WO2022054748A1 - Erythropoietin signaling inhibitor protein - Google Patents

Abstract

Provided is a drug that suppresses the growth of tumor cells by specifically targeting mutant JAK2 and inhibiting hyperactivated JAK2. The following mutant (a) or (b) of erythropoietin. (a) A mutant erythropoietin obtained by introducing a mutation into human erythropoietin comprising the amino acid sequence of SEQ ID NO: 1, said mutant comprising an amino acid sequence derived from the amino acid sequence of SEQ ID NO: 1 by substitution of the 103rd arginine by another amino acid, wherein the amino acid substituting for the 103rd arginine decreases or reverses the positive charge of arginine. (b) A mutant erythropoietin which comprises an amino acid sequence derived from the amino acid sequence of mutant (a) by deletion, substitution or addition of one to several amino acids other than the 103rd amino acid and which inhibits erythropoietin signaling.

Description

Erythropoietin signaling inhibitor protein

The present invention relates to an erythropoietin signaling inhibitor protein.

Erythropoietin (EPO) is a type of cytokine and is a hematopoietic factor that promotes the production of red blood cells. It is mainly produced in kidneys and tubular stromal cells, and acts on erythroblast progenitor cells in bone marrow to differentiate into erythrocytes. Erythropoietin binds to two extracellularly expressed erythropoietin receptors (EPORs) and promotes the phosphorylation of JAK2, which binds to the intracellular domain of the receptor, thereby transmitting cell proliferation signals downstream.

Polycythemia vera (PV) is mentioned as a disease related to this erythroid proliferation. PV is a myeloproliferative neoplasm (MPN), a cancer in which red blood cells in the blood grow abnormally. MPN is classified into chronic myelogenous leukemia (CML), polycythemia vera (PV), essential platelet disease (ET), primary myelofibrosis (PMF), etc., but 95% or more of PV, ET, Mutations in the JAK2 gene are found in about half of PMF (Non-Patent Document 1). JAK2 is a tyrosine kinase that transmits signals of erythropoietin and thrombopoietin downstream, and most of the mutations in the JAK2 gene have valine at position 617 replaced with phenylalanine (V617F) (Non-Patent Document 1). In PV patients, the JAK2 gene mutation activates the erythropoietin-independent and constitutive JAK2 signaling pathway mediated by the erythropoietin receptor, and the cells proliferate independently to develop polycythemia vera.

PV is treated by phlebotomy and administration of the antimetabolite hydroxyurea to normalize the increased blood cell count. The prognosis of PV is relatively good, and prevention of thrombosis is the main treatment. In recent years, JAK2 inhibitors have been launched on the market and have improved therapeutic effects such as alleviation of systemic symptoms such as reduction of splenomegaly, but have not improved the prognosis. In general, JAK2 inhibitors inhibit not only mutant but also normal JAK2, so it has been pointed out that side effects such as decreased platelet count and anemia and increased risk of infectious diseases associated with leukopenia.

An object of the present invention is to provide a drug that suppresses the growth of tumor cells by specifically targeting mutant JAK2 and inhibiting overactivated JAK2.

The present inventors attempted to create a mutant EPO that acts on the EPO receptor (EPOR) and inhibits downstream JAK2 signaling with structural transformation of the EPO receptor (Fig. 27). As a result, it was clarified that the following mutant EPO inhibits EPOR-mediated JAK2 signaling.
1) An EPO mutant that inhibits the EPO signal by converting the 103rd arginine (R) of erythropoietin (EPO) to aspartic acid (D) and glutamic acid (E) (R103D, R103E).
2) EPO mutants (R103E / T107E, R103E / R110E) in which the 107th threonine (T) and the 110th arginine were converted to glutamic acid in addition to R103E.

This mutant EPO gets stuck in the dimer EPOR and causes the conversion of the dimer structure of the EPOR. After that, JAK2 activity is inhibited and cell proliferation by JAK2 is inhibited by shifting the positional relationship between the dimer EPOR and the dimer JAK2 (JAK2V617F) that is complexed and activated in the cell by gene mutation. Therefore, it may be a therapeutic drug for polycythemia vera in which JAK2 gene mutation is observed in almost all cases, and EPOR is highly expressed and proliferated by the proliferation signal from wild-type EPO to JAK2. It is also expected to have a cell proliferation inhibitory effect on cancer cells of the wild-type EPO due to competition with wild-type EPO. The present invention has been completed based on these findings.

