C-terminal CDNF fragments, pharmaceutical compositions containing them, and their uses

Abstract

This invention provides C-terminal CDNF fragments, pharmaceutical compositions, and their uses. [Solution] The present invention provides a C-terminal CDNF fragment sequence or a sequence having at least 80% homology or sequence identity with said sequence. The C-terminal CDNF fragment protects ER-stressed neurons, motor neurons, and dopaminergic neurons and can permeate nerve cell membranes and the blood-brain barrier. The present invention further provides said fragment and pharmaceutical compositions containing said fragment for use in the treatment of degenerative diseases and disorders, including central nervous system diseases, diabetes, and retinal disorders. The present invention also provides a C-terminal MANF fragment sequence or a sequence having at least 80% homology or sequence identity with said sequence, and pharmaceutical compositions containing the MANF fragment for use in the treatment of degenerative diseases and disorders, including central nervous system diseases, diabetes, and retinal disorders.

Description

[Technical Field]

[0001] This invention relates to the fields of bioactive protein fragments and cell membrane-permeable peptides, as well as neuronutrition. Regarding the field of factors and proteins located in the endoplasmic reticulum (ER), more specifically, the central nervous system This relates to the treatment of degenerative diseases or disorders such as necrotic diseases, diabetes, and retinal disorders. [Background technology]

[0002] Neurotrophic factors include brain dopamine neurotrophic factor (CDNF) and midbrain astrolate cell-derived neurons. Nutritional factors (MANF) (Non-Patent Literature 1, Non-Patent Literature 2) are currently being studied in Parkinson's disease (PD). It is the most efficient protein for treating the 6-OHDA model rats. Both factors are toxic. When applied before the disease, 6-OHDA-induced behavioral symptoms and histological changes in Parkinson's disease It strongly prevents symptoms (Non-Patent Document 3, Non-Patent Document 4). More importantly, regardless of the cause Post-treatment by children (i.e., treatment after 6-OHDA induction), and 6-OHDA in Parkinson's disease When applied at a stage where the induced symptoms are already widespread, it can disrupt normal motor behavior and striatal development. - Effectively restored paminergic innervation (Non-Patent Document 3, Non-Patent Document 5). CDN F also showed dopamine in mouse and rhesus monkey MPTP models of Parkinson's disease. Protect and repair ureon. Monkey MPTP model and severe rodent 6-OHDA model. In this context, CDNF is more effective than glial cell line-derived neurotrophic factor (GDNF) in the substantia nigra pars compacta. It is efficient in the recovery of dopamine neurons and motor behavior in (SNPc) Non-patent documents 5, 6, and 7). Neuronal protection against these factors Although the underlying mechanisms are not fully understood, these promote anti-apoptotic pathways. In addition to promoting classical survival activation, it alleviates oxidative stress and ER stress, and ER stress The endoplasmic reticulum stress response (UPR) is aimed at suppressing Tres-induced apoptotic cell death. It has been suggested that the pathway may be modified (Non-Patent Document 8, Non-Patent Document 2, Non-Patent Document 9). Diabetes mellitus, as well as Parkinson's disease, Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). Many pathophysiological conditions, including neurodegenerative diseases such as LS and Huntington's disease (HD), Degenerative diseases are caused by the misidentification of proteins that trigger ER stress and activation of the UPR pathway. It is related to folding and aggregation. Therefore, the effects of CDNF and MANF are related to various central This has been demonstrated in neurological disorders (Patent Documents 1, 2, and 10). Furthermore, CDNF and MANF are involved in the pathophysiology of most, though not all, CNS diseases and injuries. It suppresses neuroinflammation involved in (Non-Patent Literature 11, Non-Patent Literature 12, Non-Patent Literature 13).

[0003] Furthermore, Patent Document 3 concerns genes that naturally encode and express functional MANF genes. The disclosure includes genetically modified non-human animals containing disrupted alleles, and these animals are Disrupted, non-functional MANF genes lead to a gradual postnatal decrease in pancreatic beta cell volume. Furthermore, MANF or CDNF for use in the intrapancreatic treatment of type 1 or type 2 diabetes. Gene therapy vectors that deliver an effective amount of lipeptides or their functional fragments have also been proposed. Furthermore, Non-Patent Document 8 states that the MANF protein is essential for the proliferation and survival of pancreatic beta cells. This discloses that it could be a therapeutic candidate for the protection and regeneration of beta cells.

[0004] Patent Document 4 describes Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, stroke, and terminal illness. Sequence CXXC for use in the treatment of peripheral neuropathy, epilepsy, diabetes, or drug addiction. This document discloses cell-permeable MANF or CDNF peptides with a length of 4 to 40 amino acids. .

[0005] Structural studies of CDNF and MANF have shown that these proteins are saposin-like N-terminal dormant It consists of two domains: In (Non-Patent Document 14) and SAP-like C-terminal (Non-Patent Document 15). It has been shown that the CXXC motif (residues 149-152 of human MANF, NC) is present. BI reference sequence: NP_006001.3) is located outside the helical core of the domain. Located in the C-terminal domain (C-MANF) of the pl region, cysteine ​​is connected by a disulfide bond. They are aligned (Non-Patent Literature 15). The corresponding motifs of CDNF are in the same position (NC BI reference sequence: NP_001025125.2). C-MANF is a sympathetic neuron. When expressed internally, it has been shown to exhibit potent anti-apoptotic activity in vitro (non-specific). (Reference 15). Non-patent document 16 describes the structural and functional determinants of MANF and CDNF. The characteristics of [the subject] are disclosed.

[0006] A cell membrane with selective permeability is similar to how the intracellular membrane operates within its internal compartments. It controls molecular exchange between the cytosol and the extracellular environment. For this reason, the cell membrane is often involved. This presents challenges in intracellular delivery of many molecules, particularly high molecular weight molecules such as full-length proteins. This becomes an obstacle. Active transport of high molecular weight molecules that pass through such barriers requires permeation through the lipid bilayer. A specific carrier capable of this is often required. Cell-permeable peptides (CPPs) are one Generally, peptides (or motifs within peptides) with a length of 5 to 30 amino acids are permeable to the cell membrane. Due to its ability to cross, proteins, plasmid DNA, RNA, oligonucleotides, liposomes, and anticancer agents are widely used for intracellular delivery (Non-Patent Document 1 7, Non-Patent Document 18, Non-Patent Document 19, Non-Patent Document 20).

Prior Art Documents

Patent Documents

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Patent Document 1

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Patent Document 8

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[0009] In this invention, the C-terminal fragment of the CDNF protein is used in vitro and in vivo in the ER It remarkably protects stressed sympathetic and dopaminergic neurons. Furthermore, in contrast to full-length CDNF, it can cross nerve cell membranes and the blood-brain barrier in vivo. We discovered that it is possible to do so.

[0010] Therefore, the object of the present invention is: Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA ATHPKTE L The sequence described above, positions 38-70 or 25-57, or positions 38-70 of sequence number 1 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 25-57. By providing a C-terminal CDNF fragment that contains or consists of consecutive amino acid residues, be.

[0011] Furthermore, the present invention relates to a C-terminal CDNF fragment and a physiologically acceptable carrier, buffer, and excipient. The present invention provides a pharmaceutical composition comprising at least one of a preservative and a stabilizer.

[0012] Furthermore, the results of the present invention are applicable to degenerative diseases including central nervous system (CNS) diseases, diabetes, or retinal diseases. The above-mentioned C-terminal CDNF fragment is provided for use in the treatment of diseases or disorders, and the above-mentioned CNS diseases Preferably, Alzheimer's disease, Parkinson's disease, Huntington's disease and other amidia. Lloyd's disease, multiple system atrophy, amyotrophic lateral sclerosis, frontotemporal lobar degeneration, Lewy body dementia A group consisting of mild cognitive impairment, traumatic brain injury, peripheral nerve injury, addiction, and stroke is selected. It can be done.

[0013] Furthermore, the present invention relates to the C-terminal fragment of MANF (C-MANF) and the mature MANF protein. In contrast, it is possible to penetrate the cell membrane of dopamine neurons, and the neurons in culture This indicates that it will protect [something].

[0014] Therefore, another object of the present invention is Sequence ID No. 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described above, positions 33-68 or 19-52, or positions 33-68 of sequence number 2 Preferably, the sequence has at least 80% homology or sequence identity with the sequence at positions 19-52. It contains or consists of at least consecutive amino acid residues of a sequence, and is administered intravenously or peripherally, or intraperitoneally. The central nervous system is administered via internal, subcutaneous, intranasal, transdermal, intramuscular, intraocular, or intra-arterial administration. (CNS) Preferably 36-78 for use in the treatment of degenerative diseases or disorders, including CNS diseases. The objective is to provide a C-terminal MANF fragment of amino acid length.

[0015] Furthermore, the above C-terminal MANF fragment, a physiologically acceptable carrier, buffer, excipient, and It contains at least one of the tranquilizers and is administered intravenously or peripherally, intraperitoneally, subcutaneously, or intranasally. This includes central nervous system (CNS) diseases, administered via transdermal, intramuscular, intraocular, or intra-arterial administration. The present invention provides pharmaceutical compositions for use in the treatment of degenerative diseases or disorders.

[0016] A further object of the present invention is Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described above, positions 33-68 or 19-52, or positions 33-68 of sequence number 2 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 19-52. It contains or consists of at least consecutive amino acid residues, preferably with a length of 36 to 78 amino acids. The C-terminal MANF fragment is provided for use in the treatment of type 1 or type 2 diabetes or retinal disease. That is what it is.

[0017] Furthermore, the present invention relates to a C-terminal MANF fragment and a physiologically acceptable carrier, buffer, and excipient. It contains at least one of the following: a preservative and a stabilizer, and is used for type 1 or type 2 diabetes or retinal disease. To provide a pharmaceutical composition for use in the treatment of patients.

[0018] The above and other advantages and benefits of the present invention are characterized in the appended claims. It is achieved using the method described. [Brief explanation of the drawing]

