgtgcaccg cgatttggcc 720 accaggaact gcctggtcgg ggagaacttg ctggtgaaaa tcggggactt tgggatgtcc 780 cgggacgtgt acagcactga ctactacagg gtcggtggcc acacaatgct gcccattcgc 840 tggatgcctc cagagagcat catgtacagg aaattcacga cggaaagcga cgtctggagc 900 ctgggggtcg tgttgtggga gattttcacc tatggcaaac agccctggta ccagctgtca 960 aacaatgagg tgatagagtg tatcactcag ggccgagtcc tgcagcgacc ccgcacgtgc 1020 ccccaggagg tgtatgagct gatgctgggg tgctggcagc gagagcccca catgaggaag 1080 aacatcaagg gcatccatac cctccttcag aacttggcca aggcatctcc ggtctacctg 1140 gacattctag gc 1152 <210> 4 <211> 1263 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding CA-TrkB <400> 4 atggcatcaa tgcagaagct gatctcagag gaggacctga agttggcaag acactccaag 60 tttggcatga aagatttctc atggtttgga tttgggaaag taaaatcaag acaaggtgtt 120 ggcccagcct ccgttatcag caatgatgat gactctgcca gcccactcca tcacatctcc 180 aatgggagta acactccatc ttcttcggaa ggtggcccag atgctgtcat tattggaatg 240 accaagatcc ctgtcattga aaatccccag tactttggca tcaccaacag tcagctcaag 300 ccagacacat ttgttcagca catcaagcga cataacattg ttctgaaaag ggagctaggc 360 gaaggagcct ttggaaaagt gttcctagct gaatgctata acctctgtcc tgagcaggac 420 aagatcttgg tggcagtgaa gaccctgaag gatgccagtg acaatgcacg caaggacttc 480 caccgtgagg ccgagctcct gaccaacctc cagcatgagc acatcgtcaa gttctatggc 540 gtctgcgtgg agggcgaccc cctcatcatg gtctttgagt acatgaagca tggggacctc 600 aacaagttcc tcagggcaca cggccctgat gccgtgctga tggctgaggg caacccgccc 660 acggaactga cgcagtcgca gatgctgcat atagcccagc agatcgccgc gggcatggtc 720. tacctggcgt cccagcactt cgtgcaccgc gattttggcca ccaggaactg cctggtcggg 780 gagaacttgc tggtgaaaat cggggacttt gggatgtccc gggacgtgta cagcactgac 840 tactacaggg tcggtggcca cacaatgctg cccattcgct ggatgcctcc agagagcatc atgtacagga aattcacgac ggaaagcgac gtctggagcc tgggggtcgt gttgtgggag attttcacct atggcaaaca gccctggtac cagctgtcaa acaatgaggt gataggtgt atcactcagg gccgagtcct gcagcgaccc cgcacgtgcc cccaggaggt gtatgagctg 1080 atgctggggt gctggcagcg agagccccac atgaggaga acatcaaggg catccatacc ctccttcaga acttggccaa ggcatctccg gtctacctgg acattctagg cgcggccgcg gccatgagca aagatggtaa aagaagaaa aagaagtcaa agacaaagtg tgtaattatg taa 1263 <210> 5 <211> 1851 <212> DNA <213> Artificial sequence <220> <223> Nucleic acid sequence encoding iSH-TrkB <400> 5 atggcatcaa tgcagaagct gatctcagag gaggacctgg tgtccaaata ccaacaggat 60 caagttgtca aagaagataa tattgaagct gtagggaaaa aattacatga atataacact 120 caatttcaag aaaaaagtcg ggaatatgat agattatatg aagactatac ccgaacttcc 180 caggaaatcc aaatgaaaag aacagctatt gaagcattta atgaaaccat aaaaatattt 240 gaagaacagt gccaaaccca ggagcggtac agcaaagaat acatagaaaa gttcaaacgt 300 gaaggcaacg agacggaaat ccaaaggatt atgcataatt atgaaaagtt aaagtctcga 360 atcagtgaaa ttgtcgatag cagaaggaga ttggaagagg acttgaagaa gcaggcggct 420 gagtatcgtg agattgacaa acgcatgaac agcattaaac cagacctcat tcagctaaga 480 aagacgagag accaatactt gatgtggttg actcagaaag gtgttcggca gaagaaattg 540 aacgagtggc tgggcaatga aaacactgaa gaccaatatt cgctggtaga agatgatgag 600 gatctgcccc accacgatga gaagactaag ttggcaagac actccaagtt tggcatgaaa 660 gatttctcat ggtttggatt tgggaaagta aaatcaagac aaggtgttgg cccagcctcc 720 gttatcagca atgatgatga ctctgccagc ccactccatc acatctccaa tgggagtaac 780 actccatctt cttcggaagg tggcccagat gctgtcatta ttggaatgac caagatccct 840 