The gist of the present invention is as follows.
[1] The following erythropoietin mutant of (a) or (b).
(a) From the amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, which is an erythropoetin variant in which a mutation is introduced into human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. And another amino acid substituted with arginine at position 103 is the variant that reduces or reverses the positive charge of arginine.
(b) In the amino acid sequence of the variant of (a), the erythropoetin variant consisting of an amino acid sequence in which one or several amino acids other than the 103rd amino acid is deleted, substituted or added, and inhibits erythropoetin signaling [ 2] The erythropoetin variant according to [1], wherein the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid.
[3] Further, in the amino acid sequence of SEQ ID NO: 1, a mutation that enhances the vicinity of the 103rd arginine to a negative electrostatic potential and / or a positively charged amino acid on the site 2 surface of human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. The erythropoetin variant according to [1] or [2], wherein a mutation that makes a residue negatively charged has been introduced.
[4] The mutation that enhances the area around the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 to a negative electrostatic potential is selected from the group consisting of the 107th threonine and the 110th arginine in the amino acid sequence of SEQ ID NO: 1. The erythropoetin variant according to [3], wherein the substitution of at least one amino acid is carried out, and another amino acid substituted with the amino acid of the site concerned reduces or reverses the positive charge of the amino acid of the site concerned.
[5] The erythropoietin variant according to [4], wherein at least one amino acid selected from the group consisting of threonine at position 107 and arginine at position 110 in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid. ..
[6] The mutation that makes a positively charged amino acid residue on the site 2 surface of human erythropoietin consisting of the amino acid sequence of SEQ ID NO: 1 negatively charged is the substitution of the 110th arginine in the amino acid sequence of SEQ ID NO: 1 and 110. The erythropoetin variant according to [3], wherein another amino acid substituted with the second arginine reduces or reverses the positive charge of the 110th arginine.
[7] The erythropoietin variant according to [6], wherein the 110th arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid.
[8] Any of the following erythropoietin mutants.
(1) An erythropoietin variant consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid.
(2) An erythropoietin variant consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with glutamic acid.
(3) An erythropoetin variant consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 107th threonine is replaced with aspartic acid or glutamic acid.
(4) In the amino acid sequence of SEQ ID NO: 1, the 103rd arginine is replaced with aspartic acid or glutamic acid, the 107th threonine is replaced with aspartic acid or glutamic acid, and the 110th arginine is replaced with aspartic acid or glutamic acid. Erythropoetin variant consisting of the amino acid sequence
(5) An erythropoetin variant consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 110th arginine is replaced with aspartic acid or glutamic acid [9] [1]. ]-[8] A polynucleotide comprising a nucleotide sequence encoding the erythropoetin variant according to any one of [8] or a sequence complementary thereto.
[10] A vector containing the polynucleotide according to [9].
[11] A cell containing the vector according to [10].
[12] A method for producing the following erythropoietin variant (a) or (b), which comprises culturing the cells according to [11].
(a) From the amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, which is an erythropoetin variant in which a mutation is introduced into human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. And another amino acid substituted with arginine at position 103 is the variant that reduces or reverses the positive charge of arginine.
(b) In the amino acid sequence of the variant of (a), the erythropoetin variant consisting of an amino acid sequence in which one or several amino acids other than the 103rd amino acid is deleted, substituted or added, and inhibits erythropoetin signaling [ 13] At least one selected from the group consisting of the erythropoetin mutant according to any one of [1] to [8], the polynucleotide according to [9], the vector according to [10], and the cell according to [11]. A composition for inhibiting erythropoetin signaling, including.
[14] At least one selected from the group consisting of the erythropoietin mutant according to any one of [1] to [8], the polynucleotide according to [9], the vector according to [10], and the cell according to [11]. A composition for inhibiting JAK2 activity, including one.
[15] At least one selected from the group consisting of the erythropoietin mutant according to any one of [1] to [8], the polynucleotide according to [9], the vector according to [10], and the cell according to [11]. A composition for inhibiting cell proliferation by JAK2, which comprises one.
[16] At least one selected from the group consisting of the erythropoietin mutant according to any one of [1] to [8], the polynucleotide according to [9], the vector according to [10], and the cell according to [11]. A composition for preventing and / or treating a disease associated with a JAK2 mutation, which comprises one.
[17] The composition according to [16], wherein the disease associated with the JAK2 mutation is a tumor.
[18] The composition according to [17], wherein the tumor is a myeloproliferative neoplasm or solid tumor.
[19] The composition according to [18], wherein the myeloproliferative neoplasm is polycythemia vera, essential platelet disease or primary myelofibrosis.
[20] At least one selected from the group consisting of the erythropoietin mutant according to any one of [1] to [8], the polynucleotide according to [9], the vector according to [10], and the cell according to [11]. Pharmaceuticals, including one.