[0019] [Figure 1] (A) CDNF has two domains: an N-terminal domain and a C-terminal domain. The N-terminal domain is a saposin-like domain that can bind to oxidized phospholipids (and at least the MANF N-terminal domain also binds to lipid sulfatides, also known as 3-O-sulfogalactosylceramide, see Non-Patent Literature 21). The C-terminal domain has a CXXC sequence and a C-terminal ER residual signal KTEL, and is a SAPLIP-like domain. CDNF can be proteolytically cleaved in vitro to produce these two domains. (B) Schematic diagrams of the structures of MANF and CDNF. The black vertical bars indicate the positions of eight conserved cysteine ​​residues. [Figure 2]CDNF and its C-terminal fragment, expressed from plasmids, rescue superior cervical ganglion (SCG) sympathetic neurons subjected to ER stress. In the experiment, SCG neurons from 7-day-old rats / mice were microinjected with a CDNF-expressing indicator plasmid, a CDNF-expressing indicator plasmid (C-CDNF), a control plasmid PCR3.1, and a positive control when nerve growth factor (10 ng / mL NGF) was added to the culture medium. The following day, ER stress-induced cell death was induced by adding 2 μM tunicamycin™, and then, three days later, the number of living fluorescent neurons was counted. The results are shown as a percentage of the initial number of neurons. [Figure 3] CDNF and CDNF fragment proteins, when microinjected into the cytoplasm, rescue SCG neurons under ER stress. In the experiment, SCG neurons were prepared from 1-day-old mice, cultured for 7 days, and then injected with recombinant human CDNF or C-CDNF protein, respectively. The following day, tunicamycin (2 μM) was added, and the number of living fluorescent neurons was counted after 3 days. The results are shown as a percentage of the initial neurons. [Figure 4] The C-terminal fragment of MANF (C-MANF) protects dopaminergic neurons in culture. Dissociated cultures of the midbrain basil of embryonic day 13 (E13) NMRI mice were grown on 96-well plates for 5 days with C-MANF, GDNF (positive control) added to the culture medium, or without growth factors as a control. The cultures were then stained for tyrosine hydroxylase (TH). Images were scanned with CellInsight™, and immunopositive neurons were counted using CellProfiler and CellProfiler analysis software. Data are expressed as a percentage of GDNF-bearing TH-positive neurons. [Figure 5]The C-terminal fragment of CDNF (C-CDNF) protects dopaminergic neurons in vitro. Dissociated cultures of the midbrain basil of E13.5 NMRI mice were grown on 96-well plates for 5 days in culture medium supplemented with CDNF or CDNF fragments at predetermined concentrations. Dopamine neurons cultured with GDNF (100 ng / mL) or without neurotrophic factors were used as controls. Cultures were immunostained for tyrosine hydroxylase (TH), and images were scanned with CellInsight™. TH-positive neurons were counted using CellProfiler and CellProfiler analysis software and expressed as a percentage of GDNF-retaining neurons. [Figure 6] The C-terminal fragment of CDNF (C-CDNF) and the C-terminal fragment of MANF (C-MANF) permeate the cell membranes of dopamine neurons and PC6 cells. A. Unlike 125I-CDNF, 125I-C-CDNF is efficiently taken up by E14 dopamine neurons in vitro, exhibiting the cell permeability of C-CDNF. Cultured E14 dopamine neurons were incubated with 30,000 cpm iodized CDNF or C-CDNF at 37°C for 2 hours. The cells were then placed on ice, washed with 0.2 M acetic acid and 0.5 M NaCl (pH 2.8), and counted with a gamma counter. Intracellular radioactivity was measured. B. Unlike full-length iodized CDNF, 125I-C-CDNF and 125I-C-MANF permeate the cell membranes of rat PC6 cells. PC6 cells, treated with or without thapsigargin for 3 hours prior to growth factor addition, were then treated with iodized CDNF or C-CDNF and C-MANF. Internalization was carried out at 37°C for 90 minutes. After placing the cells on ice, they were washed with 0.2M acetic acid and 0.5M NaCl (pH 2.8), and intracellular radioactivity was measured using a gamma counter. [Figure 7]Blood-brain barrier permeability of 125I-CDNF, 125I-C-CDNF, and 125I-C-MANF. 125I-CDNF, 125I-C-CDNF, and 125I-C-MANF were subcutaneously injected into rats. Two hours later, the rats were perfused with PBS and their brains were dissected. Radioactivity in the brain was analyzed using a gamma counter. Data are shown as mean ± SEM. p<0.05 t-test, *p>0.05. [Figure 8] Cumulative turnover at 2, 4, 6, and 8 weeks post-injury in a rat 6-OHDA model of PD. CDNF, N-terminal CDNF (N-CDNF), C-CDNF, or vehicle (PBS) were intrastriatally injected into the brains of rats 2 weeks after 6-OHDA injury. C-CDNF was more effective than full-length CDNF in the recovery of neuronal function because it significantly reduced the cumulative amount of amphetamine-induced turnover in 6-OHDA injured rats. Data are shown mean ± SEM. After one-way ANOVA, Tukey-Kramer post-hoc test, ****p<0.0001. [Figure 9] The short 4-amino acid C-terminal fragment of MANF (MANF4) was not effective in a rat 6-OHDA model of Parkinson's disease when the peptide was injected into the striatum starting 2 weeks after 6-OHDA injury and amphetamine-induced turnover was measured at 1, 4, 6, 8, 10, and 12 weeks post-injury (A) or cumulatively (B). GDNF was used as a positive control. [Figure 10] The C-terminal MANF fragment (C-MANF) stimulates the proliferation of mouse beta cells. Click EdU uptake into beta cells (n=3 wells / point) after culturing mouse pancreatic islets in vitro with placental lactogen, C-MANF, or MANF for 5 days. *p<0.05, **p<0.01, ***p<0.001. [Figure 11]Treatment with C-CDNF has a significant effect on the clinical score of the SOD1 mouse model of ALS. SOD1 mice were given a single intraventricular injection of either C-CDNF (3.75 μg) or PBS at 13 weeks of age. (A) Clinical status of female animals. C-CDNF-treated SOD1 mice showed statistically significantly better clinical scores than PBS-treated mice, indicating that C-CDNF treatment delays the onset of symptoms. (B) Balance, coordination, and muscle strength were analyzed using a rotarod. Acceleration 4-40 rpm, cutoff time 4 minutes. Time to fall (seconds) is shown on the left. SOD1-G93A female. C-CDNF treatment improves motor behavior in SOD1-G93A mice compared to PBS-treated mice. [Figure 12] Effects of 4 weeks of long-term intraventricular infusion of 1.5 μg / 24 hours of C-CDNF in a SOD1-G93A mouse ALS model. (A) Relative changes in body weight, without sex classification. 12 weeks (before minipump placement) is shown as baseline. A significant difference in body weight between treatments was detected between weeks 18 and 19 (p<0.05, two-sided unpaired t-test). (B) 4 weeks of long-term intraventricular C-CDNF infusion improves motor coordination as measured by rotorodization ability in SOD1-G93A mice. The rotorodization test used an acceleration of 4-40 rpm and a cutoff time of 4 minutes. The difference between C-CDNF treatment and PBS treatment was significant from week 13 to week 19 (p<0.01, repeated measures ANOVA). [Figure 13] Subcutaneous injection of C-CDNF reduces infarct volume in a rat model of cerebral ischemia. C-CDNF (50 μg) was administered in 100 μL volumes 30–50 minutes before distal middle cerebral artery occlusion and immediately after reperfusion. C-CDNF treatment reduced infarct volume measured from the rostral portion of the cerebral cortex (Student's t-test p<0.05). C-CDNF-treated rats showed approximately 50% less damage than vehicle-treated rats. PBS was used as a control. * indicates P<0.05. Values ​​are expressed as mean ± SEM as a percentage of PBS, n=8–9. [Figure 14]The designed C-terminal peptides of CDNF (peptides 1-7, see Table 5) rescue ER-stressed SCG neurons when microinjected into the cytoplasm. In the experiment, SCG neurons were prepared from 1-day-old mice, cultured for 7 days, and then injected with recombinant human CDNF or C-CDNF peptide. The following day, tunicamycin (2 μM) was added, and the number of viable fluorescent neurons was counted after 3 days. Results are shown as a percentage of the initial neurons. Data are shown mean ± SEM. After one-way ANOVA, Tukey-Kramer post-hoc tests were performed: *p<0.05, **p<0.01, ***p<0.001. [Figure 15] The designed C-terminal peptide of CDNF (peptides 1-7, see Table 5) protects dopamine neurons in vitro. Dissociated cultures of the midbrain basil of E13.5 NMRI mice were grown on 96-well plates for 5 days in culture medium supplemented with CDNF peptides (peptides 1-7, see Table 5) at specified concentrations. Dopamine neurons cultured with GDNF (100 ng / mL) or without neurotrophic factors were used as controls. Cultures were immunostained for tyrosine hydroxylase (TH), and images were scanned with CellInsight™. TH-positive neurons were counted using CellProfiler and CellProfiler analysis software and expressed as a percentage of GDNF-retaining neurons. Data are shown mean ± SEM. After one-way ANOVA, Tukey-Kramer post-hoc tests were performed: *p<0.05, **p<0.01, ***p<0.001. [Figure 16]Survival studies of SCG neurons after microinjection and tunicamycin treatment using designed C-terminal peptides of CDNF (peptides 8-15, see Table 6). Peptides 8 (37aa), 12 (49aa with a single amino acid substitution), 13 (49aa with a single amino acid substitution), and 15 (33aa) rescue ER-stressed SCG neurons when microinjected into the cytoplasm. In the experiment, SCG neurons were prepared from 1-day-old mice, cultured for 7 days, and then injected with recombinant human CDNF or C-CDNF peptide. The following day, tunicamycin (2 μM) was added, and the number of viable fluorescent neurons was counted after 3 days. Results are shown as a percentage of the initial neurons. Data are shown mean ± SEM. After one-way ANOVA, Tukey-Kramer post-hoc tests were performed, *p<0.05, **p<0.01. [Figure 17] Dissociated midbrain basal cultures of E13.5 NMRI mice were grown on 96-well plates for 5 days in culture medium supplemented with the C-terminal peptide of CDNF (peptides 8-15, see Table 6) at specified concentrations. Dopamine neurons cultured with GDNF (100 ng / mL) or without neurotrophic factors were used as controls. Cultures were immunostained for tyrosine hydroxylase (TH), and images were scanned with CellInsight®. TH-positive neurons were counted using CellProfiler and CellProfiler analysis software and expressed as a percentage of GDNF-retaining neurons. All C-CDNF peptides showed activity compared to uncleaved CDNF protein, but peptides 8, 12, 13, 14, and 15 showed the best protective effect on dopamine neurons in vitro. Data are shown mean ± SEM. After one-way ANOVA, Tukey-Kramer post-hoc tests were performed, with *p<0.05, **p<0.01, and ***p<0.001. [Figure 18]Modified C-terminal peptides of CDNF with N-terminal acetylation and C-terminal amidation (peptides 1, 61aa and 15, 37aa; see Tables 5 and 6) protect dopamine neurons in vitro. Dissociated cultures of the midbrain base of E13.5 NMRI mice were grown on 96-well plates for 5 days with peptides added to the culture medium at predetermined concentrations. Dopamine neurons cultured with GDNF (100 ng / mL) or without neurotrophic factors were used as controls. Cultures were immunostained for tyrosine hydroxylase (TH), and images were scanned with CellInsight®. TH-positive neurons were counted using CellProfiler and CellProfiler analysis software and expressed as a percentage of GDNF-retaining neurons. [Figure 19] C-CDNF 33aa (pep15, see Table 6), when administered repeatedly by subcutaneous injection, is effective in preventing 6-OHDA-induced impairment in motor balance as measured by amphetamine-induced rotational behavior. 6-OHDA (3 × 2 μg) was injected into the striatum. Starting two weeks after injury, animals received subcutaneous injections of C-CDNF 33aa or vehicle (PBS) twice a week for four weeks (dosage: 50 μg / subcutaneous injection, total dose after 8 subcutaneous injections: 400 μg). (A) Amphetamine-induced rotational tests were performed at 2, 4, 6, 8, 10, and 12 weeks after injury. (B) Cumulative number of rotations at 2, 4, 6, 8, 10, and 12 weeks. Values ​​are shown mean ± SEM, n=9. *** indicates a p<0.001 difference between the C-CDNF (50 μg x 8 subcutaneous injections) group and the vehicle-treated control group. [Figure 20] Chemically synthesized C-CDNF peptide (pep1, 61aa) and C-CDNF peptide (pep1, 61aa) expressed in CHO cells have identical CD spectra and retain a well-defined secondary structure with α-helices and loops. CD spectrum was measured using a JASCO J720 instrument. Peptide concentration: 30 μM. Vehicle: 20 mM PB, pH 7. Volume: 300 μL. Wavelength: 260-195 nm. [Figure 21]CD spectra of chemically synthesized C-CDNF peptides (pep1, pep8, pep9, pep12, and pep13; see Tables 5 and 6). C-CDNF peptide 8 (pep8) preserves several secondary structural elements. C-CDNF peptide 9 establishes a secondary structure similar to C-CDNF 61aa (pep1). C-CDNF peptide 12 (pep12) establishes a secondary structure but lacks several α-helix regions compared to C-CDNF 61aa (pep1). C-CDNF peptide 13 (pep13) establishes a secondary structure but lacks several α-helix regions compared to peptide 12 (pep12). CD spectra were measured using a JASCO J720 instrument. Peptide concentration: 30-100 μM. Vehicle: 20 mM PB, pH 8. Volume: 300 μL. Wavelength: 260-195 nm. [Figure 22] The CD spectrum of C-CDNF peptide 15 (pep15) indicates that the peptide conserves several secondary structural elements. CD spectrum measurement was performed using a JASCO J720 instrument. Peptide concentration: 50 μM. Vehicle: 20 mM PB, pH 8. Volume: 300 μL. Wavelength: 260-195 nm. [Figure 23] Sequence alignment and comparison of C-CDNF (SEQ ID NO: 4) and C-MANF (SEQ ID NO: 5). The C-terminal structures of both neurotrophic factors contain three α-helix motifs (helices 1, 2, and 3). C-terminal CDNF peptides 5 and 6 (see Table 5) contain only two of these motifs (helices 2 and 3). [Figure 24] Prediction of the protease cleavage site of the C-terminal CDNF fragment (SEQ ID NO: 1). [Figure 25] Prediction of the protease cleavage site of the C-terminal MANF fragment (SEQ ID NO: 2). [Modes for carrying out the invention]

[0020] This invention relates to the neurotrophic factor protein CDNF. The CDNF polypeptide is sig Full-length human CDNF with 187 amino acids containing a nal peptide, and a signal peptide. It is a mature human CDNF consisting of 161 full-length amino acids that does not contain (see Figure 1B).

[0021] Furthermore, this invention relates to the neurotrophic factor protein MANF. In particular, MANF poly The peptide is a full-length human MANF with 179 amino acids containing a signal peptide, and This is a mature human MANF with a total length of 158 amino acids that does not contain a signal peptide (see Figure 1B). ).

[0022] As used herein, the term "C-terminus" applies to CDNF or MANF polypeptides. The "fragment" is typically located at least approximately in the C-terminal SAP-like domain of the polypeptide described above. 33 pieces, 34 pieces, 35 pieces, 36 pieces, 37 pieces, 38 pieces, 39 pieces, 40 pieces, 41 pieces, 42 pieces, 43 pieces, 44 pieces, 45 pieces, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53, 54, 55, 56, or 57 adjacent or consecutive amino acids, typically fewer At most about 43 or 55 adjacent or consecutive amino acids, more typically at least about 57 Alternatively, it may contain 60 adjacent or consecutive amino acids (see Figures 1A and 1B). Also, C The terminal fragment may be longer than the length of 61 or 65 adjacent or consecutive amino acids, in some cases. Depending on the case, it may be longer than 70 adjacent or consecutive amino acids. Most preferably, C-terminus The fragments consist of 33-57, 33-61, 33-81, and 43-57 segments of the C-terminal domain. It contains 43-61, 43-81, or 60-65 adjacent or consecutive amino acids. The C-terminal fragment retains at least partially the biological activity of the complete polypeptide. These are "functional fragments" and may even possess properties that are not present in complete polypeptides.

[0023] In addition to naturally occurring allele variants of CDNF / MANF, encoded CDN S, insertions, and deletions in the amino acid sequence of F / MANF polypeptides or their C-terminal fragments. By introducing mutations into the CDNF / MANF nucleic acid sequence that bring about changes such as these, changes can be introduced. This is possible. In the sequence of CDNF / MANF polypeptides and their C-terminal domains... This involves performing nucleotide substitutions, which result in amino acid substitutions at "non-essential" amino acid residues. It is possible.

[0024] "Non-essential" amino acid residues are present in the wild-type sequence of CDNF / MANF, and their biological properties are... These are residues that can be modified without altering their activity, while "essential" amino acid residues The group is necessary for such biological activity. For example, the CDNF / MANF molecule of the present invention. The conserved amino acid residues are essential and are expected to be particularly resistant to change. Amino acids that can be substituted in a conservative manner are well known in the field.

[0025] Each amino acid is either natural or unnatural. The term "unnatural amino acid" refers to an amino acid that is either natural or unnatural. It has a structure similar to amino acids and mimics the structure and reactivity of natural amino acids, in that sense it is natural. This refers to organic compounds that are congeners of amino acids. Unnatural amino acids are modified amino acids and It may also be an amino acid analog, but it may be 20 common naturally occurring amino acids. Alternatively, it is not one of the rare natural amino acids selenocysteine ​​or pyrrolicin. Also, Non-natural amino acids may be D-isomers of natural amino acids. Examples of suitable amino acids include However, these are not limited to alanine, alloisoleucine, arginine, and asparagus. Aspartic acid, cysteine, cyclohexylalanine, 2,3-diaminopropio 4-fluorophenylalanine, glutamine, glutamic acid, glycine, histidine Homoproline, isoleucine, leucine, lysine, methionine, naphthylalanine, Norleucine, phenylalanine, phenylglycine, pipecolic acid, proline, pyrog Lutamic acid, sarcosine, serine, selenocysteine, threonine, tryptophan, Examples include rosine, valine, their derivatives, or combinations thereof.

[0026] A particular embodiment of the present invention involves at least 1, 2, 3, 4, or more consecutive amino acids. This specification includes a C-terminal CDNF fragment or a C-terminal MANF fragment that has alternating chirality. In written form, chirality refers to the "D" and "L" isomers of an amino acid. In a certain embodiment, at least 1, 2, 3, 4 or more consecutive amino acids alternate It possesses chirality, and the remaining amino acids are L-amino acids.

[0027] In this disclosure, the C-terminal CDNF fragment and the C-terminal MANF fragment of the present invention are used in neurons. Cellular uptake has been demonstrated. In certain embodiments, uptake is preferably complete Compared to the full length CDNF or MANF, it is at least 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11 or 12 times better, and for certain peptides, better than full-length CDNF or MANF. It is 13 times better. In a specific embodiment, the present invention provides full-length human CDNF and other materials. Compared to the control, the C-terminal CDNF fragment of the present invention showed improved cellular uptake efficiency. This demonstrates that, in certain embodiments, the present invention compares with controls such as full-length human MANF. In comparison, the C-terminal MANF fragment of the present invention shows improved cellular uptake efficiency.

[0028] In the context of this specification, the cell uptake efficiency is determined by the C-terminal CDNF fragment or the C-terminal MANF This refers to the ability of the fragment to permeate the cell membrane. The C-terminal CDNF fragment or C-terminal MANF fragment of the present invention. The uptake of one cell is independent of the receptor or cell type.

[0029] A person skilled in the art would know that (i) C-terminal CD incorporated into a cell type (e.g., nerve cell, endothelial cell, etc.) (ii) the amount of cell-permeable peptides such as NF fragments or C-terminal MANF fragments, and (ii) the same cell type Compare the amount of incorporated control peptides such as full-length CDNF / MANF with that amount. This allows us to test the uptake efficiency of the C-terminal CDNF fragment and / or C-terminal MANF fragment. This can be done. When measuring cell uptake efficiency, the C-terminal CDNF fragment or C-terminal MANF In the presence of cell-permeable peptides such as fragments, a specific time interval (e.g., 30 minutes, 1 hour) is used to identify a cell type. Incubate for 2 hours, etc., and then measure the amount of cell-permeable peptide taken up by the cells. Quantitative analysis may be performed. Separately, a control of the same concentration may be prepared in the presence of the cell type for the same duration. The cells are incubated, and the amount of the other peptide taken up by the cells is quantified. Quantification is performed using C Cell-permeable peptides such as terminal CDNF fragments or C-terminal MANF fragments are fluorescently labeled (for example) By measuring fluorescence intensity using techniques well known in the art (with FITC dyes), It is possible to do so.

[0030] Furthermore, the C-terminal CDNF fragment and C-terminal MANF fragment of the present invention are controlled by appropriate controls. For example, compared to Figures 14 and 15, it has a protective effect on cells, such as nerve cells. As shown herein, the protective effect refers to the C-terminal CDNF fragment or C-terminal M of the present invention. ANF ​​fragments, for example, in dopaminergic neurons or neurons under ER stress This refers to the ability to promote the survival of cells such as sympathetic nerve cells. A person skilled in the art would know that (i) cell type (e.g., sympathetic nerve cells) The C-terminal CDNF fragment or C of the present invention for the survival of (or dopaminergic neurons, etc.) (ii) the dose of the terminal MANF fragment and the survival level of the control peptide in the same cell type. By comparing the survival level with that of the same cell type without the addition of neurotrophic factors, The above protective effect can be tested. When measuring cell viability, the C-terminus of the present invention In the presence of CDNF fragments and C-terminal MANF fragments, the time required to identify cell types (e.g., 30 minutes) The cells may be incubated for 1 hour, 2 hours, etc., and then the cell viability may be quantified. Separately, control peptides at the same concentration were incubated for the same time in the presence of the cell type. Then, quantify the cell viability of the other peptide by the cells. Alternatively, the cell type can be determined. Cell viability is quantified by incubating cells for the same amount of time without transtrophic factors.

[0031] In one embodiment, when measuring cell viability, the C-terminal CDNF segment of the present invention is used for cell types. Inject a single or C-terminal MANF fragment and leave it for a specific time (e.g., 30 minutes, 1 hour, 2 hours, etc.). The cells may be incubated, and then the cell viability may be quantified. Control cells may be used. Inject buffer (i.e., without neurotrophic factors) and incubate control cells for the same amount of time. The cell viability rate is then quantified.