gtcattgaaa atccccagta ctttggcatc accaacagtc agctcaagcc agacacattt 900 gttcagcaca tcaagcgaca taacattgtt ctgaaaaggg agctaggcga aggagccttt 960 ggaaaagtgt tcctagctga atgctataac ctctgtcctg agcaggacaa gatcttggtg 1020 gcagtgaaga ccctgaagga tgccagtgac aatgcacgca aggacttcca ccgtgaggcc 1080 gagctcctga ccaacctcca gcatgagcac atcgtcaagt tctatggcgt ctgcgtggag 1140 ggcgaccccc tcatcatggt ctttgagtac atgaagcatg gggacctcaa caagttcctc 1200 agggcacacg gccctgatgc cgtgctgatg gctgagggca acccgcccac ggaactgacg 1260 cagtcgcaga tgctgcatat agcccagcag atcgccgcgg gcatggtcta cctggcgtcc 1320 cagcacttcg tgcaccgcga tttggccacc aggaactgcc tggtcgggga gaacttgctg 1380 gtgaaaatcg gggactttgg gatgtcccgg gacgtgtaca gcactgacta ctacagggtc 1440 ggtggccaca caatgctgcc cattcgctgg atgcctccag agagcatcat gtacaggaaa 1500 ttcacgacgg aaagcgacgt ctggagcctg ggggtcgtgt tgtgggagat tttcacctat 1560 ggcaaacagc cctggtacca gctgtcaaac aatgaggtga tagagtgtat cactcagggc 1620 cgagtcctgc agcgaccccg cacgtgcccc caggaggtgt atgagctgat gctggggtgc 1680 tggcagcgag agccccacat gaggaagaac atcaagggca tccataccct ccttcagaac 1740 ttggccaagg catctccggt ctacctggac attctaggcg cggccgcggc catgagcaaa 1800 gatggtaaaa agaagaaaaa gaagtcaaag acaaagtgtg taattatgta a 1851 <210> 6 <211> 356 <212> PRT <213> Mouse <400> 6 Lys Cys Gly Gln Arg Ser Lys Phe Gly Ile Asn Arg Pro Ala Val Leu 1 5 10 15 Ala Pro Glu Asp Gly Leu Ala Met Ser Leu His Phe Met Thr Leu Gly 20 25 30 Gly Ser Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser Gly Leu Gln Gly 35 40 45 His Ile Met Glu Asn Pro Gln Tyr Phe Ser Asp Thr Cys Val His His 50 55 60 Ile Lys Arg Gln Asp Ile Ile Leu Lys Trp Glu Leu Gly Glu Gly Ala 65 70 75 80 Phe Gly Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Leu Asn Asp Gln 85 90 95 Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala Ser Glu Asn 100 105 110 Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr Met Leu Gln 115 120 125 His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr Glu Gly Gly Pro 130 135 140 Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe 145 150 155 160 Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp 165 170 175 Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser 180 185 190 Gln Val Ala Ala Gly Met Val Tyr Leu Ala Ser Leu His Phe Val His 195 200 205 Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln Gly Leu Val Val 210 215 220 Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr 225 230 235 240 Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg Trp Met Pro Pro 245 250 255 Glu Ser Ile Leu Tyr Arg Lys Phe Ser Thr Glu Ser Asp Val Trp Ser 260 265 270 Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp 275 280 285 Tyr Gln Leu Ser Asn Thr Glu Ala Ile Glu Cys Ile Thr Gln Gly Arg 290 295 300 Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Asp Val Tyr Ala Ile Met 305 310 315 320 Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Ser Met Lys Asp 325 330 335 Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro Pro Ser Tyr Leu 340 345 350 Asp Val Leu Gly 355 <210> 7 <211> 1068 <212> DNA <213> Mouse <400> 7 aaatgtggac agaggagcaa atttgggatc aaccgccctg ctgtattagc tccagaggat 60 gggctggcca tgtccctaca cttcatgaca ctgggtggca gttctctttc ccctactgag 120 ggcaaaggct