The erythropoietin mutant of the present invention can inhibit JAK2 activity and inhibit cell proliferation by JAK2.
This specification includes the contents described in the Japanese patent application, Japanese Patent Application No. 2020-152102, and / or the drawings which are the basis of the priority of the present application.

The JAK2 gene sequence (SEQ ID NO: 2) and PAM sequence around the mutation introduction site are shown. The PAM sequence is underlined and the site of mutation introduction is shown in bold. The ssODN sequence (SEQ ID NO: 3) is shown. Blue letters indicate synonymous substitutions, and red letters indicate mutation introduction sites. The detection of amino acid residues and distances present on the binding surface between EPO and EPOR at site2 is shown. Red <3.0, 3.0 ≤ Orange <4.5, 4.5 ≤ Yellow <6.0, 6.0 ≤ Green <7.5, 7.5 ≤ Cyan <9.0, 9.0 ≤ Blue [Å], EPO and EPOR are shown by Space-filling. The hydrogen bonds inferred between EPO and EPOR are shown. The amino acid residues inferred to form hydrogen bonds between EPO (light red) and EPOR (light blue) at Site 2 are shown by space-filling (left) and stick (center). The state of hydrogen bonding is shown in a schematic diagram (right). It shows the inferred hydrophobic interaction between EPO and EPOR. At Site 2, the amino acid residues that were inferred to form a hydrophobic interaction between EPO (light red) and EPOR (light blue) are shown by space-filling (left) and stick (center). The hydrophobic interaction is shown in a schematic diagram (right). The electrostatic potentials of EPO and EPOR at site2 are shown. Negative charges are shown in red and positive charges are shown in blue. The location of EPO mutation introduction (site2-A and site2-B) is shown. The surface shape of EPO (left) contains site2-A (green) and site2-B (orange) selected as the location of mutation introduction, and the surface shape of EPOR (right) contains amino acid residues that interact with each other. Indicated. The direction of EPO mutation introduction (site2-A and site2-B) is shown. The direction of mutation introduction for site2-A (left) and site2-B (right) is shown. The change in electrostatic potential of the surface structure of EPO by R103A and R103E is shown. Negative charges are shown in red and positive charges are shown in blue. Candidates for introducing mutations into site2-A are shown. The substituted residues are indicated by magenta, the neighboring EPOR residues are indicated by light blue, and the charge repulsion is indicated by the red dashed line. Candidates for introducing mutations into site2-B (mutations that cause gaps in hydrophobic pockets) are shown. The residue of site2-B is green, Phe93 of EPOR is cyan, the substituted amino acid residue is magenta, and the hydrophobic interaction is shown by the dotted gray line. Candidates for introducing mutations into site2-B (mutations that cause steric hindrance with EPOR) are shown. The residue of site2-B is shown in green, the Phe93 of EPOR is shown by cyan, the substituted amino acid residue is shown by magenta, and the steric clash is shown by the red dotted line. Candidates for introducing mutations into site2-B (mutations that disrupt the hydrophobic environment of the hydrophobic pocket of the EPO) are shown. The residue of site2-B is shown in green, the residue of EPOR Phe93 is shown in cyan, and the substituted amino acid residue is shown in magenta. The direction of EPO mutation introduction (site2-A and site2-B) is shown. The direction of mutation introduction for site2-A (left) and site2-B (right) is shown. Amino acid residues show an example of mutagenesis. The comparison of cell proliferation ability by mEPO1 ~ 10 (a.UT-7 / EPO, b. UT-7 / EPO, 0.03 U / mL in the presence of EPO, c. UT-7 / EPO / JAK2V617F) is shown. The electrostatic potentials of EPO and EPOR at site2 are shown. Negative charges are shown in red and positive charges are shown in blue. a: The electrostatic potential was mapped to the surface shapes of EPO and EPOR. Areas involved in the interaction are shown in circles. b: The electrostatic potential of site 2 of wild-type EPO (WT) and mutant EPO (R103D, R103E) is shown. In the lower row, the replaced residue is shown by magenta, the neighboring EPOR residue is shown by light blue, and the charge repulsion is shown by the red dashed line. The interaction between EPO and EPOR near site2-A is shown. A two-residue mutant EPO with a negatively charged amino acid residue introduced near R103 is shown. Negative charges are shown in red and positive charges are shown in blue. a: Mutation introduction position (top) and one-residue substitution R103E Electrostatic potential map of surface shape (bottom). b: Electrostatic potential map of the surface shape of the mutant EPO with two-residue substitutions. The peripheral structure of the mutant EPO is shown. a: Peripheral structure of S100E / R103E, b: Peripheral structure of R103E / T107E. The mutation of the two-residue substitution in which the positive charge of EPO site2 is replaced with the negative charge is shown. Negative charges are shown in red and positive charges are shown in blue. a: Mutation introduction position (top) and one-residue substitution R103E Electrostatic potential map of surface shape (bottom). b: Electrostatic potential map of the surface shape of the mutant EPO with two-residue substitutions. The peripheral structure of the mutant EPO is shown. a: Peripheral structure of R14E / R103E, b: Peripheral structure of K97E / R103E, c: Peripheral structure of R103E / R110E. The peripheral structure (R4E / R103E) of the mutant EPO is shown. a: Peripheral structure of R4E / R103E. R4 and R103 are shown by Ball & Stick. b: The side chain structures of R4 and E4 (R / E4) were merged. R4 is indicated by the blue side chain and E4 is indicated by the red side chain. The three-dimensional structure of EPO and the position of candidate for introduction of mutation in the amino acid sequence are shown. The comparison of cell proliferation ability by mEPO4.1 ~ 4.11 (a.UT-7 / EPO, b.UT-7 / EPO, 0.03 U / mL in the presence of EPO, c.UT-7 / EPO / JAK2V617F) is shown. The comparison of cell proliferation ability by mEPO 4.4.10 (a.UT-7 / EPO, b. UT-7 / EPO, 0.03 U / mL in the presence of EPO, c. UT-7 / EPO / JAK2V617F) is shown. We show EPOR-mediated inhibition of the JAK2-STAT5 signaling system by mEPO4, mEPO4.4, mEPO4.10, and mEPO4.4.10. It is a concept diagram of this invention.