[0032] In certain embodiments, the protective effect of the C-terminal CDNF fragment of the present invention (as cell viability) The measurement was performed on cells incubated without growth factors, or in a buffer solution that did not contain growth factors. Compared to cells injected with, at least 1.01 times, at least 1.02 times, and at 1.03 times, at least 1.04 times, at least 1.05 times, at least 1.05 times, less At least 1.06 times, at least 1.07 times, at least 1.08 times, at least 1.0 9 times, at least 1.1 times, at least 1.11 times, at least 1.12 times, at 1.13 times, at least 1.14 times, at least 1.15 times, at least 1.16 times, less At least 1.17 times, at least 1.19 times, at least 1.2 times, at least 1.21 times times, at least 1.23 times, at least 1.25 times, at least 1.26 times, at least 1.27 times, at least 1.28 times, at least 1.29 times, at least 1.3 times, less At least 1.31 times, at least 1.32 times, at least 1.33 times, at least 1.34 times, at least 1.35 times, at least 1.37 times, at least 1.4 times, at least 1 0.42 times, at least 1.43 times, at least 1.45 times, at least 1.48 times, less At least 1.49 times, at least 1.5 times, at least 1.55 times, at least 1.6 times, At least 1.65 times, at least 1.7 times, at least 1.75 times, at least 1.8 times, at least 1.85 times, at least 1.89 times, at least 1.9 times, at least 1 0.94 times, at least 1.95 times, at least 2.0 times, at least 2.05 times, less Both are 2.10 times, at least 2.11 times, at least 2.14 times, and at least 2.15 times. , at least 2.16 times, at least 2.19 times, at least 2.2 times, at least 2. 21 times, at least 2.23 times, at least 2.24 times, at least 2.25 times, less Both are 2.26 times, at least 2.28 times, at least 2.3 times, at least 2.32 times, At least 2.35 times, at least 2.37 times, at least 2.39 times, at least 2. 4 times, at least 2.42 times, at least 2.45 times, at least 2.5 times, at 2.52 times, at least 2.55 times, at least 2.6 times, at least 2.65 times, less At least 2.7 times, at least 2.75 times, at least 2.8 times, at least 2.85 times, At least 2.9 times, at least 3.0 times, at least 3.1 times, at least 3.2 times, At least 3.3 times, at least 3.4 times, at least 3.5 times, at least 3.6 times, At least 3.7 times, at least 3.8 times, at least 3.9 times, and at least 4.0 times be.

[0033] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.01 times.

[0034] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.02 times.

[0035] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.03 times.

[0036] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.04 times.

[0037] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.05 times.

[0038] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.05 times.

[0039] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.06 times.

[0040] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.07 times.

[0041] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.08 times.

[0042] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.09 times.

[0043] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.1 times.

[0044] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.11 times.

[0045] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.12 times.

[0046] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.13 times.

[0047] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.14 times.

[0048] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.15 times.

[0049] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.16 times.

[0050] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.17 times.

[0051] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.19 times.

[0052] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.2 times.

[0053] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.21 times.

[0054] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.23 times.

[0055] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.25 times.

[0056] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.26 times.

[0057] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.27 times.

[0058] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.28 times.

[0059] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.29 times.

[0060] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.3 times.

[0061] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.31 times.

[0062] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.32 times.

[0063] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.33 times.

[0064] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.34 times.

[0065] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.35 times.

[0066] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.37 times.

[0067] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.4 times.

[0068] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.42 times.

[0069] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.43 times.

[0070] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.45 times.

[0071] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.48 times.

[0072] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.49 times.

[0073] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.5 times.

[0074] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.55 times.

[0075] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.6 times.

[0076] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.65 times.

[0077] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.7 times.

[0078] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.75 times.

[0079] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.8 times.

[0080] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.85 times.

[0081] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.89 times.

[0082] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.9 times.

[0083] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.94 times.

[0084] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 1.95 times.

[0085] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.0 times.

[0086] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.05 times.

[0087] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.10 times.

[0088] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.11 times.

[0089] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.14 times.

[0090] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.15 times.

[0091] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.16 times.

[0092] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.19 times.

[0093] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.2 times.

[0094] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.21 times.

[0095] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.23 times.

[0096] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.24 times.

[0097] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.25 times.

[0098] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.26 times.

[0099] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.28 times.

[0100] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.3 times.

[0101] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.32 times.

[0102] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.35 times.

[0103] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.37 times.

[0104] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.39 times.

[0105] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.4 times.

[0106] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.42 times.

[0107] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.45 times.

[0108] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.5 times.

[0109] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.52 times.

[0110] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.55 times.

[0111] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.6 times.

[0112] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.65 times.

[0113] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.7 times.

[0114] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.75 times.

[0115] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.8 times.

[0116] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.85 times.

[0117] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 2.9 times.

[0118] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.0 times.

[0119] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.1 times.

[0120] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.2 times.

[0121] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.3 times.

[0122] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.4 times.

[0123] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.5 times.

[0124] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.6 times.

[0125] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.7 times.

[0126] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.8 times.

[0127] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 3.9 times.

[0128] In one embodiment, the protective effect is compared to cells incubated without growth factors. It is at least 4.0 times.

[0129] In certain embodiments, the protective effect of the C-terminal MANF fragment of the present invention is achieved by adding growth factors. Compared to cells incubated without growth factors, or cells injected with a buffer that does not contain growth factors. , at least 1.05 times, at least 1.1 times, at least 1.2 times, at least 1.3 times, at least 1.4 times, at least 1.5 times, at least 1.6 times, at least 1.7 times double, at least 1.8 times, at least 1.9 times, at least 2.0 times, at least 2.1 times 0x, at least 2.2x, at least 2.3x, at least 2.4x, at least 2. 5 times, at least 2.6 times, at least 2.7 times, at least 2.8 times, at least 2. 9 times, at least 3.0 times, at least 3.1 times, at least 3.2 times, at least 3. 3 times, at least 3.4 times, at least 3.5 times, at least 3.6 times, at least 3. It is 7 times, at least 3.8 times, at least 3.9 times, and at least 4.0 times.

[0130] Therefore, the present invention relates to Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA ATHPKTE L The sequence described above, positions 38-70 or 25-57, or positions 38-70 of sequence number 1 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 25-57. A C-terminal CDNF fragment containing or consisting of at least a series of amino acid residues, which permeates the cell membrane. It is a perfusion peptide that has a protective effect on nerve cells and is preferably used as a pharmaceutical. Provides a fragment for this purpose. In one embodiment, the C-terminal CDNF fragment is 38 of SEQ ID NO: 1. Sequences with positions up to 70 or 25-57 and at least 81%, 82%, 83%, 84%, 85% 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , including sequences having 96%, 97%, 98%, or 99% homology or sequence identity, It consists of.

[0131] In a preferred embodiment, the fragments have a length of 33 to 81 amino acids. The fragment is located at positions 38-70 or 25-57 of the sequence described in Sequence ID 1, or Sequence ID 1. 1. Ranks 38-70 or 25-57 and at least 80%, 81%, 82%, 83% 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Having 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity. The sequence includes at least consecutive amino acid residues, and if present, adjacent to the consecutive amino acid residues. The resulting sequence has at least 80% homology or sequence identity with the sequence at the corresponding position of sequence number 1. It preferably possesses the characteristic of "flanking sequence". This refers to an amino acid that extends both or at least one of the terminal ends of the aforementioned consecutive amino acid residues. In one embodiment, the sequence adjacent to the consecutive amino acid residues is the corresponding position of SEQ ID NO: 1 The arrangement of the placements and at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Preferably, it has % or 99% homology or sequence identity.

[0132] Although we do not wish to be limited by theory, the results in Figure 15 show cell membrane permeability and The amino acid sequence motifs important for the protective effect on nerve cells are located at approximately position 38 and approximately This indicates that it is located between amino acid residues at position 70. This region has two helical structures ( It includes helix 2 and helix 3, with a CXXC motif in between (see Figure 23). Furthermore, the results in Figures 16 and 17 include C with helix 1 and 2 and the CXXC motif. -CDNF peptide 15(33aa) demonstrates a protective effect on nerve cells. Therefore, the present invention relates to the C-terminal CDNF fragment being located at positions 38-70 of the sequence described in Sequence ID No. 1, 3 7th to 70th, 36th to 70th, 35th to 70th, 34th to 70th, 33rd to 70th, 32nd to 70th , 31st to 70th, 38th to 73rd, 37th to 73rd, 36th to 73rd, 35th to 73rd, 34th to 7th 3 positions, positions 33 to 73, positions 32 to 73, positions 31 to 73, positions 25 to 57, positions 24 to 57, 23 ~57, positions 22 to 57, positions 21 to 57, positions 20 to 57, positions 25 to 58, positions 25 to 59, 25 to 60, 25 to 61, 25 to 62, 25 to 63, 25 to 64, 25 to 65 positions, 25 to 66, 25 to 67, 25 to 68, 25 to 69, or 25 to 70, of or, positions 38 to 70, 37 to 70, 36 to 70, 35 to 70, 34 to 70, 33 to 70, 32 to 70, 31 to 70, 38 to 73, 37 to 73, 3 6 to 73, 35 to 73, 34 to 73, 33 to 73, 32 to 73, 31 to 73 , positions 25 to 57, 24 to 57, 23 to 57, 22 to 57, 21 to 57, 20 to 5 [[ID=十六]]7, 25 to 58, 25 to 59, 25 to 60, 25 to 61, 25 to 62, 25[[ID=十七]] ~63, 25 to 64, 25 to 65, 25 to 66, 25 to 67, 25 to 68, 25 to 69, or having at least 80% homology or sequence identity with a sequence of 25 to 70 comprising, or consisting of, at least contiguous amino acid residues of a sequence having, and where present, the sequence adjacent to the contiguous amino acid residues preferably has at least 80% [[ID=二十五]] homology or sequence identity with the sequence at the corresponding position of SEQ ID NO: 1. In one embodiment, the C-terminal " CDNF fragment is positions 38 to 70, 3 to 70, 36 to 70, 35 to 7 0, 34 to 70, 33 to 70, 32 to 70, 31 to 70, 38 to 73, 37 ~73, 36 to 73, 35 to 73, 34 to 73, 33 to 73, 32 to 73, " 31 to 73, positions 25 to 57, 24 to 57, 23 to 57, 2to 57, 21 to 57 positions, 20 to 57, 25 to 58, 25 to at 60, 25 to 61, 25 to 62nd place, 25th to 63rd place, 25th to 64th place, 25th to 65th place, 25th to 66th place, 25th to 67th place, 2 The sequences 5-68th, 25-69th, or 25-70th, and at least 81%, 82%, 83% %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 It has 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity. The sequence includes or consists of the following: In one embodiment, adjacent to the continuous amino acid residue. If the array exists, at least 81%, 82%, the array at the corresponding position of array index 1. 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity Preferably has.

[0133] In another preferred embodiment, the C-terminal CDNF fragment is 3 of the sequence described in SEQ ID NO: 1. Positions 1-73 (Peptide 6; SEQ ID NO: 15), Positions 25-73 (Peptide 4; SEQ ID NO: 13) , positions 21-73 (Peptide 3; SEQ ID NO: 12), positions 21-70 (Peptide 7; SEQ ID NO: 1) 6), positions 31-81 (peptide 5; sequence number 14), positions 25-81 (peptide 2; sequence number 14), No. 11), positions 25-57 (peptide 15; SEQ ID NO: 24), or positions 37-73 (peptide 15). Tydo 8; Sequence ID 17), or positions 31-73, 25-73, 21-7 3rd place, 21st-70th place, 31st-81st place, 25th-81st place, 25th-57th place, or 37th-73rd place At least one consecutive amino acid sequence having at least 80% homology or sequence identity with the sequence. If a sequence contains or consists of residues, and is present, the sequence adjacent to the consecutive amino acid residues is Preferably, the arrangement at the corresponding position in column number 1 has at least 80% homology or sequence identity. It has. In one embodiment, the C-terminal CDNF fragment is located at positions 31-73 of SEQ ID NO: 1, 25 ~73rd place, 21~73rd place, 21~70th place, 31~81st place, 25~81st place, 25~57th place, Or sequences with positions 37-73 and at least 81%, 82%, 83%, 84%, 85%, 86% 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , containing or not containing sequences having 97%, 98%, or 99% homology or sequence identity In one embodiment, the sequence adjacent to the consecutive amino acid residues corresponds to Sequence ID No. 1. The array of positions and at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Preferably, it has 98% or 99% homology or sequence identity.

[0134] In another preferred embodiment, the C-terminal CDNF fragment is 3 of the sequence described in SEQ ID NO: 1. At least 80% homology or correspondence between the sequence at positions 1-73, or the sequence at positions 31-73 of sequence number 1. The sequence contains or consists of at least consecutive amino acid residues having sequence identity, and is present in In total, the sequence adjacent to the aforementioned consecutive amino acid residues is less than the sequence at the corresponding position in Sequence ID No. 1. Both preferably have 80% homology or sequence identity. In one embodiment, C-terminal C The DNF fragment contains at least 81%, 82%, and 83% of the sequence at positions 31-73 of sequence number 1. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, Having 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity. It includes or consists of an array.

[0135] In another preferred embodiment, the C-terminal CDNF fragment is 2 of the sequence set forth in SEQ ID NO: 1 at positions 5 to 57, or at least 80% homology or sequence identity with the sequence at positions 25 to 57 of SEQ ID NO: 1, and includes, or consists of, at least contiguous amino acid residues of a sequence having such homology or sequence identity, and where present, the sequence adjacent to the contiguous amino acid residues preferably has at least 80% homology or sequence identity with the sequence at the corresponding position of SEQ ID NO: 1. In one embodiment, the C-terminal C DNF fragment includes, or consists of, a sequence having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homology or sequence identity with the sequence at positions 25 to 57 of SEQ ID NO: 1.

[0136] In a preferred embodiment, the present invention relates to a C-terminal CDNF fragment consisting of at least 50 contiguous amino acid residues of the sequence set forth in SEQ ID NO: 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA ATHPKTE L or a sequence that is at least 90% homologous to the sequence set forth in SEQ ID NO: 1. In one embodiment, the sequence is at least 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% homologous to the sequence of SEQ ID NO: 1.

[0137] Also, the present invention relates to SEQ ID NO: 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL​​​​ At least one of the sequences described above, or a sequence that is at least 90% homologous to the sequence of sequence number 2. This relates to a C-terminal MANF fragment consisting of 50 consecutive amino acid residues. In one embodiment, The sequence is at least 91%, 92%, 93%, 94%, 95%, 96% of the sequence at sequence number 2. They are 97%, 98%, or 99% homologous.

[0138] As used herein and in the following sections of the claims, the term “fragment” means “heavenly.” Natural peptides (either degradation products, synthetically synthesized peptides, or recombinant peptides) The compound includes, and modified peptides, the modified peptides for example, to make the peptides more stable. Alternatively, it may have modifications that result in lower immunogenicity. Such modifications include While not limited to these, cyclization, N-terminal modification, C-terminal modification, peptide bond modification, skeletal modification, and Residual modification is one example. Furthermore, the fragment may also include elongation, deletion, substitution, or insertion. That's good too.

[0139] In one embodiment, the fragment exhibits resistance to protease cleavage. In one embodiment, The fragments undergo elongation, deletion, or insertion, destroying at least one protease cleavage site. Includes substitution or modification.

[0140] As used herein, the "protease cleavage site" is recognized by the protease. This refers to the amino acid sequence to be cleaved. In some embodiments, a C-terminal CDNF fragment or The C-terminal MANF fragment is a cysteine ​​protease, metalloprotease, or serine protease. It contains one or more protease cleavage sites that can be cleaved by thease. In several embodiments, the protease cleavage site is, for example, shown in Figure 24 or Figure 25, or These are the protease cleavage sites shown in Tables 7 and 8.

[0141] As used herein, the terms “protease-resistant fragment” or “fragment that is protease-cleaved” "Shows resistance to" means destroying at least one natural protease cleavage site, or natural Contains a modified amino acid sequence that alters the sequence near or adjacent to the natural protease cleavage site. Furthermore, compared to the corresponding natural C-terminal CDNF or C-terminal MANF fragments, the protea C-terminal CDNF fragments or C-terminal MANF fragments that prevent, inhibit, reduce, or slow down cleavage To point.

[0142] In some embodiments, a suitable modification involves at least one protease cleavage site. Destroy it.

[0143] In some embodiments, appropriate modifications involve at least two protease cleavage sites. Destroy it.

[0144] In some embodiments, appropriate modifications involve at least three protease cleavage sites. Destroy it.

[0145] In some embodiments, appropriate modifications involve at least four or more protease cleavages. Destroy the body part.

[0146] In one embodiment, all cysteine ​​protease cleavage sites are destroyed.

[0147] In one embodiment, all metalloproteinase cleavage sites are destroyed.

[0148] In one embodiment, all serine protease cleavage sites are destroyed.

[0149] Modifications can include amino acid substitution, deletion, insertion, elongation, or modification.

[0150] For example, residues 1-75 of sequence number 1 (for example, residues 1-8, 16-23, 26-33, 32-39, 37-44, 38-45, 43-50, 46-53, 57-64, 59-6 Any one amino acid or sequence within the regions corresponding to 6, 60-67, and 68-75) Replace the corresponding amino acids numbered 3, 4, and 11-24 with any other amino acid. They can be deleted or modified, for example, adjacent to the protease cleavage site. Positional displacement may affect the recognition of cleavage sites by proteases. Substitution or insertion of one or more additional amino acids within one or more proteas This can potentially destroy the ze cleavage site. Deletion of one or more residues at the degenerate position is also possible. This could potentially destroy both protease cleavage sites.

[0151] In some embodiments, the protease-resistant fragment is located at positions 46 and 49 of SEQ ID NO: 1 , 35th, 63rd, 4th, 71st, 19th, 40th, 41st, 60th, 62nd, 29th At the corresponding position, or at the corresponding amino acids of sequence numbers 3, 4, and 11-24, the amino acids Includes substitution or modification.