ccggactcca gggccacatc atggagaacc cacagtactt cagtgatacc 180 tgtgtccatc acatcaagcg ccaggacatc attctcaagt gggagctagg ggagggagcc 240 tttggaaagg tctttctcgc tgagtgctac aaccttctga atgatcagga caagatgctc 300 gtggctgtca aggcactgaa ggaggcatcc gagaatgctc ggcaggactt tcagcgtgag 360 gccgagctgc tcaccatgct acagcaccag cacatcgtgc gcttctttgg agtctgcacc 420 gagggtggcc cgttgctcat ggtctttgag tacatgcgcc acggggacct caaccgtttc 480 ctccggtccc acggacctga tgcaaagctg ctggctggtg gtgaggatgt ggctcctggt 540 cctctgggcc ttgggcagct tctggctgtg gctagccagg tggctgctgg tatggtatat 600 ctagccagcc tgcactttgt gcaccgggat ctggccacac gcaactgtct ggtgggtcag 660 ggactagtgg tgaagattgg agactttggc atgagcagag acatctatag cacagactat 720 taccgagtgg gaggtcggac catgctgccc atccgctgga tgccacccga gagcatcctc 780 taccgcaagt tcagcaccga gagcgatgtg tggagctttg gggtggtgct ctgggagatc 840 ttcacctatg gaaagcagcc ctggtaccag ctctccaaca ctgaggcgat cgagtgtatc 900 acgcagggcc gggagctgga gcggccgcgc gcctgccctc ctgatgtcta cgccatcatg 960 cgaggctgct ggcagcgaga accgcagcaa cgcctcagca tgaaggatgt gcacgcgcgt 1020 ctgcaggccc tggcacaggc gccacccagt tacctggacg ttctgggc 1068 <210> 8 <211> 1182 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding CA-TrekA <400> 8 atggcatcaa tgcagaagct gatctcagag gaggacctgg tcgacaaatg tggacagagg 60 agcaaatttg ggatcaaccg ccctgctgta ttagctccag aggatgggct ggccatgtcc ctacacttca tgacactggg tggcagttct ctttccccta ctgagggcaa aggctccgga ctccagggcc acatcatgga gaacccacag tacttcagtg atacctgtgt ccatcacatc aagcgccagg acatcattct caagtggggag ctaggggagg gagccttttgg aaaggtcttt 300 ctcgctgagt gctacaacct tctgaatgat caggacaaga tgctcgtggc tgtcaaggca ctgaaggagg catccgagaa tgctcggcag gactttcagc gtgaggccga gctgctcacc atgctacagc accagcacat cgtgcgcttc tttggagtct gcaccgaggg tggcccgttg 480 ctcatggtct ttgagtacat gcgccacggg gacctcaacc gtttcctccg gtcccacgga 540 cctgatgcaa agctgctggc tggtggtgag gatgtggctc ctggtcctct gggccttggg 600 cagcttctgg ctgtggctag ccaggtggct gctggtatgg fatherctagc cagcctgcac 660 tttgtgcacc gggatctggc cacacgcaac tgtctggtgg gtcagggact agtggtgaag 720 attggagact ttggcatgag cagagacatc tatagcacag actattaccg agtgggaggt cggaccatgc tgcccatccg ctggatgcca cccgagagca tcctctaccg caagttcagc 840 accgagagcg atgtgtggag ctttggggtg gtgctctggg agatcttcac ctatggaaag 900 cagccctggt accagctctc caacactgag gcgatcgagt gtatcacgca gggccgggag 960 ctggagcggc cgcgcgcctg ccctcctgat gtctacgcca tcatgcgagg ctgctggcag 1020 cgagaaccgc agcaacgcct cagcatgaag gatgtgcacg cgcgtctgca ggccctggca 1080 caggcgccac ccagttacct ggacgttctg ggctctagag ccatgagcaa agatggtaaa 1140 aagaagaaaa agaagtcaaa gacaaagtgt gtaattatgt aa 1182 <210> 9 <211> 371 <212> PRT <213> Mouse <400> 9 Lys Tyr Gly Arg Arg Ser Lys Phe Gly Met Lys Gly Pro Val Ala Val 1 5 10 15 Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro Leu His His Ile Asn His 20 25 30 Gly Ile Thr Thr Pro Ser Ser Leu Asp Ala Gly Pro Asp Thr Val Val 35 40 45 Ile Gly Met Thr Arg Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Arg 50 55 60 Gln Gly His Asn Cys His Lys Pro Asp Thr Tyr Val Gln His Ile Lys 65 70 75 80 Arg Arg Asp Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly 85 90 95 Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Ser Pro Thr Lys Asp Lys 100 105 110 Met Leu Val Ala