Hereinafter, embodiments of the present invention will be described in more detail.

The present invention provides the following erythropoietin mutants (a) or (b).
(a) From the amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, which is an erythropoetin variant in which a mutation is introduced into human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. And another amino acid substituted with arginine at position 103 is the variant that reduces or reverses the positive charge of arginine.
(b) In the amino acid sequence of the variant of (a), the erythropoetin variant consists of an amino acid sequence in which one or several amino acids other than the 103rd amino acid are deleted, substituted or added, and inhibits erythropoetin signaling.

In the erythropoietin variant of the present invention, the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid that reduces or reverses the positive charge of arginine. An example of another amino acid substituted with arginine at position 103 is aspartic acid, glutamic acid.

In the erythropoetin variant of (b), one or several (for example, two or three) amino acids other than the 103rd amino acid were deleted, substituted or added in the amino acid sequence of the variant of (a). It consists of an amino acid sequence and can inhibit erythropoetin signaling. Inhibition of erythropoietin signaling by EPO mutants is that by adding EPO mutants to cells that proliferate in an EPO-dependent manner, EPO-dependent proliferation mediated by EPO receptors is suppressed, and JAK2 phosphorylation by EPO addition is suppressed. It can be confirmed by investigating that the subsequent phosphorylation of STAT5 and ERK1 / 2 is suppressed. Phosphorylation of STAT5 and ERK1 / 2 can be measured by immunodetection methods using phosphorylation-specific antibodies (Western blotting, immunoprecipitation, immunohistochemistry, ELISA, flow cytometry, etc.).

The erythropoetin variant of (b) is preferably one having at least 80% or more sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably 96% or more sequence identity, and more preferably 98% or more sequence. Have identity.

The erythropoietin mutant of (a) or (b) shall be produced by incorporating the gene encoding the EPO into which the mutation has been introduced into an appropriate vector, introducing it into an appropriate host cell, and producing it as a recombinant protein. Can be done.

In the erythropoetin variant of the present invention, a human erythropoetin site 2 consisting of a mutation in the amino acid sequence of SEQ ID NO: 1 that enhances the vicinity of the 103rd arginine to a negative electrostatic potential and / or the amino acid sequence of SEQ ID NO: 1 Mutations may be introduced that make the surface positively charged amino acid residues negatively charged.

An example of a mutation in the amino acid sequence of SEQ ID NO: 1 that enhances the area around arginine at position 103 to a negative electrostatic potential is selected from the group consisting of threonine at position 107 and arginine at position 110 in the amino acid sequence of SEQ ID NO: 1. It is a substitution of at least one amino acid, and another amino acid substituted with the amino acid at the site reduces or reverses the positive charge of the amino acid at the site.