[0152] In some embodiments, protease-resistant fragments suitable for the present invention may be further modified. It may contain. For example, 20% or more of the residues of SEQ ID NOs: 1, 3, 4, or 11-24. You may change up to (for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%). %, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , by changing or altering 19% or more of the residues (Also possible). Therefore, protease-resistant fragments suitable for the present invention are SEQ ID NOs: 1, 3, 4, or 11-24 and at least 80% (at least 80, 81, 82, 83, 84, 85, 86) 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% They may have matching amino acid sequences, for example.

[0153] As used herein, the term "cysteine ​​protease cleavage site" ("cysteine") The "cysteine ​​protease cleavage site" or "cysteine ​​protease cleavage sequence" is , Peptide functions as a recognition sequence for enzyme protease cleavage by cysteine ​​protease This refers to the amino acid sequence of a protein or protein. In some embodiments, cysteine ​​cleavage is used. The part is QILH|SWGE or HSWG|EECR in sequence number 1, or sequence number 3 It may contain 4, or 11-24 corresponding amino acids (in the sequence, cleavage) (The body part is indicated by "|").

[0154] As used herein, the term "metalloproteinase cleavage site" ("metalloproteinase") is used in this specification. The "cleavage site" or "metalloproteinase cleavage sequence" is formed by metalloproteinases. The amino acid sequence of a peptide or protein that functions as a recognition sequence for enzyme protease cleavage. Refers to the column. In some embodiments, the metalloproteinase cleavage site is the sequence number 1. DLRK|MRVA, DYVN|LIQE, MPAM|KICE, QICE|LKYE, LAPK|YAAT, EKTD|YVNL, or RVAE|LKQI, or sequence number It may contain 3, 4, or corresponding amino acids 11-24 (in the sequence, The point of cross-section is indicated by "|".

[0155] As used herein, the term "serine protease cleavage site" ("serine protease") is used in this specification. The "cleavage site" or "serine protease cleavage sequence" is formed by serine protease The amino acid sequence of a peptide or protein that functions as a recognition sequence for enzyme protease cleavage. Refers to a column. In some embodiments, the serine cleavage site is VAEL|K of SEQ ID NO: 1 QIL, LDLA|SVDL, or TDYV|NLIQ, or sequence numbers 3, 4, or It may contain 11-24 corresponding amino acids (in the sequence, the cleavage site is "| (Indicated by ").

[0156] In one embodiment, the protease is a cysteine ​​protease, a metalloprotease Selected from the group consisting of, and serine proteases.

[0157] In one embodiment, the cysteine ​​protease is cathepsin K.

[0158] In one embodiment, the metalloproteinase is MMP-9 or MMP-3.

[0159] In one embodiment, the serine protease is chymotrypsin A or elastase-2. That is the case.

[0160] For example, residues 11-70 of sequence number 2 (for example, residues 11-18, 18-25, 21- 28, 24-31, 33-40, 52-59, 54-61, 59-66, or 63-70 Any one amino acid in the region corresponding to ), or the corresponding amino acid of SEQ ID NO: 5 or 6 The ano acid can be substituted, deleted, or modified with any other amino acid. For example, substitution at a position adjacent to the protease cleavage site is performed at the site of protease cleavage. This may affect recognition. One or more additional amino acids within each recognition site Substitution or insertion may destroy one or more protease cleavage sites. Degenerate position Deletion of one or more residues in the region can also disrupt both protease cleavage sites. It is possible.

[0161] In some embodiments, the protease-resistant fragment is located at positions 62 and 27 of SEQ ID NO: 2 , a position equivalent to 14th, 66th, 36th, 21st, 55th, 57th, or 24th place, The corresponding amino acid in SEQ ID NO: 5 or 6 contains an amino acid substitution.

[0162] In some embodiments, protease-resistant fragments suitable for the present invention may be further modified. It may contain, for example, the residue of SEQ ID NO: 2, or the corresponding residue of SEQ ID NO: 5 or 6. The amount of amino acids may be changed by up to 20% or more (for example, 1%, 2%, 3%, 4%). 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 1 (You may change or modify up to 6%, 17%, 18%, 19%, or more of the residues.) Therefore, a protease-resistant fragment suitable for the present invention is SEQ ID NO: 2, or SEQ ID NO: 5 or 6 corresponding amino acids and at least 80% (at least 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 9 They may have matching amino acid sequences (e.g., 8, 99%).

[0163] As used herein, the term "metalloproteinase cleavage site" ("metalloproteinase") is used in this specification. The "cleavage site" or "metalloproteinase cleavage sequence" is formed by metalloproteinases. The amino acid sequence of a peptide or protein that functions as a recognition sequence for enzyme protease cleavage. Refers to the column. In some embodiments, the metalloproteinase cleavage site is the sequence number 2. KINE|LMPK, KINE|LMPK, STVD|LKKL, QICE|LKYD, EKSD|YIRK, or LMPK|YAPK, or the corresponding A in sequence number 5 or 6. It may contain amino acids (in the sequence, the cleavage site is indicated by "|").

[0164] As used herein, the term "serine protease cleavage site" ("serine protease") is used in this specification. The "cleavage site" or "serine protease cleavage sequence" is formed by serine protease The amino acid sequence of a peptide or protein that functions as a recognition sequence for enzyme protease cleavage. Refers to a column. In some embodiments, the serine cleavage site is VKEL|K in SEQ ID NO: 2 KIL, DKQI|DLST, SDYI|RKIN, or IDLS|TVDL, It may contain the corresponding amino acid of SEQ ID NO: 5 or 6 (in the sequence, the cleavage site is " (Indicated by |)

[0165] In one embodiment, the protease is a metalloprotease and a serineprotease. Selected from the following group.

[0166] In one embodiment, the metalloproteinase is MMP-9 or MMP-2.

[0167] In one embodiment, the serine protease is chymotrypsin A or elastase-2. That is the case.

[0168] In embodiments of the present invention, the lengths of the fragments are 33-81, 43-81, 43-61, and It is in the range of 50-81 amino acids. Preferably, the length of the fragment is 55-75, 55-7 It is in the range of 0, 55-61, 61-65, or 61-70 amino acids. More preferably, The lengths of the fragments are 49-61, 50-61, 51-61, 53-61, 57-61, 55- 69, 55-68, 55-67, 55-66, 56-69, 56-68, 56-67, 5 6-61, 57-69, 57-68, 57-67, 57-61, 58-69, 58-68 , 58-67, 58-61, 59-69, 59-68, 59-67, 59-61, 60- 69, 60-68, 60-67, 60-66, 60-64, 60-63, 61-62, 6 It is in the range of amino acids 1-63, 61-64, 61-65, 61-66, or 61-67. For example, a preferred fragment is at least 33, 34, 35, 36, 37, 38, 39, 4 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, A compound consisting of 67, 68, 69, 70, 71, 72, 73, 74, or 75 amino acids. This can be done. In one embodiment, the C-terminal CDNF fragment is 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 It consists of the amino acids. In one embodiment, the C-terminal MANF fragment is 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 4 9, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61 It consists of the amino acids. The fragments are alanine, arginine, asparagine, aspartic acid, Cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine Lysine, methionine, phenylalanine, proline, serine, threonine, tryptose Not only any of the naturally occurring amino acids such as phan, tyrosine, and valine, but also non-adjuncts. It may contain original or modified amino acids. Preferably, the fragment is human CDNF or M The sequence of the C-terminal domain of the ANF protein and at least 100%, 99%, 98%, 9 7%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% It has homology or sequence identity. More preferably, the fragment is human CDNF or MANF. It has at least 80% homology or sequence identity with the C-terminal domain of the protein. As used herein, “homology” means that at least a fragment of a reference sequence and another sequence are identical. This refers to sequence similarity between sequences. As described below, BLAST defines percent identical. The sequences are compared based on sex and similarity.

[0169] The term "identical" or percentage "identity" is used in the context of two or more amino acid sequences. This refers to two or more identical arrays or subarrays. One of the following array comparison algorithms is used. By using or by manual alignment and visual inspection, the comparison window or specified When comparing and sorting for the greatest match across a region, if the two sequences have a certain percentage of matching If the amino acid residues are the same as the area (i.e., across a specific region, or in a specific area) If not found, 29% identity is applied across the entire sequence, and 30%, 40%, and 45% as needed. 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% (99%, or 100% identity), the two sequences are "substantially identical." Therefore, identity extends over a region that is at least about 10 amino acids long, or more preferably or over a region that is 10, 15, 20, 25, 30, or more amino acids long It exists.

[0170] In sequence comparison, typically one sequence acts as the reference sequence and is compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are entered into the computer. If necessary, specify the coordinates of the sub-arrays and the array algorithm program parameters. Specify the default program parameters or specify alternative parameters. This is possible. Next, the array comparison algorithm is performed based on the program parameters. Calculate the percentage sequence identity of the test sequence to the reference sequence. When making comparisons, the sequences do not need to be contiguous, but if there are gaps, the overall percentage... This will likely come with a penalty that reduces identity.

[0171] As used herein, the "comparison window" optimally aligns the two arrays and then... A column can be compared to an array of references with the same number of consecutive positions, any one of the number of consecutive positions. Includes references to segments. The sorting method for the arrays for comparison is ClustalW or F ASTA and other such technologies are well-known in the relevant technical field.

[0172] Two examples of algorithms suitable for determining percent sequence identity and sequence similarity. There are the BLAST and BLAST2.0 algorithms, respectively, as described by Altschul et al. (1997) Nucleic Acids Res 25(17):3389-3402 and Altschul et al. (1990) J. Mol Bio It is described in l 215(3)-403-410. Regarding the amino acid sequence, the BLASTP program By default, the word length is 3, and the expected value (E) is 10, and BLOSUM62 is... Coring matrix (Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA 89) (See 22):10915-10919), alignment of 50 (B), expected value of 10 (E), M=5, N=-4 and a comparison of both chains are used. For short amino acid sequences, PAM30 score is used. A ng matrix can be applied.

[0173] Furthermore, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (for example) See Karlin and Altschul, (1993) Proc Natl Acad Sci USA 90(12):5873-5877. One measure of similarity provided by the BLAST algorithm is between two amino acid sequences. This is the minimum sum probability (P(N)), which provides an indicator of the probability that the agreement may occur by chance.

[0174] Preferably, the sequence of sequence number 1 and at least 90%, 91%, 92%, 93%, 94% C-terminal CDNFs that are 95%, 96%, 97%, 98%, or 99% homologous are SEQ ID NOs. The sequence CXXC (where X is any amino acid) is present at positions 52-55 of 1. , the sequence of sequence number 1 and at least 90%, 91%, 92%, 93%, 94%, 95%, 9 The above sequences that are 6%, 97%, 98%, or 99% homologous are sequence number 3: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECXXCAEKTD YVNLIQELAP KYA ATHPKTE L (In the formula, X is any amino acid) It consists of at least 50 consecutive amino acid residues in the sequence.

[0175] In another preferred embodiment, the sequence of sequence number 1 and at least 90%, 91%, 92% The above sequences are homologous by %, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. This sequence contains the CKGC sequence at positions 52-55 of sequence number 1.

[0176] In the most preferred embodiment, the above sequence is: Sequence ID 4: KYEKTLDLAS VDLRKMRVAE LKQILHSWGE ECRACAE KTD YVNLIQELAP KYAATHPKTE L The sequence of, or the sequence of sequence number 4, and at least 90%, 91%, 92%, 93%, 94%, The sequences are 95%, 96%, 97%, 98%, or 99% homologous.

[0177] In one embodiment, the C-terminal CDNF fragment has its native C-terminal amino acid, namely ER It does not contain residual signal. Therefore, in a preferred embodiment, the fragment is 7 of SEQ ID NO: 1 It lacks the ER retention signal KTEL, which corresponds to ranks 8th to 81st.

[0178] Furthermore, the present invention relates to a fragment that is bound to a detectable chemical or biochemical portion such as a FITC label. This indicates that it is possible to detect a chemical or biochemical "Part" refers to the amino acid sequence or a detectable chemical or for the purpose of facilitating peptide detection. This refers to tags that display a biochemical component, such as visible, fluorescent, chemiluminescent, or other fluorescent materials. A pigment that can be extracted; an enzyme that can be detected in the presence of a substrate, e.g., NBT+BCIP and alkaline hydroxylase Sphatase, or peroxidase with a suitable substrate, etc.; detectable proteins, e.g. Examples of detectable molecules selected from green fluorescent protein, etc. Preferably, the tag is It does not prevent or interfere with the transmission of fragments to target cells.

[0179] Furthermore, a C-terminal CDNF fragment or C-terminal fragment to enhance the stability and / or cell permeability of the fragment. N-terminal and / or C-terminal modifications of terminal MANF fragments are also preferred. CDNF fragments or MANF fragments The acetylation-amidation of the end of the compound (i.e., N-terminal acetylation and C-terminal amidation) This is one of the options known in the art (see, for example, Non-Patent Document 22).

[0180] Both the C-terminal CDNF fragment and the C-terminal MANF fragment kill dopamine neurons. In order to provide strong protection (see Figures 4 and 5), prior art such as Patent Documents 1 and 5, Fragments of Alzheimer's disease, Parkinson's disease (PD), multiple system atrophy, and amyotrophic lateral sclerosis (ALS) ALS, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, Huntington's disease (HD), used in the treatment of traumatic brain injury, drug addiction, and central nervous system (CNS) disorders such as stroke. This demonstrates that it can be used. Further results supporting the present invention are shown in the PD model C -Figure 8 shows the effect of CDNF, and Figure 1 shows the effect of C-CDNF in the ALS model. Provided in 1 and 12. In addition, the short MANF peptide (MANF4) is Parkinson's In a rat 6-OHDA model of disease, tests were conducted in a more clinically oriented setting for nerve repair. It is also important to note that if added after 6-OHDA, it is ineffective (see Figure 9). ).

[0181] The effects of C-terminal CDNF or C-terminal MANF fragments in the CNS are on neurons. In addition, other CNS cells such as microglia, astrocytes, and neural stem cells or neural progenitor cells. Cell type, and other characteristics they possess besides survival, such as migration, proliferation, differentiation, and maturation. This includes targeting ripe or mature plants.

[0182] The results shown in Figure 10 indicate that the C-terminal MANF fragment is effective in treating type 1 and type 2 diabetes. This supports the point. Furthermore, Patent Document 6 states that CDNF and MANF are also effective in retinal damage. It is disclosed that it is active. Therefore, the present invention relates to the aforementioned central nervous system (CNS) diseases, glucose Regarding the treatment of urinary tract diseases and retinal disorders. Also, ER stress-induced apoptotic cell death is related to disease. It also contributes to other degenerative diseases in which the function or structure of the affected tissue or organ gradually deteriorates over time. (See Non-Patent Document 23). Some further examples of such degenerative diseases include age-related macular degeneration. Stargardt disease, glaucoma, retinitis pigmentosa, and optic nerve degeneration; Niemann-Pick disease; A Therotropic arteriosclerosis; progressive supranuclear palsy; cancer; Tay-Sachs disease; keratoconus; inflammatory bowel disease IBD; prostatitis; osteoarthritis; osteoporosis; and rheumatoid arthritis, as well as traumatic brain injury. More acute conditions such as injury or ischemia-reperfusion injury, for example, myocardial ischemia, renal ischemia, or This is a stroke. Therefore, the present invention also relates to the treatment of degenerative diseases or disorders.

[0183] In the treatment method, a pharmaceutically effective amount of the C-terminal fragment is administered to the patient. In other words, the present invention The relevant fragments include Alzheimer's disease, Parkinson's disease (PD), and non-motor symptoms of PD (constipation, urinary tract infection). (Severe heart disease and hallucinations, etc.), multiple system atrophy, amyotrophic lateral sclerosis, ischemic stroke, peripheral neuropathy Frontotemporal dementia, Lewy body dementia, mild cognitive impairment, Huntington's disease, epilepsy, Traumatic brain injury, peripheral nerve injury, hemorrhagic stroke, or addiction (e.g., cocaine, morphine, Central nervous system (CNS) disorders and other neurological disorders (such as amphetamine or alcohol abuse) For use in the treatment of harm, and degenerative diseases or disorders including type 1 and type 2 diabetes or retinal disorders. It is for the treatment of Parkinson's disease or amyotrophic lateral sclerosis. It is intended for medical use.

[0184] The actual dose (e.g., effective dose) of the C-terminal fragment of CDNF or MANF administered to the patient. This includes weight, severity of condition, type of disease being treated, previous or simultaneous therapeutic interventions, and patient characteristics. The administration method can be determined by the underlying disease and physical and physiological factors such as the route of administration. The physician responsible for this shall determine the concentration of the active ingredient(s) in the composition and its effects on each individual subject. It is possible to determine an appropriate dosage(s).