Val Lys Ala Leu Lys Asp Pro Thr Leu Ala Ala Arg 115 120 125 Lys Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu 130 135 140 His Ile Val Lys Phe Tyr Gly Val Cys Gly Asp Gly Asp Pro Leu Ile 145 150 155 160 Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg 165 170 175 Ala His Gly Pro Asp Ala Met Ile Leu Val Asp Gly Gln Pro Arg Gln 180 185 190 Ala Lys Gly Glu Leu Gly Leu Ser Gln Met Leu His Ile Ala Ser Gln 195 200 205 Ile Ala Ser Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg 210 215 220 Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Ala Asn Leu Leu Val Lys 225 230 235 240 Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr 245 250 255 Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu 260 265 270 Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Phe 275 280 285 Gly Val Ile Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Phe 290 295 300 Gln Leu Ser Asn Thr Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val 305 310 315 320 Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr Asp Val Met Leu 325 330 335 Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Asn Ile Lys Glu Ile 340 345 350 Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr Pro Ile Tyr Leu Asp 355 360 365 Ile Leu Gly 370 <210> 10 <211> 1113 <212> DNA <213> Mouse <400> 10 aagtatggtc gccggtccaa atttggaatg aagggtcctg tggctgttat cagtggagag 60 gaggactcag ccagcccact gcatcacatc aaccatggca tcactacacc atcatcgttg 120 gatgctgggc ctgacacagt ggtcattggc atgacccgca tcccagtcat tgagaacccc 180 cagtacttcc gtcagggtca caattgccac aagccagaca catatgttca gcacatcaag 240 aggagagaca tcgtgttgaa gagagaattg ggtgagggag cctttgggaa ggtcttcctg 300 gctgagtgct acaatctaag ccccaccaaa gacaagatgc tagtggcagt gaaggccctg 360 aaggatccca ccttggctgc caggaaggat ttccagaggg aggctgagct gctcacgaac 420 ctgcagcatg agcatattgt caagttctat ggggtgtgtg gtgatggtga cccactcatc 480 atggtctttg aatacatgaa gcatggagac cttaacaagt tcctcagggc ccatgggcca 540 gatgccatga tcctcgtgga tggacagcca cgtcaggcca agggggagct agggctctct 600 cagatgctcc acatcgccag tcagatagcc tcgggcatgg tgtacctggc ttcccagcac 660 tttgtacacc gggacctggc cacgaggaac tgcctggttg gagccaatct actagtgaag 720 attggagatt ttggcatgtc cagggacgtc tacagtactg attactacag ggtgggagga 780 cacaccatgc tccccatccg ctggatgccc cctgaaagca taatgtaccg gaagttcacc 840 acagagagtg atgtctggag cttcggggtt attctttggg agatctttac ctatgggaag 900 caaccatggt tccagctttc caacacggag gtcattgaat gcatcaccca aggccgtgtc 960 ttggagagac ccagagtctg ccctaaagaa gtgtatgatg tcatgctggg gtgctggcag 1020 agggaaccac agcagcggct gaatattaag gagatctaca aaatcctcca tgctttgggg 1080 aaggccaccc cgatctacct ggacattctt ggc 1113 <210> 11 <211> 1227 <212> DNA <213> Artificial Sequence <220> <223> Nucleic acid sequence encoding CA-TrkC <400> 11 atggcatcaa tgcagaagct gatctcagag gaggacctgg tcgacaagta tggtcgccgg 60 tccaaatttg gaatgaaggg tcctgtggct gttatcagtg gagaggagga ctcagccagc 120 ccactgcatc acatcaacca tggcatcact acaccatcat cgttggatgc tgggcctgac 180 acagtggtca ttggcatgac ccgcatccca gtcattgaga acccccagta cttccgtcag 240 ggtcacaatt gccacaagcc agacacatat gttcagcaca tcaagaggag agacatcgtg 300 ttgaagagag aattgggtga gggagccttt gggaaggtct tcctggctga gtgctacaat 360 ctaagcccca ccaaagacaa gatgctagtg gcagtgaagg