Specifically, an erythropoetin variant in which at least one amino acid selected from the group consisting of threonine at position 107 and arginine at position 110 in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid is exemplified. be able to.

An example of a mutation that makes a positively charged amino acid residue on the surface of human erythropoetin site 2 consisting of the amino acid sequence of SEQ ID NO: 1 negatively charged is the 110th arginine substitution in the amino acid sequence of SEQ ID NO: 1. The amino acid substituted with arginine in No. 110 reduces or reverses the positive charge of arginine at position 110.

The site 2 of human erythropoietin is the surface of binding to the erythropoietin receptor present in human erythropoietin, and erythropoietin has two sites, site 1 having high affinity with the receptor and site 2 having low affinity with the receptor. Each of site1 and site2 interacts with one erythropoietin receptor to dimerize the receptor. Two JAK2s located in the cytoplasmic domain of the dimerized receptor approach and phosphorylate each other to activate and activate downstream signal transduction. (Syed RS, Reid SW, et al .: Efficiency of signaling through cytokine receptors depends critically on receptor orientation. Nature. 1998; 395 (6701): 511-516.)
Specifically, an erythropoietin variant in which the 110th arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid can be exemplified.

Preferred specific examples of the erythropoietin mutant of the present invention are listed below.
(1) An erythropoietin variant (mEPO3: R103D) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid.
(2) An erythropoietin variant (mEPO4: R103E) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with glutamic acid.
(3) An erythropoetin variant (mEPO 4.4:) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 107th threonine is replaced with aspartic acid or glutamic acid. R103E / T107E, R103D / T107D, R103D / T107E, mEPO4.3: R103E / T107D)
(4) In the amino acid sequence of SEQ ID NO: 1, the 103rd arginine is replaced with aspartic acid or glutamic acid, the 107th threonine is replaced with aspartic acid or glutamic acid, and the 110th arginine is replaced with aspartic acid or glutamic acid. Erythropoetin variants consisting of amino acid sequences (mEPO4.4.10: R103E / T107E / R110E, R103D / T107E / R110E, R103D / T107E / R110D, R103D / T107D / R110E, R103D / T107D / R110D, R103E / T107E / R110D, R103E / T107D / R110E, R103E / T107D / R110D)
(5) An erythropoetin variant (mEPO 4.10:) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 110th arginine is replaced with aspartic acid or glutamic acid. R103E / R110E, R103D / T110D, R103D / T110E, mEPO4.9: R103E / T110D)

The present invention provides a polynucleotide containing a nucleotide sequence encoding the above-mentioned erythropoietin variant or a sequence complementary thereto.

The polynucleotide of the present invention may be single-stranded or double-stranded. In the case of a double strand, it consists of a polynucleotide containing a nucleotide sequence encoding the above-mentioned erythropoietin variant and a complementary strand thereof.

The polynucleotide may be any of DNA, RNA, and a chimera of DNA and RNA, and the nucleotides constituting the polynucleotide may be modified.

The polynucleotide of the present invention can be produced by artificial gene synthesis or a site-specific mutagenesis method.

A recombinant vector is prepared by incorporating a polynucleotide containing a nucleotide sequence encoding the erythropoetin mutant of the present invention into a vector, and this is introduced into a host cell for transformation, and the transformed cell is cultured to obtain the present invention. Erythropoetin mutants can be produced. Accordingly, the present invention comprises culturing cells comprising a vector containing a polynucleotide comprising a nucleotide sequence encoding the erythropoietin variant of (a) or (b), the erythropoietin variant of (a) or (b). Provide a method for producing. The present invention also provides a vector (recombinant vector) containing a polynucleotide containing a nucleotide sequence encoding the erythropoietin variant of a) or (b). The present invention also provides cells comprising a vector containing a polynucleotide comprising a nucleotide sequence encoding the erythropoietin variant of (a) or (b).

The recombinant vector of the present invention can be prepared by inserting a polynucleotide containing a nucleotide sequence encoding the erythropoietin variant of (a) or (b) and a sequence complementary thereto into an appropriate vector.

As the vector, pcDNA3.1, pcDNA3.3, pcDNA.3.4, etc. can be used.

A promoter, enhancer, splicing signal, poly A addition signal, selectable marker, SV40 replication origin, or the like may be added to the expression vector.

Further, the expression vector may be a fusion protein expression vector. Various fusion protein expression vectors are commercially available, pcDNA3.1 + C-6His, pcDNA3.1 + C-HA, pcDNA3.1 + C-DYK, pcDNA3.1 + C-Myc, pcDNA3.1 + N- 6His, pcDNA3.1 + N-HA, pcDNA3.1 + N-DYK, pcDNA3.1 + N-Myc and the like can be exemplified.