[0185] In one embodiment of the present invention, a C-terminal CDNF fragment or a MANF fragment is incorporated into a pharmaceutical composition. Such compositions of the present invention include peptides having a desired purity and, Physiologically acceptable carriers (such as nanocarriers), excipients, or stabilizers of choice (Remington's A mixture of Pharmaceutical Sciences, 22nd edition, Allen, Loyd V., Jr, Ed., (2012)) By doing so, it is prepared for storage in the form of a freeze-dried cake or aqueous solution. Acceptable carriers Excipients or stabilizers are harmless to the recipient at the dose and concentration used, for example, Buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid; low Polypeptides with a molecular weight (less than approximately 10 residues); serum albumin, gelatin, or immunoglobulin Proteins such as phosphorus; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamine , amino acids such as asparagine, arginine, or lysine; glucose, mannose, or Monosaccharides, disaccharides, and other carbohydrates including dextrin; chelating agents such as EDTA; mannitol Sugar alcohols such as sorbitol or other sugar alcohols; salt-forming counterions such as sodium; and / or tungsten. Tween, Pluronic, or polyethylene glyco Examples include nonionic surfactants such as PEG.

[0186] Furthermore, the fragments were prepared, for example, by coacervation technology or interfacial polymerization. Capsules (for example, hydroxymethylcellulose or gelatin-microcapsules, respectively) Colloidal drug delivery system in cells and poly-(methyl methacrylate) microcapsules (For example, liposomes, albumin spheres, microemulsions, nanoparticles, and nano It may be encapsulated in a capsule or in a macroemulsion. Such a technique is used by Remi This information is disclosed in ngton's Pharmaceutical Sciences (see above).

[0187] In one embodiment, the pharmaceutical composition contains, for example, at least about 0.1% of the active compound In other embodiments, the active compound may be, for example, about 2 units of the unit's weight. Including a percentage of approximately 75%, or approximately 25% to 60%, and any range that can be derived from there. It is possible.

[0188] In other non-limiting examples, the dose of a pharmaceutical composition or preparation is approximately 1 ng per dose unit. From a C-terminal CDNF or C-terminal MANF fragment at a concentration of / kg / body weight, approximately 5 ng / kg / body weight can be obtained. , about 10ng / kg / body weight, about 50ng / kg / body weight, about 100ng / kg / body weight, about 2 00ng / kg / body weight, approx. 350ng / kg / body weight, approx. 500ng / kg / body weight, 1μg / kg / body weight, approx. 5μg / kg / body weight, approx. 10μg / kg / body weight, approx. 50μg / kg / body weight Weight, approximately 100μg / kg / body weight, approximately 200μg / kg / body weight, approximately 350μg / kg / body weight Approximately 500 μg / kg / body weight, approximately 1 milligram / kg / body weight, approximately 5 milligrams / kg / body weight Weight, approximately 10 mg / kg / body weight, approximately 50 mg / kg / body weight, approximately 100 mg mg / kg / body weight, approximately 200 mg / kg / body weight, approximately 350 mg / kg / body weight, From approximately 500 mg / kg / body weight to approximately 1000 mg / kg / body weight, C-terminal CDNF cleavage Including fragments or C-terminal MANF fragments or more, and any range that can be derived therefrom. It is possible. In non-limiting examples that can be derived from the numbers listed herein, From approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, from approximately 5 μg / kg / body weight to approximately 50 C-terminal CDNF fragments or C-terminal MANF fragments, etc., in the range of 0 milligrams / kg / body weight, It can be administered based on the numbers indicated.

[0189] Furthermore, the present invention is characterized by a pharmaceutical composition that may further contain nerve cells. These can be, for example, neurons, neural stem cells, or neural progenitor cells.

[0190] In another embodiment, the pharmaceutical composition is a nucleus encoding the C-terminal fragment defined above. Recombinant vector containing the ocid sequence, nucleoci encoding the C-terminal fragment defined above. A recombinant viral vector containing the C-terminal sequence, or a host expressing the C-terminal fragment defined above. The viral vector contains a therapeutically effective amount of cells. The viral vector preferably has a C-terminus as defined above. Adenoviruses, adeno-associated viruses, and lentigines containing polynucleotides that code for fragments. A group consisting of retroviruses such as thiviruses, herpesviruses, and papillomaviruses. Selected from. Typically, recombinant vectors and recombinant viral vectors are used in Tissue-specific methods for directing the expression of the polynucleotides of the present invention in various in vitro and in vivo systems. Alternatively, it may contain expression regulatory sequences such as cell-type-specific promoters. Furthermore, the vector may contain multiple These may also be hybrid vectors containing the regulatory elements necessary for expression in the system. Vectors containing such regulatory systems are commercially available, and those skilled in the art will know the C-terminal fragment as defined herein. It will be possible to easily clone it into such a vector. Selection of a suitable recombinant viral vector, nucleic acid sequence for expressing the C-terminal fragment in the vector Methods for inserting a viral vector into a cell and methods for delivering a viral vector to a target cell are relevant to the technical field. This falls within the scope of the technology. See, for example, Non-Patent Document 24.

[0191] The routes of administration include known methods, as well as intravenous or peripheral administration, intraperitoneal, subcutaneous, and subarachnoid administration. Intravenous, intranasal, transdermal, intracerebral, intramuscular, intraocular, intraarterial, or intralesional means, or the following sustained release methods. The system follows the general route of injection or infusion. The C-terminal fragment or the pharmaceutical composition containing the fragment It can be administered continuously by injection or bolus injection. Generally, the impairment allows In such cases, the fragments should be formulated and administered for site-specific delivery. Administration should be continuous or periodic. This can be done by using an embedded pump with a constant flow rate or a programmable flow rate. This can be done by regular injections. The present invention relates to the C-terminal MANF fragment and CDNF fragment. Does this indicate that both can permeate the nerve cell membrane and the blood-brain barrier? Therefore, peripheral or systemic administration is preferred (see Figures 6 and 7). Other preferred routes of administration include dermatological administration. The administration methods are intra-abaradermal, intraventricular, intranasal, or transdermal. Figure 13 shows the results of stroke induction. This study demonstrates the effects of subcutaneous injection of C-CDNF protein in rats.

[0192] A suitable example of a sustained-release formulation is a semipermeable matrix of a solid hydrophobic polymer containing fragments. This matrix can be used to create molded products, such as films or microcapsules. This is the state. Examples of sustained-release matrices include polyester, Langer et al., J. Biomed . Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982) Hydrogels, or polyvinyl alcohol, polylactide (Patent Documents 7, 8) Examples include ), or non-degradable ethylene vinyl acetate (see Langer et al. above).

[0193] Gene therapy vectors use the corresponding administration methods defined above for peptide fragments. Preferably, for example, by intravenous injection, or intraperitoneal, subcutaneous, subarachnoid, or It can be delivered to the target via intracerebroventricular administration. Gene therapy vector pharmaceutical formulations are permitted It may contain a tolerable diluent, or a sustained-release matrix with an embedded gene delivery vehicle. It can include a pocket.

[0194] The sequence alignment in Figure 23 shows a high degree of sequence identity between the C-terminal CDNF and MANF peptides. This demonstrates the properties. Therefore, based on the results shown herein, the inventors have determined that the C-terminal MANF section In one case, amino acid sequence motifs are important for cell membrane permeability and protective effects on nerve cells. However, amino acid residues at approximately positions 33 and 68 of SEQ ID NO: Inter-base, and positions approximately 19 and 52 of sequence number 2, which correspond to positions 25-57 of sequence number 1. It is presumed to be located between anoacid residues. Therefore, the present invention relates to Sequence ID No. 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described above, positions 33-68 or 19-52, or positions 33-68 of sequence number 2 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 19-52. At least containing or consisting of consecutive amino acid residues, and sequences adjacent to said consecutive amino acid residues It is preferred that the sequence at the corresponding position of sequence number 2 has at least 80% homology or sequence identity. It is a cell-permeable peptide and has a protective effect on nerve cells, preferably This concerns C-terminal MANF fragments with a length of 36-78 amino acids.

[0195] In a preferred embodiment, the present invention relates to Sequence ID No. 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL At least 50 sequences of the sequence described above, or sequences that are at least 90% homologous to sequence number 2. It consists of a series of amino acid residues and can be administered intravenously or peripherally, intraperitoneally, subcutaneously, intranasally, percutaneously, or intramuscularly. Degenerative diseases, including central nervous system (CNS) diseases, administered via intraocular, intraocular, or intra-arterial administration. Or relating to a C-terminal MANF fragment for use in the treatment of disorders.

[0196] Based on the results shown in Figure 10, the present invention further relates to Sequence ID No. 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL At least 5 sequences described in the sequence, or sequences that are at least 90% homologous to sequence number 2. Consists of 0 consecutive amino acid residues, C-terminal MA for use in the treatment of type 1 or type 2 diabetes. Regarding NF fragments.

[0197] For all of the above embodiments, the sequence of Sequence ID No. 2 is at least 90% homologous to the above The sequence contains the sequence CXXC (where X is any amino acid) at positions 47-50 of SEQ ID NO: 2. It is preferable.

[0198] More preferably, the sequence which is at least 90% homologous to the sequence of sequence number 2 is sequence number No. 6: QICELKYDKQ IDLSTVDLKK LRVKELKKIL DDWGETC XXC AEKSDYIRKI NELMPKYAPK AASARTDL (In the formula, X is any amino acid) It consists of at least 50 consecutive amino acid residues in the sequence.

[0199] Most preferably, the above MANF fragment is sequence number 5: KYDKQ IDLSTVDLKK LRVKELKKIL DDWGETCKGC A EKSDYIRKI NELMPKYAPK AASARTDL It has a sequence that is at least 90% homologous to the sequence of sequence number 5.

[0200] In one embodiment, the C-terminal MANF fragment has its native C-terminal amino acid, namely ER It does not contain residual signal. Therefore, in a preferred embodiment, the fragment is 7 of SEQ ID NO: 2 It lacks the ER retention signal RTDL, which corresponds to positions 5th to 78th.

[0201] The C-terminal MANF fragment is modified in the same way as the C-terminal CDNF fragment described above. It is possible.

[0202] Furthermore, the present invention relates to a C-terminal MANF fragment and a physiologically acceptable carrier, buffer, and excipient. A central nervous system (CNS) disease, type 1 or type 2, comprising, and at least one of the following: This relates to a pharmaceutical composition for use in the treatment of type 1 diabetes or retinal disorders. (C-terminal MANF segmentation) The above pharmaceutical composition containing the flakes is preferably administered peripherally to the patient, and therefore preferably by the Suitable for peripheral administration.

[0203] Furthermore, this specification refers to a pharmaceutically effective amount of a C-terminal CDNF fragment or C-terminal as defined herein. End-MANF fragments are administered to patients with central nervous system (CNS) disorders, type 1 or type 2 diabetes. Or the present invention relates to a method for treating degenerative diseases or disorders, including retinal disorders. Preferably, the fragment is peripheral It is administered to the target.

[0204] Furthermore, this specification does not apply to central nervous system (CNS) diseases, type 1 or type 2 diabetes, or retinal disorders. C-terminal C as defined herein for the manufacture of pharmaceuticals for the treatment of degenerative diseases or disorders including Regarding the use of DNF fragments or C-terminal MANF fragments.

[0205] Furthermore, the present invention relates to Sequence ID No. 4: KYEKTLDLAS VDLRKMRVAE LKQILHSWGE ECRACAE KTD YVNLIQELAP KYAATHPKTE L C-CDNF having the sequence of sequence , or a sequence that is at least 90% homologous to the sequence of sequence number 4. This provides an isolated polynucleotide containing a nucleotide sequence encoding a fragment.

[0206] Furthermore, the present invention relates to an expression vector encoding the above-mentioned isolated polynucleotide and the vector We provide host cells transformed with the above-mentioned isolated polynucleotides. Select a recombinant vector and insert a nucleic acid sequence into the vector to express the C-CDNF fragment. Methods for introducing and delivering recombinant vectors to target cells are part of the art in this field. It is within the range. See, for example, Tuschl, T. (2002), Nat. Biotechnol, 20: 446-448.

[0207] In order to clarify the background of the present invention, and in particular to provide additional details regarding its implementation, The publications and other materials used herein are incorporated herein by reference. The invention will be further described in the following embodiments, which are not intended to limit the scope of the invention. [Examples]

[0208] Tests using superior cervical ganglion cells For culturing sympathetic nerve neurons (Non-Patent Document 15, Non-Patent Document 25, Non-Patent Document 26) (Non-patent documents 27, 28), the superior cervical ganglion of mice 0-3 days postnatally (P) Lagenase (2.5 mg / mL, Worthington), Dispase (5 mg / mL, Roche) Molecular Biochemicals, and trypsin (10 mg / mL, Worthington) Digestion is performed at 37°C for 45 minutes, and then mechanically using a silicon-coated glass Pasteur pipette. It dissociated. Extensive pre-plating removed non-neuronal cells. Almost pure nuclei 35mm plastic coated with polyornithine / laminin (Sigma) In a diced dish, neurobasal medium and B27 supplement (in vitro) Small standard microislands in (Gibco) containing 30 ng / mL of mouse neurogenic factor The cells were cultured for 5-6 days in the presence of promega (NGF). Thorough washing and functional inhibition of N NGF was removed by adding a GF antibody (Roche). Neurons were treated with a special neuronal matrix. Microinjection device (Non-patent document 15, Non-patent document 25, Non-patent document 26, Non-patent document Pressure microinjection was performed using (Non-Patent Document 27, Non-Patent Document 29) Non-patent document 30). For survival analysis, all neurons on the microisland were experimentally... The count was recorded at the start (initial number) and at the end (3 days), and expressed as a percentage of the initial count.

[0209] Microinjection of sympathetic neurons was performed as described above (Non-patent document) 29). The plasmid for CDNF was mentioned earlier. To put it simply, it is a plasmid for neonatal mouse SCG After growing the ureon in NGF (Promega) for 5-6 days, the full length (FL) - CDNF and The expression plasmid for C-CDNF is used in the repository for highly sensitive green fluorescent protein (EGFP). Along with the vector plasmid, a vector concentration of 10 ng / uL was used in each experiment to introduce a micro-micron into the nucleus. Loin injection was performed. Similar results were obtained at a plasmid concentration of 50 ng / uL. In protein microinjection, recombinant protein in 200 ng / uL PBS is fully absorbed. Identification of neurons that successfully received full-length (FL)-CDNF and C-CDNF proteins. Dextran Texas Red (molecular weight 70,000 Da) is a fluorescent reporter that facilitates this process. (Invitrogen, molecular probes) along with microinjectors directly into the cytoplasm An inspection was performed. The next day, tunicamycin (2 μM) was added, and three days later, the living fluorescent ni... The eurons were counted. Living fluorescence (expressing EGFP or dextran Texas) was used. Count neurons (including red) "blindly" after 3 days, Early living fluorescent neurons counted 2-3 hours after clo injection The results were expressed as a percentage (%). Experiments using plasmids were conducted using a single method for plasmid experiments. The process was repeated five times in a standing culture, while four independent protein injection experiments were performed. On average, In each experimental group, injection was successful in 50 to 80 neurons. The results are shown as mean ± SEM. The data from each experimental group were analyzed using one-way ANOVA and a post-hoc Dunnett t-test. , control plasmid PCR3.1 (vector) or PBS (for protein injection experiments) We compared it with (the previous case). The null hypothesis was rejected when p < 0.05.

[0210] CDNF expression plasmid Constructs encoding full-length (FL) or carboxy-terminal (C) domains are classified as TOPO / Using a TA cloning system (Invitrogen) or restriction endonucleases, It was inserted into the pCR3.1 vector (Invitrogen). Long CDNF consists of 537 bp (179 amino acids) and 561 bp (187 amino acids), respectively. It has the amino acid length of ) and possesses an N-terminal signal sequence for ER targeting. C- CDNF is 186 bp long and corresponds to amino acids 127-187 of FL-CDNF. ru.

[0211] E511: Human CDNF / bidirectional TOPO TA in pCR3.1. Includes stop codon. Full-length cDNA (untagged). Ampicillin selected. DH5α. Verified by sequencing. Done. E811: pCR3.1 hCDNF C-human CDNF C-terminal with signal sequence End sequence. Cloned by PCR and Invitrogen TA cloning system. Insertion Input size 207 bp. Transformed into DH5a cells. Ampicillin selection. Sequencing... Verified.