ccctgaagga tcccaccttg 420 gctgccagga aggatttcca gagggaggct gagctgctca cgaacctgca gcatgagcat 480 attgtcaagt tctatggggt gtgtggtgat ggtgacccac tcatcatggt ctttgaatac 540 atgaagcatg gagaccttaa caagttcctc agggcccatg ggccagatgc catgatcctc 600 gtggatggac agccacgtca ggccaagggg gagctagggc tctctcagat gctccacatc 660 gccagtcaga tagcctcggg catggtgtac ctggcttccc agcactttgt acaccgggac 720 ctggccacga ggaactgcct ggttggagcc aatctactag tgaagattgg agattttggc 780 atgtccaggg acgtctacag tactgattac tacagggtgg gaggacacac catgctcccc 840 atccgctgga tgccccctga aagcataatg taccggaagt tcaccacaga gagtgatgtc 900 tggagcttcg gggttattct ttgggagatc tttacctatg ggaagcaacc atggttccag 960 ctttccaaca cggaggtcat tgaatgcatc acccaaggcc gtgtcttgga gagacccaga 1020 gtctgcccta aagaagtgta tgatgtcatg ctggggtgct ggcagaggga accacagcag 1080 cggctgaata ttaaggagat ctacaaaatc ctccatgctt tggggaaggc caccccgatc 1140 tacctggaca ttcttggctc tagagccatg agcaaagatg gtaaaaagaa gaaaaagaag 1200 tcaaagacaa agtgtgtaat tatgtaa 1227 <210> 12 <211> 357 <212> PRT <213> Homo sapiens <400> 12 Lys Cys Gly Arg Arg Asn Lys Phe Gly Ile Asn Arg Pro Ala Val Leu 1 5 10 15 Ala Pro Glu Asp Gly Leu Ala Met Ser Leu His Phe Met Thr Leu Gly 20 25 30 Gly Ser Ser Leu Ser Pro Thr Glu Gly Lys Gly Ser Gly Leu Gln Gly 35 40 45 His Ile Ile Glu Asn Pro Gln Tyr Phe Ser Asp Ala Cys Val His His 50 55 60 Ile Lys Arg Arg Asp Ile Val Leu Lys Trp Glu Leu Gly Glu Gly Ala 65 70 75 80 Phe Gly Lys Val Phe Leu Ala Glu Cys His Asn Leu Leu Pro Glu Gln 85 90 95 Asp Lys Met Leu Val Ala Val Lys Ala Leu Lys Glu Ala Ser Glu Ser 100 105 110 Ala Arg Gln Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr Met Leu Gln 115 120 125 His Gln His Ile Val Arg Phe Phe Gly Val Cys Thr Glu Gly Arg Pro 130 135 140 Leu Leu Met Val Phe Glu Tyr Met Arg His Gly Asp Leu Asn Arg Phe 145 150 155 160 Leu Arg Ser His Gly Pro Asp Ala Lys Leu Leu Ala Gly Gly Glu Asp 165 170 175 Val Ala Pro Gly Pro Leu Gly Leu Gly Gln Leu Leu Ala Val Ala Ser 180 185 190 Gln Val Ala Ala Gly Met Val Tyr Leu Ala Gly Leu His Phe Val His 195 200 205 Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Gln Gly Leu Val Val 210 215 220 Lys Ile Gly Asp Phe Gly Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr 225 230 235 240 Tyr Arg Val Gly Gly Arg Thr Met Leu Pro Ile Arg Trp Met Pro Pro 245 250 255 Glu Ser Ile Leu Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser 260 265 270 Phe Gly Val Val Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp 275 280 285 Tyr Gln Leu Ser Asn Thr Glu Ala Ile Asp Cys Ile Thr Gln Gly Arg 290 295 300 Glu Leu Glu Arg Pro Arg Ala Cys Pro Pro Glu Val Tyr Ala Ile Met 305 310 315 320 Arg Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg His Ser Ile Lys Asp 325 330 335 Val His Ala Arg Leu Gln Ala Leu Ala Gln Ala Pro Pro Val Tyr Leu 340 345 350 Asp Val Leu Gly Glx 355 <210> 13 <211> 1071 <212> DNA <213> Homo sapiens <400> 13 aaatgtggac ggagaaacaa gtttgggatc aaccgcccgg ctgtgctggc tccagaggat 60 gggctggcca tgtccctgca tttcatgaca ttgggtggca gctccctgtc ccccaccgag 120 ggcaaaggct ctgggctcca aggccacatc atcgagaacc cacaatactt cagtgatgcc 180 tgtgttcacc acatcaagcg ccgggacatc gtgctcaagt gggagctggg ggagggcgcc 240 tttgggaagg tcttccttgc tgagtgccac aacctcctgc