Transformed cells can be obtained by introducing the recombinant vector of the present invention into a host cell.

Examples of the host include CHO cells, BHK cells, HT-1080 cells, NS0 cells, SP2 / 0 cells, HEK293 cells, and the like.

To introduce the recombinant vector into the host, Molecular Cloning 2nd Edition, J. Mol. Sambrook et al. , Cold Spring Harbor Lab. The method described in Press, 1989 (eg, calcium phosphate method, DEAE-dextran method, transfection method, microinjection method, lipofection method, electroporation method, transduction method, scrape loading method, shotgun method, etc.) or infection. Can be done by.

Transformed cells can be cultured in a medium, and the erythropoietin mutant of (a) or (b) can be collected from the culture. When the erythropoietin variant of (a) or (b) is secreted into the medium, the medium may be recovered, and the erythropoietin variant of (a) or (b) may be separated and purified from the medium. If the erythropoietin variant of (a) or (b) is produced in the transformed cell, the cell is lysed and the erythropoietin variant of (a) or (b) is isolated from the lysate. , Purify.

If the erythropoietin variant of (a) or (b) is expressed in the form of a fused erythropoietin variant with another protein (which functions as a tag), the factor Xa is after isolation and purification of the fused erythropoietin variant. Or enzyme (enterokinase) treatment can cleave another protein to obtain the desired erythropoietin mutant of (a) or (b).

Separation and purification of the erythropoietin mutant of (a) or (b) can be performed by a known method. As known separation and purification methods, differences in molecular weight such as methods using solubility such as salting out and solvent precipitation, dialysis method, ultrafiltration method, gel filtration method, and SDS-polyacrylamide gel electrophoresis can be used. A method of utilizing, a method of utilizing a charge difference such as ion exchange chromatography, a method of utilizing a specific affinity such as affinity chromatography, a method of utilizing a hydrophobic difference such as reverse-phase high-speed liquid chromatography, etc. A method that utilizes the difference in isoelectric points, such as electrophoretography, is used.

The erythropoetin variant of the present invention, the polynucleotide of the present invention, the vector of the present invention and the cell of the present invention can be used to inhibit erythropoetin signaling. Erythropoietin signaling involves activation of JAK2 (ie, erythropoietin binds to the extracellularly expressed erythropoietin receptor and promotes the phosphorylation of JAK2 bound to the intracellular domain of the receptor. The proliferation signal is transmitted downstream.) Can be exemplified. Therefore, the present invention comprises a polynucleotide containing the erythropoietin variant of (a) and / or (b), a nucleotide sequence encoding the erythropoietin variant or a sequence complementary thereto, a vector containing the polynucleotide, and the vector. Provided are a composition for inhibiting erythropoietin signaling, comprising at least one selected from the group consisting of containing cells. The present invention also comprises a polynucleotide containing the erythropoetin variant of (a) and / or (b), a nucleotide sequence encoding the erythropoetin variant or a sequence complementary thereto, a vector containing the polynucleotide, and the vector. Provided are a composition for inhibiting JAK2 activity, comprising at least one selected from the group consisting of containing cells. Further, the present invention comprises a polynucleotide containing the erythropoetin variant of (a) and / or (b), a nucleotide sequence encoding the erythropoetin variant or a sequence complementary thereto, a vector containing the polynucleotide and the vector. Provided is a composition for inhibiting cell proliferation by JAK2, which comprises at least one selected from the group consisting of containing cells. If the vector contains a polynucleotide comprising a nucleotide sequence encoding the erythropoetin variant of (a) and / or (b), the vector is a nucleotide sequence encoding the erythropoetin variant of (a) and / or (b). It is preferable that the polynucleotide containing the above can be introduced into cells, and examples thereof include gene therapy vectors such as adenovirus, retrovirus, lentivirus, adeno-associated virus, Sendai virus, liposome, and plasmid. Further, the polynucleotide or vector of the present invention introduced into cells (self, allogeneic) can also be used for cell therapy. A method for introducing a gene of interest into a vector, a method for introducing a recombinant vector into a cell, a method for administering a recombinant vector or a gene-introduced cell to a human, and an administration site are known, and can be used in the present invention as it is or as it is. It can be modified and applied. In gene therapy and cell therapy, genome editing technology may be used. In genome editing, artificial nucleases such as ZFN (zinc-finger nuclease), TALEN (transcription activator-like effector nuclease), and CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR-associated protein 9) can be used.