[0212] Plasmids that express proteins and peptide fragments Human recombinant CDNF (26-amino acid signal sequence and 161-amino acid mature CDNF) A full-length pre-CDNF consisting of 187 amino acids, which has an NF sequence, human N-CD NF (Human CDNF signaling sequence of 26 amino acids and amino acids 1-1 of mature CDNF) (consisting of 100 parts), and human C-CDNF (amino acids 101 to 161) A 26-amino acid CDNF signal sequence fused with the C-terminal domain of mature CDNF. (consists of)

[0213] Human recombinant MANF (21-amino acid signal sequence and 158-amino acid mature MA) A full-length pre-MANF (179 amino acids) having an NF sequence, human NM ANF ​​(21 amino acid human MANF signal sequence and mature MANF amino acids 1-1) (consisting of the part with acid 95), and human C-MANF (from amino acid 96 to amino acid 158) Is it a 21-amino acid CDNF signal sequence fused with the C-terminal domain of mature MANF? (Ranaru).

[0214] The codons optimize the cDNA synthesis of hMANF and hCDNF, and their domains We ordered the necessary components from GeneWith and constructed the respective pQMCF expression vectors. N-CDN F, C-CDNF, N-MANF, and C-MANF constructs have a histidine tag at the C-terminus. It possessed [the characteristic]. The cDNA was verified by sequencing with the final vector. hMA NF and hCDNF proteins were obtained using the CHOEBNALT85 suspension cell line derived from CHO. The cells were produced and cultured in a serum-free medium of known composition.

[0215] CHOEBNALT85 cells were transfected with 1 μg of expression plasmid. 48 hours after infection, 700 μg / mL of G418 was added to the cell population. Plasmids containing the protein were selected. Protein expression and secretion were transfected in 48 steps. After a certain period, the cell lysates and supernatant were analyzed under reducing conditions.

[0216] hMANF and hCDNF proteins are purified by two-step ion exchange chromatography. The samples were then gel-filtered into PBS (pH 7.4). CDNF antibody and MANF antibody (MANF 4E12-HRP and CDNF-7D6-HRP, Icosagen Tartu, Estonia Based on the results of SDS-PAGE and Western blot analysis using ), the protein used was The purity was over 99%.

[0217] The domains of CDNF and MANF were purified using a Ni affinity column, and the protein Quality, anti-His tag mouse monoclonal antibody (catalog number A00186, Jensk Analysis was performed using SDS-PAGE and Western blotting with LP(Lipto). The produced protein had the following sequence:

[0218] Mature human CDNF: QEAGGRPGADCEVCKEFLNRFYKSLIDRGVNFSLDTIEKE LISFCLDTKGKENRLCYYLGATKDAATKILSEVTRPMSVH MPAMKICEKLKKLDSQICELKYEKTLDLASVDLRKMRVAE LKQILHSWGEECRACAEKTDYVNLIQELAPKYAATHPKTE L (Sequence ID 7)

[0219] Human N-CDNF: QEAGGRPGADCEVCKEFLNRFYKSLIDRGVNFSLDTIEKE LISFCLDTKGKENRLCYYLGATKDAATKILSEVTRPMSVH MPAMKICEKLKKLDSQICEL (Sequence ID 8)

[0220] Human C-CDNF: KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTD YVNLIQELAPKYAATHPKTEL (Sequence ID 4)

[0221] Mature Human MANF: LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFC REARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEK ICEKLKKKDSQICELKYDKQIDLSTVDLKKLRVKELKKIL DDWGETCKGCAEKSDYIRKINELMPKYAPKAASARTDL Column number 9)

[0222] Human N-MANF: LRPGDCEVCISYLGRFYQDLKDRDVTFSPATIENELIKFC REARGKENRLCYYIGATDDAATKIINEVSKPLAHHIPVEK ICEKLKKKDSQICEL (Sequence ID 10)

[0223] Human C-MANF: KYDKQIDLSTVDLKKLRVKELKKILDDWGETCKGCAEKSD YIRKINELMPKYAPKAASARTDL(Sequence ID 5)

[0224] Tests using dopamine neurons To test dopamine neurons (Non-Patent Document 31, Non-Patent Document 32), 13.5 The ventral midbrain and midbrain base of NMRI mouse embryos were excised. The tissue was then treated with 0.5% trypsin. After incubation using ICN Biochemical, a large tip thermal processing pass tool Neurons were mechanically dissociated using a pipette. On a 96-well culture plate coated with ), N2 supplement (in vitro) Using DMEM / F12 medium (Invitrogen) containing GDNF (100ng In or out of the presence of CDNF, MANF, C-CDNF ( / mL), or in various concentrations of CDNF, MANF, C-CDNF They were grown for 5 days with C-MANF polypeptide. At the start of the experiment, the same amount of nucleotides were used. Ron was plated into each well. Cultures without added neurotrophic factors were negatively affected. A control group was used. Since the midbrain culture contains several neuronal species, the culture was fixed. Furthermore, tyrosine hydroxylase (T), a specific marker of dopaminergic neurons, Immunostaining was performed with an antibody against H) (Millipore). Images of each well were taken using CellInsight. t(trademark) scans and identifies immunopositive neurons with CellProfiler and Cell The data was counted using Profiler analysis software. Expressed as a percentage of ureon. All experiments were performed at least three times in independent cultures. The process was repeated. The results were expressed as mean SEM, and significance was examined using one-way ANOVA and Tukey ANOVA. The null hypothesis was tested by either a post-hoc test or a 2-two Student's t-test. The hypothesis was rejected when p ≤ 0.05.

[0225] Iodination of CDNF, C-CDNF, and C-MANF Using the lactoperoxidase method, CDNF, C-CDNF, and C-MANF were extracted. 1 25 The protein in question was iodinated with I-Na. Dissolve in pH 7.5) 125 I-Na(1mCi / 2.8μL, 1mCi = 37mBq) Mixed with GE Healthcare. 10 μL of 50 μg / mL lactoperoxidase and 0 The reaction was initiated by adding 0.05% H2O2. The mixture was left at room temperature for 20 minutes. The solution was curated and mixed with three volumes of 0.1M phosphate solution containing 0.1M NaI and 0.4M NaCl. After stopping the reaction by adding a buffer solution (pH 7.5), 25 μL of 2.5% BSA was added. Gel filtration on an ephadex G-25 column (PM10, GE Healthcare) was used to separate free iodine and iodinated proteins. For column equilibration and elution, 0.1 M phosphate buffer (pH 7.5) containing 1% BSA was used. In some cases, iodinated growth factors were concentrated using a YM-10 Centricon column (Millipore). Free iodine and iodinated proteins were separated by gel filtration on an ephadex G-25 column (PM10, GE Healthcare). For column equilibration and elution, 0.1 M phosphate buffer (pH 7.5) containing 1% BSA was used. In some cases, iodinated growth factors were concentrated using a YM-10 Centricon column (Millipore). The specific activities of I-labeled CDNF, C-CDNF, and C-MANF were measured with a Wizard3 1480 automatic 125 I-labeled gamma counter (PerkinElmer, Wallac) and were approximately 10 cpm 8 / μg protein. The labeled proteins were kept at 4°C and used within 3 weeks after labeling. The labeled proteins were kept at 4°C and used within 3 weeks after labeling. .

[0226] Internalization experiments in El3.5 dopaminergic neurons Mouse cultured El3.5 dopaminergic neurons grown on 24-well plates were incubated with iodinated CDNF or C-CDNF at 30,000 cpm per well at 37°C for 2 hours. The cells were transferred to ice and washed once with 0.5 mL of ice-cold medium. Next the cells were transferred to Eppendorf tubes and washed once with 0.2 M acetic acid, 0.5 M NaCl (p H2.8) at 4°C. After centrifugation at 1000 g for 10 minutes, the cells were dissolved in 0.5 mL of 0.5 N NaOH and counted with a gamma counter. .

[0227] Blood-brain barrier permeability test in rats 125 I-CDNF, 125 IC-CDNF, or 125 IC-MANF (all at 10 cpm in 10 μL per total protein) were subcutaneously injected into adult male Wistar rats. 6 . Two hours later, the animals were perfused with PBS. Radioactivity was detected in various brain regions using a gamma counter. The analysis was performed. The data is shown as mean ± SEM. After analyzing the differences between groups using ANOVA, Tuk We performed the ey-Kramer post-hoc test.

[0228] Internalization experiment of PC6.3 cells Rat PC6.3 pheochromocytoma cells were divided into 10% FCS and 5% FCS on 24-well plates. The cells were grown in a DMEM culture medium containing horse serum. The cells were washed with PBS and 30 per well. Incubate with iodized CDNF or C-CDNF at 37°C for 90 minutes. The cells were then placed on ice and washed once with 0.5 mL of ice-cold medium. Next, the cells were etched. Transfer to a Pendorf tube and wash once with 0.2M acetic acid and 0.5M NaCl (pH 2.8). The cells were purified. After centrifuging at 1000g for 10 minutes, the cells were treated with 0.5 mL of 0.5N NaOH. The samples were dissolved and counted using a Wallach gamma counter.

[0229] Nerve repair studies in a rat 6-OHDA model of Parkinson's disease (PD). In the PD nerve repair model, rats were damaged with 6-OHDA as described above (unpatented). References 4, Non-Patent Document 5, Non-Patent Document 33). In short, to give rats isoflu Under Lang's anesthesia, 3 × 2 μg of 6-OHDA was administered as a unilateral stereotactic injection (at a 10° angle) into the left striatum. (Coordinates relative to Bregma and dura mater: A / P +1.6; L / M -2.8; D / V -6) , A / P 0.0:L / M -4.1;D / V -5.5, and A / P -1.2;L / M -4.5; D / V -5.5). Two weeks later, the results of amphetamine-induced rotation (injury) Rats were divided into groups based on size. Subsequently, CDNF (10 μg), C-CDN F (equimolar to 10 μg of CDNF) and N-CDNF (equimolar to 10 μg of CDNF) The rats were injected intrastriatically using the same coordinate system as 6-OHDA. After dividing the rats into groups... In the reference experiment, an osmotic minipump was inserted subcutaneously, and a cannula was placed into the injured striatum. MANF4 (i.e., MANF peptide CKGC, see Patent Document 4) and GD are produced by the ni-pump. After delivering NF or vehicle solution to the striatum for two weeks, the minipump and cannula are removed. Within neurons, 6-OHDA has the following two synergistic effects: 1) They accumulate in the cytosol and form free radicals that cause oxidative stress. 2) It is a potent inhibitor of mitochondrial respiratory chain complexes I and IV. Sex neurons were treated with the NAT inhibitor desipramine (15 mg / kg, intraperitoneal administration, 6 -30 minutes before OHDA injection, protected. Unilateral injury size and therapeutic effect, CDNF, In experiments using C-CDNF, N-CDNF, and PBS-treated rats, the injury rate was 2. At 4, 6, and 8 weeks, the reference experiment targeting MANF4 and GDNF showed 1, 4, and 8 At 10 and 12 weeks, the behavior was measured using amphetamine-induced spinning behavior. Discharge (2.5 mg / kg, intraperitoneal administration) Complete (360°) ipsilateral and contralateral rotation The number was recorded for 120 minutes after a 30-minute acclimatization period. The results were measured using net ipsilateral rotation toward the injured side. The exclusion criteria were set to average (net rotational speed) ± 2 × STDEV.

[0230] Tyrosine hydroxylase (TH) - Immunohistochemistry Perfusion and tissue treatment. Immediately after nerve repair testing, an overdose of pentobarbital sodium ( Rats were anesthetized with 90 mg / kg intraperitoneal administration (Orion Pharma) and then given PBS. Next, 4% paraformaldehyde in 0.1M sodium phosphate buffer (pH 7.4) Intracardiac perfusion was performed. The brain was removed and post-fixed for 4 hours, and sodium phosphate containing 20% ​​sucrose was used. The samples were stored in Lime buffer at 4°C. Serial coronal frozen sections, 40 μm thick, were processed using a slide micrometer. The tissue was excised using a sieve. As described elsewhere (Non-Patent Document 4), immunohistochemistry was performed. The perfused brain was post-fixed overnight in paraformaldehyde at 4°C, and then in 20% sucrose. The brain was preserved in phosphorus. The brain was cut into six consecutive sections 40 μm thick. The floating sections were then treated with phosphorus. Wash with acid-buffered saline (PBS) and remove endogenous peroxidase activity with 0.3% aqueous peroxide. Quenching was performed with Sigma-Aldrich solution to block nonspecific antibody binding. Sections are placed in blocking buffer (4% bovine serum albumin and 0.1% triglycerides in 1×PBS). The sections were incubated at ton X-100 for 1 hour. The sections were then cooled at 4°C with loose blocking. Mouse monoclonal anti-tyrosine hydroxylase (TH) antibody in cirrhosis fluid (1:2.00 0, Catalog number MAB318, RRID: AB_2201528, Millipore, Billerica After incubation overnight in Massachusetts, biotinylated secondary antibody (1:200) Incubate in anti-rat or anti-mouse vector, Burlingame, California. The staining was enhanced with the avidin-biotin-enzyme complex (ABC kit, vector). The signal was visualized using 3',3'-diaminobenzidine as a chromogen.

[0231] TH-positive cell count from substantia nigra Regarding TH-positive cells in the substantia nigra pars compacta (SNpc), the ratio to bregma is approximately A / P - Analysis was performed on six sections spanning an SNpc range of 4.5 to -6.0. 3DHistechs From the image obtained with the scanner, Matlab(RRID:nlx_153890, MassW The cells were counted using the algorithm (Natic, Massachusetts). The scanner resolution was set to 0.24 μm / pixel with a ×20 NA 0.8 objective lens.

[0232] Absorbance analysis of TH-positive neurites in the striatum The absorbance of TH-positive neurites in the striatum is approximately A / P+ relative to the bregma of each rat. Measurements were taken on three striatal sections with values ​​of 2.2, +0.84, and -0.12. Background To reduce the signal, sections are scanned using an automated scanner (3DHistech, Budapest, Ha (Scanning service provided by the Institute of Biotechnology, University of Helsinki) The image was converted to 16-bit grayscale. The corpus callosum does not have TH signals. Therefore, this was used as a measure of nonspecific background staining. From the obtained images, the area The total density (integrated densities) per unit is used in ImageJ (NIH The data was analyzed using the following method. The data is expressed as a percentage (%) relative to the healthy side.

[0233] Beta cell proliferation assay Pancreatic islets were isolated from 8-week-old female virgin C57bl6Rcc mice. The islets were then placed in growth medium. Allow to recover overnight, then the next day, add the same number of pancreatic islets / well (70 / well) to placental lactogen (PL). Treated with 500 ng / mL, C-MANF, or MANF for 5 days. Half of the culture medium was used. The culture medium was changed daily with fresh medium containing growth factors. Edu( Click-iT (registered trademark) Edu proliferation kit (Invitrogen) for 4 pancreatic islet collections. It was added 8 hours prior. Pancreatic islets were destroyed with trypsin and placed on a glass slide of cell centrifuge. Centrifugation was performed. Cytospin and proliferating cells were examined using Click-iT AlexaFluor. After staining with ZID chromogenic reagent, fix and incubate overnight at 4°C with insulin staining (guinea pig 1:200). (Abcam, Cambridge, UK) was used to detect beta cells. The cells were washed and Alex a Fluor(registered trademark) 488 (1:400, Molecular Probes, LifeTech) The slides were stained with a secondary antibody conjugated to Nology (California, USA). This includes the API (Vector Laboratories, Burlingame, California, USA). Vectashield mounting medium Mounted with ium. 12 images (10x magnification) were mounted at 40x / Plan-Apoc. hromat / 0.95 Corr M27, and 63× / Plan-Apochrom Equipped with at / 1.40 Oil / M27 and 483 AxioCam HRm camera. Using a Zeiss AxioImager M2 482 reflected fluorescence microscope, AxioV Acquired using ision4 software, Image Pro Plus software (Analyzed by Media Cybernetics, Bethesda, Maryland, USA) The number of I-positive nuclei was quantified. The relative number of proliferating beta cells was quantified, and 3-5 replicates / treatment were performed. I compared it to El.

[0234] ALS mouse model Transgenic SOD1 G93A mice were used in this study to transfect ALS. Transgenic mouse models were used. Transgenic mice containing various human SOD1 mutations were used. S develops progressive neurodegeneration and motor neuron (MN) death, and is generally observed in preclinical studies. This provides an animal model that has been used and has greatly contributed to understanding the pathogenesis of FALS (non-patent document). Reference 34). Transgenic SOD1 mice exhibit an ALS-like disease transmitted in an autosomal dominant manner. These mice exhibit the following clinical characteristics. In these mice, hindlimb weakness and tremor-like movements are the initial symptoms. The symptoms appear around 10 weeks of age, followed by progressive motor paralysis and neurogenic muscle atrophy. (Non-patent document 35). These mice subsequently showed impairments in walking and eating, and for several weeks They usually die within 14-16 weeks of age. Human SOD with glycine 93 mutated to alanine. Transgenic mice possessing gene 1 were originally developed at The Jackson Laboratories. tory(http: / / www.jax.org, Bar Harbor, Maine, System B6) Obtained from SJL-TgN(SOD1-G93A)1Gur). Transgenic. Currently, DNA tail testing and PCR can detect specific oligonucleotides previously used by others. The analysis was performed using the specified conditions (see Jackson Lab's website). All experiments In this study, wild-type B6SJL-TgN(SOD1)2Gur was included as a control. .