ctgagcagga caagatgctg 300 gtggctgtca aggcactgaa ggaggcgtcc gagagtgctc ggcaggactt ccagcgtgag 360 gctgagctgc tcaccatgct gcagcaccag cacatcgtgc gcttcttcgg cgtctgcacc 420 gagggccgcc ccctgctcat ggtctttgag tatatgcggc acggggacct caaccgcttc 480 ctccgatccc atggacctga tgccaagctg ctggctggtg gggaggatgt ggctccaggc 540 cccctgggtc tggggcagct gctggccgtg gctagccagg tcgctgcggg gatggtgtac 600 ctggcgggtc tgcattttgt gcaccgggac ctggccacac gcaactgtct agtgggccag 660 ggactggtgg tcaagattgg tgattttggc atgagcaggg atatctacag caccgactat 720 taccgtgtgg gaggccgcac catgctgccc attcgctgga tgccgcccga gagcatcctg 780 taccgtaagt tcaccaccga gagcgacgtg tggagcttcg gcgtggtgct ctgggagatc 840 ttcacctacg gcaagcagcc ctggtaccag ctctccaaca cggaggcaat cgactgcatc 900 acgcagggac gtgagttgga gcggccacgt gcctgcccac cagaggtcta cgccatcatg 960 cggggctgct ggcagcggga gccccagcaa cgccacagca tcaaggatgt gcacgcccgg 1020 ctgcaagccc tggcccaggc acctcctgtc tacctggatg tcctgggcta g 1071 <210> 14 <211> 372 <212> PRT <213> Homo sapiens <400> 14 Lys Tyr Gly Arg Arg Ser Lys Phe Gly Met Lys Gly Pro Val Ala Val 1 5 10 15 Ile Ser Gly Glu Glu Asp Ser Ala Ser Pro Leu His His Ile Asn His 20 25 30 Gly Ile Thr Thr Pro Ser Ser Leu Asp Ala Gly Pro Asp Thr Val Val 35 40 45 Ile Gly Met Thr Arg Ile Pro Val Ile Glu Asn Pro Gln Tyr Phe Arg 50 55 60 Gln Gly His Asn Cys His Lys Pro Asp Thr Tyr Val Gln His Ile Lys 65 70 75 80 Arg Arg Asp Ile Val Leu Lys Arg Glu Leu Gly Glu Gly Ala Phe Gly 85 90 95 Lys Val Phe Leu Ala Glu Cys Tyr Asn Leu Ser Pro Thr Lys Asp Lys 100 105 110 Met Leu Val Ala Val Lys Ala Leu Lys Asp Pro Thr Leu Ala Ala Arg 115 120 125 Lys Asp Phe Gln Arg Glu Ala Glu Leu Leu Thr Asn Leu Gln His Glu 130 135 140 His Ile Val Lys Phe Tyr Gly Val Cys Gly Asp Gly Asp Pro Leu Ile 145 150 155 160 Met Val Phe Glu Tyr Met Lys His Gly Asp Leu Asn Lys Phe Leu Arg 165 170 175 Ala His Gly Pro Asp Ala Met Ile Leu Val Asp Gly Gln Pro Arg Gln 180 185 190 Ala Lys Gly Glu Leu Gly Leu Ser Gln Met Leu His Ile Ala Ser Gln 195 200 205 Ile Ala Ser Gly Met Val Tyr Leu Ala Ser Gln His Phe Val His Arg 210 215 220 Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Ala Asn Leu Leu Val Lys 225 230 235 240 Ile Gly Asp Phe Gly Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr 245 250 255 Arg Val Gly Gly His Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu 260 265 270 Ser Ile Met Tyr Arg Lys Phe Thr Thr Glu Ser Asp Val Trp Ser Phe 275 280 285 Gly Val Ile Leu Trp Glu Ile Phe Thr Tyr Gly Lys Gln Pro Trp Phe 290 295 300 Gln Leu Ser Asn Thr Glu Val Ile Glu Cys Ile Thr Gln Gly Arg Val 305 310 315 320 Leu Glu Arg Pro Arg Val Cys Pro Lys Glu Val Tyr Asp Val Met Leu 325 330 335 Gly Cys Trp Gln Arg Glu Pro Gln Gln Arg Leu Asn Ile Lys Glu Ile 340 345 350 Tyr Lys Ile Leu His Ala Leu Gly Lys Ala Thr Pro Ile Tyr Leu Asp 355 360 365 Ile Leu Gly Glx 370 <210> 15 <211> 1116 <212> DNA <213> Homo sapiens <400> 15 aaatatggtc gacggtccaa atttggaatg aagggtcccg tggctgtcat cagtggtgag 60 gaggactcag ccagcccact gcaccacatc aaccacggca tcaccacgcc ctcgtcactg 120 gatgccgggc ccgacactgt ggtcattggc atgactcgca tccctgtcat tgagaacccc 180 cagtacttcc gtcagggaca caactgccac aagccggaca cgtatgtgca gcacattaag 240 aggagagaca tcgtgctgaa gcgagaactg ggtgagggag cctttggaaa ggtcttcctg 