The composition of the present invention can be used for pharmaceuticals, experimental reagents, and the like.

Pharmaceuticals include prevention and prevention of diseases associated with JAK2 mutations, specifically tumors (eg, myeloproliferative neoplasms such as polycythemia vera, essential platelet disease, and primary myelofibrosis, and solid tumors). / Or can be used for treatment. EPO and EPOR are head and neck tumors, breast cancers, colon cancers, prostate cancers, ovarian cancers, uterine cancers and cervical cancer cells, neuroblastomas, stellate cell tumors and other solid nervous system tumors, as well as numerous. It is known to be expressed in malignant cell lines (Henke, M. et al. (2003) Lancet 362: 1255; Arcasoy, MO et al. (2003) Biochem; Biophys. Res. Communi. 307: 999. Yasuda, Y. et al. (2002) Cancerogenesis 23: 1797; Acs, G. et al. (2003) Am J. Physiol. 162: 1789; Batra, S. et al. (2003) Lab. Invest. 83 1477; Yasuda, Y. et al. (2003) Cancerogenesis 24: 1021), and it has also been reported that they function in these cells and tissues (Biophys. Res. Communi. 307: 999; Acs, G. et al. (2003) Am J. Physiol. 162: 1789; Batra, S. et al. (2003) Lab. Invest. 83: 1477; Yasuda, Y. et al. (2003) Carcinogenesis 24: 1021) Therefore, the EPO variants of the present invention may be effective in the prevention and / or treatment of these cancers.

Select from the group consisting of the erythropoetin variant of (a) and / or (b), a polynucleotide containing a nucleotide sequence encoding the variant or a sequence complementary thereto, a vector containing the polynucleotide, and a cell containing the vector. At least one (hereinafter referred to as "active ingredient") is used alone or in combination with a pharmaceutically acceptable carrier, diluent or excipient as a pharmaceutical composition of a suitable dosage form for mammals. It can be administered orally or parenterally to (eg, humans, mice, rats, rabbits, guinea pigs, dogs, cats, monkeys, chimpanzees, sheep, goats, pigs, cows). The dose varies depending on the subject, target disease, symptoms, administration route, etc., but for example, for the prevention and treatment of myeloproliferative neoplasms (for example, true erythrocytosis, essential thrombosis or primary myelofibrosis). When the active ingredient is a protein, the dose is usually about 10 μg to 200 mg / kg body weight, preferably about 0.1 mg to 100 mg / kg body weight, and when the active ingredient is a polynucleotide. , Usually about 10 ng to 100 mg / kg body weight, preferably about 0.1 mg to 20 mg / kg body weight, and when the active ingredient is a vector containing a polynucleotide, usually about 1x10 ¹⁰ to 1x10 ¹⁴ genome copy / kg body weight, preferably about 1x10 ¹¹ ~ 1x10 ¹³ Genome copy / kg body weight, daily ~ once every 10 years, preferably twice a week ~ once every 5 years, by oral, intramuscular injection, subcutaneous injection, intravenous injection It should be administered. When the active ingredient is a cell containing a vector, the dose of the active ingredient is usually about 1x10 ⁴ to 1x10 ¹² cells, preferably 1x10 ⁷ to 1x10 ⁸ cells, preferably about once every day to 10 years. It is recommended to administer by intramuscular injection, subcutaneous injection, intravenous injection, preferably intravenous injection, at a frequency of about twice a week to once every five years. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms. The same applies when used for the prevention / treatment of solid tumors.

Examples of the composition for oral administration include solid or liquid dosage forms, specifically tablets, pills, granules, powders, capsules, syrups, emulsions, suspensions and the like. Such a composition can be produced by a conventional method, and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. For example, examples of carriers and excipients for tablets include lactose, starch, sucrose, magnesium stearate and the like.

Examples of the composition for parenteral administration include injections, suppositories, etc., and the injections include intravenous injections (liquid or lyophilized preparations), subcutaneous injections, intradermal injections, intramuscular injections, etc. It is preferable to use a dosage form such as a drip injection. Such injections are prepared by conventional methods, that is, by dissolving, suspending or emulsifying the active ingredient in a sterile aqueous or oily solution normally used for injections. Aqueous solutions for injection include physiological saline, isotonic solutions containing glucose and other adjuvants, and suitable solubilizing agents such as alcohols (eg, ethanol), polyalcohols (eg, propylene glycol, etc.). It may be used in combination with polyethylene glycol), a nonionic surfactant (for example, polysorbate 80, HCO-50 (polyoxythetylene (50 mol) added-of-hydrogenated castor oil)) and the like. Examples of the oily liquid include sesame oil and soybean oil, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is usually filled in a suitable ampoule. Suppositories used for rectal administration can be prepared by mixing the active ingredient with a conventional suppository base.