[0235] ALS mouse experimental setup In single-dose experiments, mice approximately 13 weeks old were given PBS or C- under isoflurane anesthesia. CDNF (3.75 μg, which is equimolar to 10 μg of full-length CDNF diluted in PBS) A single injection was administered into the ventricles. Subsequently, the mice were evaluated twice a week for signs of disease and changes in body weight. The evaluation involved tests such as rotorods, designed to assess the motor activity of mice. This was completed through a series of behavioral tests, including the following:

[0236] In long-term injection experiments, 12-week-old SOD1 mice were given brain injections under isoflurane anesthesia. The nule (connected to an Alzet osmotic minipump via a catheter tube) is inserted into the right ventricle. It was inserted. C-CDNF (1.5 μg / 24h) was injected for 28 days. Motor behavior was slowed. The mice were evaluated using a tarot test. Clinical signs and weight changes in the mice were assessed.

[0237] Clinical scoring of ALS mice We performed clinical scoring of SOD1 mice using instructions from Jackson Laboratory. The mice were carefully examined twice a week after they reached 12 weeks of age. The animals were gently held at the base of their tails. The ability to lift, tremble, stiffness, and extend limbs was assessed and scored. Coring is a hindlimb nerve scoring system developed by ALSTDI (ALS Therapy Development Institute). It is a 1-5 scale based on the system.

[0238] Rotor rod In the Rotor Rod, the mouse was placed on a rotating rod (acceleration 4-40 rpm / min) (Ugo Basile, Italy). The cutoff time was set to 4 minutes. In the rotarod test, mice were 12 After they reached a certain age, we did it twice a week.

[0239] Distal occlusion of the middle cerebral artery as a model for stroke Male Sprague Dawley rat (weight 230-270g, Envigo) The experiment used animals from the Netherlands. This experiment complies with EU Directive 201 on the management and use of laboratory animals. Implemented in accordance with the 0 / 63 / EU 3R principles (local laws and regulations), Finnish National Museum Approved by the Experimental Committee. All experiments were conducted blindly, with rats being assigned to different treatment groups. Assigned to Ndam. Anesthetized rats with chloral hydrate (0.4 g / kg, administered intraperitoneally). I did. Cortical stroke, as described above (Non-Patent Document 36), involves the distal middle cerebral artery (dMCA). It was induced by occluding both common carotid arteries (CCA) for 60 minutes. Brief explanation Then, both CCAs were identified and isolated by ventral midline cervical incision. The rats were then placed in a stereotactic device. The procedure was performed by inserting the tube and performing a craniotomy in the right hemisphere. The right (MCA) was ligated with 10-0 sutures, and the bilateral common cervical artery was opened. The cardiopulmonary bypass (CCA) was ligated with a non-traumatic arterial clamp for 60 minutes. 60 minutes after ischemia, the area around the MCA was ligated. The surrounding sutures and arterial clips of the CCA were removed to induce reperfusion injury. After recovery, the rats were returned to their home cages. Their body temperature was maintained at 37°C during and after surgery.

[0240] To test the neuroprotective effects of subcutaneous C-CDNF, 50 μg of C-CDNF was administered to dM Phosphate was administered subcutaneously in a volume of 100 μL 30-50 minutes before CA occlusion and immediately after reperfusion. Buffered saline (PBS) was used as a vehicle control. Two days after dMCAo. The rats were euthanized using 2% 2,3,5-triphenyltetrazolium chloride (TTC, s Infarct volume was measured by staining (Guma-Aldrich, St. Louis, Missouri) in rats. The rat was decapitated, its brain was removed, and sliced ​​into 2.0 mm thick sections using an acrylic rat brain block. Rice was used. Brain slices were treated with 2% TTC solution (Sigma, St. Louis, Missouri, USA). After incubation at room temperature for 15 minutes, fixation is performed using a 4% paraformaldehyde solution. The slices were then transferred. The infarcted areas of each slice were scanned using a digital scanner and ImageJ software. The following measurements were taken: the infarct volume of each animal was measured using the average slice thickness (2 mm) of the rostral brain slices examined. The result was obtained by multiplying it by the total infarct area. Student's t-test was used for statistical analysis.

[0241] Determination of the shortest length of the active C-CDNF fragment C-CDNF peptides having deletions at the N-terminus and / or C-terminus (see Tables 5 and 6) , produced by conventional peptide synthesis (Stawikowski and Fields, Curr Protoc Protein) Sci.; 2002 February CHAPTER: Unit-18.1.). The above bioassay was performed on each peptide. The experiment was conducted using the following method. The results are shown in Tables 1-4 and Figures 14-18.

[0242] Table 1: Data from Figures 14(A) and 16(B). Data were obtained from plants grown in the presence of NGF. This represents the percentage of surviving neurons compared to other neurons. [Table 1A]

[0243] [Table 1B]

[0244] Table 2: Change in the ratio of surviving neurons compared to neurons injected with PBS (data from Table 1) (The amount of PBS was divided by the amount of t.) [Table 2]

[0245] Table 3: Data from Figures 15(A) and 17(B). The data were obtained in the presence of GDNF. This is the percentage of surviving neurons compared to the depleted neurons. [Table 3A-1]

[0246] [Table 3A-2]

[0247] [Table 3A-3]

[0248] [Table 3A-4]

[0249] [Table 3B-1]

[0250] [Table 3B-2]

[0251] [Table 3B-3]

[0252] [Table 3B-4]

[0253] [Table 3B-5]

[0254] Table 4: Multiplier change in surviving neurons compared to neurons grown without growth factors (Table 3) (The data was divided by the value for "no factor".) [Table 4-1]

[0255] [Table 4-2]

[0256] [Table 4-3]

[0257] [Table 4-4]

[0258] Table 5: Designed C-terminal CDNF peptides with disulfide bonds in the CRAC sequence. Peptide 1 (pep1;61aa): KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTD YVNLIQELAPKYAATHPKTEL (Sequence ID 4) Peptide 2 (pep2;57aa): TLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNL IQELAPKYAATHPKTEL (Sequence ID 11) Peptide 3 (pep3;53aa): KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTD YVNLIQELAPKYA (Sequence ID 12) Peptide 4 (pep4;49aa): TLDLASVDLRKMRVAELKQILHSWGEECRACAEKTDYVNL IQELAPKYA (Sequence ID 13) Peptide 5 (pep5;51aa): VDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAP KYAATHPKTEL (Sequence ID 14) Peptide 6 (pep6;43aa): VDLRKMRVAELKQILHSWGEECRACAEKTDYVNLIQELAP KYA (Sequence ID 15) Peptide 7 (pep7;50aa): KYEKTLDLASVDLRKMRVAELKQILHSWGEECRACAEKTD YVNLIQELAP (Sequence ID 16) Table 6: Designed C-terminal CDNF peptides 8-15. Peptides 9-11 were disrupted. It has a CRAC sequence (mutant amino acids are shown in bold). Peptides 12-14 are amino acids Includes substitutions (mutated amino acids are shown in bold).

[0259] [Table 5]

[0260] Table 7: Predicted cleavage sites for each protease in Sequence ID No. 1 [Table 6]

[0261] Table 8: Predicted cleavage sites for each protease in Sequence ID No. 2 [Table 7]

[0262] Prediction of protease cleavage sites Predicting the protease cleavage sites of C-terminal CDNF and C-terminal MANF fragments, https: This was done using the PROSPER software at / / prosper.erc.monash.edu.au / home.html The predicted protease cleavage sites are shown in Tables 7 and 8, and Figures 20 and 21.

[0263] To design protease-resistant C-terminal CDNF fragments and / or C-terminal MANF fragments In vitro protease assay A fragment library from C-terminal CDNF fragments and / or C-terminal MANF fragments is protected Based on information from the ase cleavage database, for example, https: / / www.ebi.ac.uk / merops Using the MEROPS database at / index.shtml, natural amino acids, for example, P4-P3- One or more natural amino acids at the P2-P1-P1' or P2-P1-P1' positions, Unused amino acids, amino acids known to inhibit proteases, or modifications It can be designed by substituting amino acids. A fragment library is, for example, 96U It may be synthesized in a 384-well format. For example, a fragment for a protease assay is , fluorescent dyes, for example, 7-amino-4-methylcoumarin or amino-4-trifluoromethyl A protea is a group of 4-14 amino acids (AMC or AFC) with a C-terminal amino acid attached to it. It may consist of a -se and assay form. The fragment design is P1' -P2'-P3'-P4'-P5'-P6'-AMC, P1'-P2'-P3'-P4' -P5'-AMC, P1'-P2'-P3'-P4'-AMC, P1'-P2'-P3' -AMC, P1'-P2'-AMC, or P6-P5-P in combination with P1'-AMC 4-P3-P2-P1, P5-P4-P3-P2-P1-, P4-P3-P2-P1, P 3-P2-P1- or P2-P1- may be used (the amino acid residue is outside the cleavage site). The sides are numbered sequentially. The cleavable bond is located between the P1 and P1' positions. Recombinant human MMP-2, MMP-3, MMP-9, and elastase-2 protein For example, purchase the components from R&D Systems. Follow the manufacturer's instructions to install them. Test and activate the rotease. Dissolve the synthetic fragment in the appropriate buffer to the appropriate concentration. To resolve. In a reaction mixture containing the fragment and protease in a protease-specific buffer. Then, the protease is reacted. The reaction is carried out at +37°C, and a certain amount of the reaction mixture is different. Samples are collected at a specific point in time, and protease cleavage activity is measured. The fragments are then subjected to, for example, MALDI-MS or The protease cleavage rate is measured by HPLC analysis.

[0264] In another in vitro protease assay, the above fragments are optionally labeled with chromogenic tags. The N-terminus or C-terminus is attached to a solid support, and then exposed to recombinant protease to obtain The fragments may be analyzed as described above.

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Arumae , M. Saarma and T.I. Heino, 2013. Characterization of the Structural and Functio nal Determinants of MANF / CDNF in Drosophila In Vivo Model. PLoS One 8(9),e73928. Marino, Giada, Ulrich Eckhard, and Christopher M. Overall, Protein Termini and T heir Modifications Revealed by Positional Proteomics. 2015, ACS Chem. Biol. 10:1 754-1764 Nadella R, Voutilainen MH, Saarma M, Gonzalez-Barrios JA, Leon-Chavez BA, Jimene z JM, Jimenez SH, Escobedo L, Martinez-Fong D. Transient transfection of human C DNF gene reduces the 6-hydroxydopamine-induced neuroinflammation in the rat subs tantia nigra. J. Neuroinflammation. 11: 209, 2014. Neves J, Zhu J, Sousa-Victor P, Konjikusic M, Riley R, Chew S, Qi Y, Jasper H, L amba DA, Immune modulation by MANF promotes tissue repair and regenerative succe ss in the retina. Science 2016 Jul 1;353(6294). Oakes and Papa, Annu. Rev. Pathol. Mech. Dis. 2015. 10:173-94. Parkash, V., P. Lindholm, J. Peranen, N. Kalkkinen, E. Oksanen, M. Saarma, V.M. 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Claims

1. Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA AHTPKTE L The sequence described above, positions 38-70 or 25-57, or positions 38-70 of sequence number 1 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 25 to 57. It contains or consists of at least consecutive amino acid residues, is a cell membrane-permeable peptide, and is nerve cell permeable. A C-terminal CDNF fragment for pharmaceutical use that has a protective effect on cells.

2. A fragment for use according to claim 1 in the treatment of a degenerative disease or disorder.

3. A fragment for use according to claim 2, wherein the degenerative disease is a neurodegenerative disease.

4. The aforementioned neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, multiple system atrophy, and muscular atrophy. Chlorotic sclerosis, frontotemporal lobar degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, Central nervous system (CNS) disorders selected from the group consisting of traumatic brain injury, drug intoxication, and stroke. A fragment for use according to claim 3.

5. A fragment for use according to claim 4, wherein the CNS disease is Parkinson's disease.

6. A fragment for use according to claim 2 in the treatment of type 1 or type 2 diabetes.

7. A fragment for use according to claim 3 in the treatment of amyotrophic lateral sclerosis.

8. A fragment for use according to claim 2 in the treatment of retinal disorders such as retinitis pigmentosa.

9. Intravenous administration, intraperitoneal, subcutaneous, subarachnoid, ventricular, nasal, percutaneous, intracerebral, intramuscular, ocular It is administered by intra-arterial or intra-arterial administration, or via a viral expression vector. A fragment for use according to any one of claims 1 to 8.

10. A fragment for use according to claim 9, wherein the intravenous administration is peripheral administration.

11. The sequence described in Sequence ID No. 1, positions 38-70 or 25-57, or sequence ID No. 3 It has at least 80% homology or sequence identity with the sequence at positions 8-70 or 25-57. It includes at least consecutive amino acid residues of the sequence, and if present, the consecutive amino acid residues Adjacent sequences have at least 80% homology or sequence with the sequence at the corresponding position of sequence number 1. Preferably having identity, preferably having a length of 33 to 81 amino acids, claims 1 to 1 A fragment for use as described in any one of item 0.

12. Positions 37-70, 36-70, 35-70, and 34-70 of the sequence described in Sequence ID No. 1 , ranks 33-70, 32-70, or 31-70, or numbers 37-70 of sequence number 1 1st place, 36th-70th place, 35th-70th place, 34th-70th place, 33rd-70th place, 32nd-70th place, or This is a sequence that has at least 80% homology or sequence identity with the sequence at positions 31 to 70. Both contain or consist of consecutive amino acid residues, and if present, adjacent to the consecutive amino acid residues The adjacent sequence has at least 80% homology or sequence similarity with the sequence at the corresponding position of sequence number 1. A fragment for use according to claim 11, preferably having oneness.

13. Positions 25-57, 25-58, 25-59, and 25-60 of the sequence described in Sequence ID No. 1 , 25th to 61st, 25th to 62nd, 25th to 63rd, 25th to 64th, 25th to 65th, 25th to 6th 6th place, 25th to 67th place, 25th to 68th place, 25th to 69th place, or 25th to 70th place, or arrangement Number 1: 25th-57th, 25th-58th, 25th-59th, 25th-60th, 25th-61st, 2 5th to 62nd, 25th to 63rd, 25th to 64th, 25th to 65th, 25th to 66th, 25th to 67th , with at least 80% homology to the sequences at positions 25-68, 25-69, or 25-70. It contains or consists of at least consecutive amino acid residues of a sequence having sex or sequence identity, If present, the sequence adjacent to the consecutive amino acid residues is the sequence at the corresponding position in Sequence ID 1. Preferably having at least 80% homology or sequence identity, the use according to claim 11 A fragment for use.

14. Positions 31-73 (peptide 6; sequence number 15) and 25-73 of the sequence described in Sequence ID No. 1 (Peptide 4; SEQ ID NO: 13), positions 21-73 (Peptide 3; SEQ ID NO: 12), positions 21-7 0th position (peptide 7; SEQ ID NO: 16), 31st to 81st positions (peptide 5; SEQ ID NO: 14), 25 ~81st place (Peptide 2; SEQ ID NO: 11), 25th to 57th place (Peptide 15; SEQ ID NO: 24) , or positions 37-73 (peptide 8; SEQ ID NO: 17), or positions 31-73 of SEQ ID NO: 1 25th to 73rd, 21st to 73rd, 21st to 70th, 31st to 81st, 25th to 81st, 25th to 73rd It has at least 80% homology or sequence identity with the sequence at position 57, or between positions 37 and 73. The sequence includes or consists of at least consecutive amino acid residues, and if present, the consecutive A The sequence adjacent to the mino acid residue is at least 80% in phase with the sequence at the corresponding position in SEQ ID NO:

1. A fragment for use according to claim 12 or 13, preferably having the same sex or sequence identity. 。

15. The sequence described in Sequence ID No. 1, positions 31-73 or 25-57, or sequence ID No. 3 It has at least 80% homology or sequence identity with the sequence at positions 1-73 or 25-57. The sequence includes or consists of at least consecutive amino acid residues, and if present, the consecutive The sequence adjacent to the amino acid residue is at least 80% of the sequence at the corresponding position in SEQ ID NO:

1. A fragment for use according to claim 14, preferably having homology or sequence identity.

16. A length of 43 to 81 amino acids, preferably a length of 43 to 61 amino acids, claim A fragment for use as described in any one of paragraphs 1 to 15.

17. Any of claims 1 to 16, comprising further modifications at the terminal to protect the fragment from enzymatic degradation. A fragment for use as described in item 1.

18. Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA AHTPKTE L The sequence described, or the sequence of Sequence ID No. 1, has at least 90% homology or sequence identity. A sequence consisting of at least 50 consecutive amino acid residues, any one of claims 1 to 17 Fragments for use as described in the section.