300 gccgagtgct acaacctcag cccgaccaag gacaagatgc ttgtggctgt gaaggccctg 360 aaggatccca ccctggctgc ccggaaggat ttccagaggg aggccgagct gctcaccaac 420 ctgcagcatg agcacattgt caagttctat ggagtgtgcg gcgatgggga ccccctcatc 480 atggtctttg aatacatgaa gcatggagac ctgaataagt tcctcagggc ccatgggcca 540 gatgcaatga tccttgtgga tggacagcca cgccaggcca agggtgagct ggggctctcc 600 caaatgctcc acattgccag tcagatcgcc tcgggtatgg tgtacctggc ctcccagcac 660 tttgtgcacc gagacctggc caccaggaac tgcctggttg gagcgaatct gctagtgaag 720 attggggact tcggcatgtc cagagatgtc tacagcacgg attattacag ggtgggagga 780 cacaccatgc tccccattcg ctggatgcct cctgaaagca tcatgtaccg gaagttcact 840 acagagagtg atgtatggag cttcggggtg atcctctggg agatcttcac ctatggaaag 900 cagccatggt tccaactctc aaacacggag gtcattgagt gcattaccca aggtcgtgtt 960 ttggagcggc cccgagtctg ccccaaagag gtgtacgatg tcatgctggg gtgctggcag 1020 agggaaccac agcagcggtt gaacatcaag gagatctaca aaatcctcca tgctttgggg 1080 aaggccaccc caatctacct ggacattctt ggctag 1116

Claims

1. A nucleic acid construct encoding a fusion polypeptide comprising an intracellular region of Trk and a membrane localization sequence, wherein the number of amino acids in the fusion polypeptide is in the range of 300 to 650, the membrane localization sequence induces lipid modification of amino acids, and the intracellular region of Trk is selected from a polypeptide represented by an amino acid sequence recorded in any one of SEQ ID NOs: 2, 6 and 9.

2. The nucleic acid construct according to claim 1, wherein The membrane localization sequence is a farnesylation sequence.

3. The nucleic acid construct according to claim 1 or 2, wherein The nucleic acid construct is a vector.

4. The nucleic acid construct according to claim 3, wherein The nucleic acid construct is an adeno-associated virus vector. 5 . A pharmaceutical composition comprising the nucleic acid construct according to claim 1 .

6. The pharmaceutical composition according to claim 5, wherein The pharmaceutical composition is a drug for protecting or regenerating visual nerves.

7. A nervous system cell into which the nucleic acid construct according to any one of claims 1 to 4 is expressibly introduced.

8. The nervous system cell according to claim 7, wherein The intracellular region of Trk is localized to the cell membrane through the membrane localization sequence in the cell, thereby activating cell activity.

9. Use of any one of the nucleic acid construct according to any one of claims 1 to 4, the pharmaceutical composition according to any one of claims 5 to 6, and the nervous system cell according to claim 7 or 8 in the preparation of a medicament for treating visual nerve protection or visual nerve regeneration.

10. A fusion polypeptide comprising an intracellular region of Trk and a membrane localization sequence, wherein the number of amino acids in the fusion polypeptide is in the range of 300 to 650, the membrane localization sequence induces lipid modification of amino acids, and the intracellular region of Trk is selected from a polypeptide represented by an amino acid sequence recorded in any one of sequence numbers 2, 6 and 9.

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