19. The sequence having at least 90% homology or sequence identity with the sequence of sequence number 1 is distributed Claim 1, wherein the sequence CXXC (wherein X is any amino acid) is located at positions 52 to 55 of column number 1. Fragments for use as described in 8.

20. The sequence having at least 90% homology or sequence identity with the sequence of sequence number 1 is distributed Column number 3: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECXXCAEKTD YVNLIQELAP KYA AHTPKTE L (X is any amino acid) For use according to claim 19, comprising at least 50 consecutive amino acid residues of the sequence A fragment of it.

21. The sequence having at least 90% homology or sequence identity with the sequence of sequence number 1 is distributed A fragment for use according to claim 19, comprising the sequence CKGC at positions 52-55 of column number 1.

22. The sequence number 1 contains the C-terminal amino acid L at position 81, as described in any one of claims 17 to 21. Fragments for use in publication.

23. Claim 17, lacking the ER residual signal KTEL corresponding to positions 78-81 of sequence number 1, A fragment for use as described in 18 or 20.

24. Claims 17-23 further include C-terminal amidation and N-terminal acetylation as modifications. A fragment for use as described in any one of the items.

25. Any one of claims 1 to 24, further bonded to a detectable chemical or biochemical portion Fragments for use as described in the section.

26. A cell-permeable peptide that can cross the human blood-brain barrier, claims 1 to 25 A fragment for use as described in any one of the items.

27. Resistant to protease cleavage, for use according to any one of claims 1 to 26 piece.

28. Extension, deletion, insertion, or replacement that destroys at least one protease cleavage site, A fragment for use according to claim 27, including or modifications.

29. The aforementioned proteases include cysteine ​​protease, metalloprotease, and serine protease. A fragment for use according to claim 27 or 28, selected from the group consisting of theases.

30. The cysteine ​​protease is cathepsin K, any one of claims 27 to 29. Fragments for use as described.

31. The metalloproteinase is MMP-9, as described in any one of claims 27 to 29. Fragments for use.

32. The metalloproteinase is MMP-3, as described in any one of claims 27 to 29. Fragments for use.

33. Claim 27, the serine protease is chymotrypsin A or elastase-2. A fragment for use as described in any one of paragraphs 29.

34. Residues 1-8, 16-23, 26-33, 32-39, 37-44, 38- of SEQ ID NO: 1 45, 43-50, 46-53, 57-64, 59-66, 60-67, or 68-75 Any one amino acid in the region corresponding to is substituted, deleted, or modified, according to the claim. A fragment for use as described in any one of paragraphs 27 to 33.

35. Sequence ID 1, positions 46, 49, 35, 63, 4, 71, 19, 40, 41 Any amino acid at the position corresponding to the 62nd, 69th, or 10th position is substituted or modified. A fragment for use according to any one of claims 27 to 34.

36. A C-terminal CDNF fragment according to any one of claims 1 and 11 to 35, and a physiologically permitted It comprises at least one of a carrier, buffer, excipient, preservative, and stabilizer. Pharmaceutical composition.

37. Intravenous administration, preferably peripheral administration, intraperitoneal, subcutaneous, subarachnoid, intraventricular, intranasal, transcatheter For administration via skin, brain, muscle, eye, or arterial channels, or for use with viral expression vectors. The pharmaceutical composition according to claim 36, administered via a mediator.

38. A pharmaceutical composition according to claim 36 or 37 for use as a pharmaceutical.

39. A pharmaceutical composition for use according to claim 38 in the treatment of a degenerative disease or disorder.

40. The aforementioned degenerative disease or disorder is Alzheimer's disease, Parkinson's disease, multiple system atrophy, muscular atrophy Lateral sclerosis, frontotemporal dementia, Lewy body dementia, mild cognitive impairment, Huntington's disease Central nervous system (CNS) conditions selected from the group consisting of traumatic brain injury, drug intoxication, and stroke. A pharmaceutical composition for use according to claim 39, which is a disease.

41. The pharmaceutical composition for use according to claim 40, wherein the CNS disease is Parkinson's disease. 。

42. The pharmaceutical combination for use according to claim 40, wherein the CNS disease is amyotrophic lateral sclerosis. A finished product.

43. The pharmaceutical composition according to claim 40 for use in the treatment of type I or type II diabetes.

44. A pharmaceutical composition according to claim 40 for use in the treatment of retinal disorders such as retinitis pigmentosa. 。

45. Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA AHTPKTE L The sequence described above, positions 38-70 or 25-57, or positions 38-70 of sequence number 1 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 25 to 57. It contains or consists of at least consecutive amino acid residues, is a cell membrane-permeable peptide, and is nerve cell permeable. A C-terminal CDNF fragment that has a protective effect on cells.

46. The sequence described in Sequence ID No. 1, positions 31-70 or 25-57, or sequence ID No. 3 It has at least 80% homology or sequence identity with the sequence at positions 1-70 or 25-57. The sequence includes at least consecutive amino acid residues, and the sequence adjacent to the consecutive amino acid residues is , having at least 80% homology or sequence identity with the sequence at the corresponding position of sequence number 1. Claims for lengths up to 57, 53, 51, 50, 49, 43, 37, or 33 amino acids Fragment described in 45.

47. Positions 37-70, 36-70, 35-70, and 34-70 of the sequence described in Sequence ID No. 1 , ranks 33-70, 32-70, or 31-70, or numbers 37-70 of sequence number 1 1st place, 36th-70th place, 35th-70th place, 34th-70th place, 33rd-70th place, 32nd-70th place, or This is a sequence that has at least 80% homology or sequence identity with the sequence at positions 31 to 70. The fragment according to claim 46, comprising or including consecutive amino acid residues.

48. Positions 25-57, 25-58, 25-59, and 25-60 of the sequence described in Sequence ID No. 1 , 25th to 61st, 25th to 62nd, 25th to 63rd, 25th to 64th, 25th to 65th, 25th to 6th 6th place, 25th to 67th place, 25th to 68th place, 25th to 69th place, or 25th to 70th place, or arrangement Number 1: 25th-57th, 25th-58th, 25th-59th, 25th-60th, 25th-61st, 2 5th to 62nd, 25th to 63rd, 25th to 64th, 25th to 65th, 25th to 66th, 25th to 67th , with at least 80% homology to the sequences at positions 25-68, 25-69, or 25-70. It contains or consists of at least consecutive amino acid residues of a sequence having sex or sequence identity, If present, the sequence adjacent to the consecutive amino acid residues is the sequence at the corresponding position in Sequence ID 1. Preferably having at least 80% homology or sequence identity, the cross-section according to claim 46. Piece.

49. Positions 31-73 (peptide 6) and 25-73 (peptide 4) of the sequence described in Sequence ID No. 1 , 21st to 73rd place (Peptide 3), 21st to 70th place (Peptide 7), 31st to 81st place (Peptide 5) Positions 25-81 (peptide 2), 25-57 (peptide 15), or 37-7 Position 3 (peptide 8), or positions 31-73, 25-73, and 21-73 of SEQ ID NO: 1, Distribution for ranks 21-70, 31-81, 25-81, 25-57, or 37-73 At least the continuous amino acid residue of a sequence having at least 80% homology or sequence identity with the column A fragment according to claim 47 or 48, comprising or consisting of a base.

50. The sequence from positions 31 to 73 of sequence number 1, or the sequence from positions 31 to 73 of sequence number 1 and a small portion of it. It contains at least 80% homology or sequence identity of sequences, including at least one consecutive amino acid residue. A fragment according to claim 49, comprising or.

51. Claims for lengths up to 57, 53, 51, 50, 49, 43, 37, or 33 amino acids A fragment as described in any one of items 45 to 50.

52. Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described above, positions 33-68 or 19-52, or positions 33-68 of sequence number 2 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 19 to 52. At least containing or consisting of consecutive amino acid residues, and sequences adjacent to said consecutive amino acid residues It is preferable that the sequence at the corresponding position of sequence number 2 has at least 80% homology or sequence identity. It is a cell membrane-permeable peptide, has a protective effect on nerve cells, and is preferably This is a C-terminal MANF fragment with a length of 36 to 78 amino acids.

53. A fragment according to claim 52, having a length of 36 to 59 amino acids.

54. A fragment according to claim 52 or 53 for use as a pharmaceutical.

55. Preferably administered intravenously, more preferably peripherally, intraperitoneally, subcutaneously, intranasally, percutaneously, or intramuscularly. , administered intraocularly or intra-arterially, for use in the treatment of degenerative diseases or disorders, A fragment according to claim 54.

56. Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described, or the sequence of Sequence ID No. 2, has at least 90% homology or sequence identity. The use according to claim 55, consisting of at least 50 consecutive amino acid residues of the sequence The C-terminal MANF fragment of the eye.

57. Sequence ID 5: KYDKQ IDLSTVDLKK LRVKELKKIL DDWGETCKGC A EKSDYIRKI NELMPKYAPK AASARTDL A sequence, or a sequence having at least 90% homology or sequence identity with the sequence of sequence number 5. A fragment for use according to claim 56, having the above characteristics.

58. The use according to claim 56 or 57, wherein the C-terminal amino acid L is located at position 78 of SEQ ID NO: 2 A fragment of the eye.

59. Claim 56 or This is a fragment for use as described in 57.

60. Preferably, an enzyme selected from the group consisting of C-terminal amidation and N-terminal acetylation. The claim according to any one of claims 52 to 59, further modifications to protect the fragment from the solution. Fragments for use.

61. For use according to any one of claims 52 to 60, exhibiting resistance to protease cleavage. A fragment of it.

62. Extension, deletion, insertion, or replacement that destroys at least one protease cleavage site, A fragment for use according to claim 61, including or modifications.

63. The aforementioned protease is selected from the group consisting of metalloproteases and serineproteases. A fragment for use according to claim 61 or 62.

64. The use according to claim 63, wherein the metalloproteinase is MMP-9 or MMP-2. A fragment for.

65. Claim 63, the serine protease is chymotrypsin A or elastase-2. Fragments for use as described.

66. Residues 11-18, 18-25, 21-28, 24-31, 33-40, 5 of SEQ ID NO: 2 Any one A within the region corresponding to 2-59, 54-61, 59-66, or 63-70 The amino acid is substituted, deleted, or modified according to any one of claims 52 to 65. Fragments for use.

67. The 62nd, 27th, 14th, 66th, 36th, 21st, 55th, and 57th positions of sequence number 2, also The amino acid at the position corresponding to the 24th position is substituted or modified, according to claims 52 to 66. A fragment for use as described in any one of the items.

68. Any of claims 52 to 67, further bonded to a detectable chemical or biochemical portion. A fragment for use as described in paragraph one. A fragment for use.

69. Claims 52-6, which are cell-permeable peptides that can cross the human blood-brain barrier. A fragment for use as described in any one of paragraphs 8.

70. The aforementioned neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, multiple system atrophy, and muscular atrophy. Chlorotic sclerosis, frontotemporal lobar degeneration, Lewy body dementia, mild cognitive impairment, Huntington's disease, Central nervous system (CNS) disorders selected from the group consisting of traumatic brain injury, drug intoxication, and stroke. A fragment for use according to any one of claims 52 to 69.

71. A C-terminal MANF fragment according to any one of claims 52 to 69, and a physiologically acceptable It comprises a carrier, a buffer, an excipient, a preservative, and a stabilizer, and the said The method of administration is intravenous, preferably peripheral, intraperitoneal, subcutaneous, nasal, percutaneous, intramuscular, intraocular, Alternatively, it is administered by intra-arterial injection, and is used for the treatment of central nervous system (CNS) diseases. A pharmaceutical composition.

72. Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described above, positions 33-68 or 19-52, or positions 33-68 of sequence number 2 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 19 to 52. At least containing or consisting of consecutive amino acid residues, and sequences adjacent to said consecutive amino acid residues It is preferable that the sequence at the corresponding position of sequence number 2 has at least 80% homology or sequence identity. It is a cell membrane-permeable peptide and is used in the treatment of type 1 or type 2 diabetes or retinal diseases. A C-terminal MANF fragment, preferably 36 to 78 amino acids in length, for use.

73. A fragment for use according to claim 72, having a length of 36 to 59 amino acids.

74. Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequence described, or the sequence of Sequence ID No. 2, has at least 90% homology or sequence identity. The use according to claim 73, comprising at least 50 consecutive amino acid residues of the sequence A fragment of the eye.

75. Sequence ID 5: KYDKQ IDLSTVDLKK LRVKELKKIL DDWGETCKGC A EKSDYIRKI NELMPKYAPK AASARTDL A sequence, or a sequence having at least 90% homology or sequence identity with the sequence of sequence number 5. A fragment for use according to claim 74, having the above characteristics.

76. Preferably, an enzyme selected from the group consisting of C-terminal amidation and N-terminal acetylation. The following is a further modification of any one of claims 72 to 75, including further modifications to protect the fragment from the solution. Fragments for use.

77. A fragment for use according to claim 76, which exhibits resistance to protease cleavage.

78. Extension, deletion, insertion, or replacement that destroys at least one protease cleavage site, A fragment for use according to claim 77, including or modifications.

79. The aforementioned protease is selected from the group consisting of metalloproteases and serineproteases. A fragment for use according to claim 77 or 78.

80. The use according to claim 79, wherein the metalloproteinase is MMP-9 or MMP-2. A fragment for.

81. Claim 79, the serine protease is chymotrypsin A or elastase-2. Fragments for use as described.

82. Residues 11-18, 18-25, 21-28, 24-31, 33-40, 5 of SEQ ID NO: 2 Any one A within the region corresponding to 2-59, 54-61, 59-66, or 63-70 The amino acid is substituted, deleted, or modified according to any one of claims 72 to 81. Fragments for use.

83. The 62nd, 27th, 14th, 66th, 36th, 21st, 55th, and 57th positions of sequence number 2, also The amino acid at the position corresponding to the 24th position is substituted or modified, according to claims 72 to 82. A fragment for use as described in any one of the items.

84. Claims 72-8, which are cell-permeable peptides that can cross the human blood-brain barrier. A fragment for use as described in any one of paragraphs 3.

85. A C-terminal MANF fragment according to any one of claims 72 to 84, and a physiologically acceptable It comprises at least one of a carrier, buffer, excipient, preservative, and stabilizer, and is of type 1 or This is a pharmaceutical composition for use in the treatment of type 2 diabetes or retinal disease.

86. A pharmaceutical composition for use according to claim 85, for peripheral administration.

87. Intravenous, intraperitoneal, subcutaneous, subarachnoid, intraventricular, nasal, percutaneous, intracerebral, intramuscular, intraocular, Alternatively, it may be administered by intra-arterial administration or via a viral expression vector, claim 86. A pharmaceutical composition for use as described above.

88. Positions 31-73 (peptide 6) and 25-73 (peptide 4) of the sequence described in Sequence ID No. 1 , 21st to 73rd place (Peptide 3), 21st to 70th place (Peptide 7), 31st to 81st place (Peptide 5) or positions 25-81 (peptide 2), positions 25-57 (peptide 15), or Positions 37-73 (peptide 8), or positions 31-73, 25-73, and 21- of SEQ ID NO:

1. 73rd, 21st-70th, 31st-81st, 25th-81st, 25th-57th, or 37th-7th A sequence of amino acid residues having at least 80% homology or sequence identity with the sequence at position 3. Isolated polynucleotides containing a nucleotide sequence encoding a C-terminal CDNF fragment consisting of Do.

89. An expression vector encoding an isolated polynucleotide as described in claim 88.

90. A host cell transformed with the vector described in claim 89.

91. Sequence ID 1: MPAMKICEKL KKLDSQICEL KYEKTLDLAS VDLRKMR VAE LKQILHSWGE ECRACAEKTD YVNLIQELAP KYA AHTPKTE L The sequence described above, positions 38-70 or 25-57, or positions 38-70 of sequence number 1 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 25 to 57. At the very least, administer an effective amount of a C-terminal CDNF fragment containing or consisting of consecutive amino acid residues. A method of treating a degenerative disease or disorder, including the following.

92. Sequence ID 2: ICEKLKKKDS QICELKYDKQ IDLSTVDLKK LRVKELK KIL DDWGETCKGC AEKSDYIRKI NELMPKYAPK AAS ARTDL The sequences described above, positions 33-68 or 19-52, or positions 38-70 of sequence number 2 Or, a small number of sequences that have at least 80% homology or sequence identity with the sequence at positions 19 to 52. Even if not present, it contains or consists of consecutive amino acid residues, and if present, the consecutive amino acid residues The adjacent sequence has at least 80% homology or correspondence with the sequence at the corresponding position of sequence number 2. A C-terminal MANF section preferably having sequence identity and a length of 36 to 78 amino acids. Treatment of degenerative diseases, type 1 or type 2 diabetes, or retinal diseases, including administering an effective dose of one of the tablets. How to do it.

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