The oral or parenteral pharmaceutical compositions described above may be prepared in dosage form in dosage form that is compatible with the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampols), suppositories, etc., and the content of the active ingredient in each dosage unit dosage form is such that the active ingredient is a protein. In the case, usually about 5 μg to 100 mg, when the active ingredient is a polynucleotide, usually about 0.1 mg to 1000 mg, when the active ingredient is a vector containing a polynucleotide, usually about 1x10 ¹⁰ to 1x10 ¹⁵ genome copy, the active ingredient is a vector. In the case of cells containing, it is usually good to have about 2x10 ⁵ to 3x10 ⁸ cells. These contents can be changed as appropriate.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.
[Example 1]
Method
1. 1. Preparation of UT-7 / EPO / JAK2V617F cells
1) Genome editing-Design of sgRNA and ssODN CRISPR direct, a gRNA design software, was used to design single guide RNA (sgRNA). First, off-target prediction was performed on the upstream 20 bases of the PAM sequence (NGG) existing around the 1849th guanine (G1849), which is the mutation introduction site of the JAK2 gene (Fig. 1), and four sgRNAs were designed (4 types of sgRNAs). table 1). Of these, three types of sgRNAs (# 1, # 3, # 4) were commissioned and synthesized, excluding # 2 sgRNA, which has a high probability of 3-base mismatch. Since the efficiency of genome editing increases when cleavage by Cas9 (3 bases upstream of the PAM sequence) occurs near the mutation introduction site, # 3 sgRNA was used for the genome editing experiment.

Single-stranded oligonucleic acid (ssODN) was used as the knock-in donor. G1849T, which corresponds to the introduction of the V617F mutation in JAK2, and 93-base-long ssODN (Fig. 2) containing synonymous substitutions of T1848C to prevent re-cleavage by Cas9 were synthesized by contract.

# 1: SEQ ID NO: 4
# 2: SEQ ID NO: 5
# 3: SEQ ID NO: 6
# 4: SEQ ID NO: 7

2) Preparation of homozygous mutants of UT-7 / EPO / JAK2V617F cells by genome editing
2) -1 Culture of UT-7 / EPO cells Culture UT-7 / EPO cells in IMDM / 10% FCS containing 1 unit / mL EPO under 37 ° C, 5% CO ₂ conditions, every 3-4 days. Was subcultured at a density of 1x10 ⁵ cells / mL.

2) -2 Introducing mutations into the JAK2 gene by genome editing A complex consisting of 1.5 μg, Cas9 protein, 0.3 μg sgRNA, and 0.5 μg donor oligonucleic acid was introduced into UT-7 / EPO cells of 2x10 ⁵ cells by electroporation. did. Electroporation was performed using the Neon Transfection System (Thermo Fisher) under the conditions of 1115 V, 30 ms, and 1 pulse. The cells were cultured in EPO-containing IMDM / 10% FCS, 24-well plate, 37 ° C., 5% CO ₂ conditions for 24 hours, and in EPO-free IMDM / 10% FCS for 20 days.

A cell population having a cell viability of 90% or more was seeded on 10 96-well plates so as to become a single cell by the limiting dilution method, and cloning was performed. Clone grown in EPO-free IMDM / 10% FCS was transferred to a 6-well plate and expanded-cultured.

Genomic DNA was extracted from cell pellet of 2x10 ⁵ cells, and the region containing the mutation introduction site was PCR-amplified. A 598 bp PCR product was purified and directly sequenced, and the mutation transfer efficiency was quantified by the ABC-PCR method for clones in which the mutation was introduced at the target site.

2) -3 Quantification of mutation introduction efficiency by ABC-PCR (Alternately Binding Probe Competitive PCR) Using a fluorescent probe, a standard curve for allelic Baden of JAK2V617F mutation was created, and the genomic DNA of the clone obtained above was obtained. Allele Baden was quantified.

The fluorescent probe sequence and PCR primer sequence are shown below.

Fluorescent probe (ABQP-JAKd-1):
TAMRA-cctgtagttttacttactctcgtctccacaga-BODYPY-FL (SEQ ID NO: 8)
PCR primer:
Forward primer (F-JAKd-1): 5'-atctatagtcatgctgaaagtaggagaaag-3'(SEQ ID NO: 9)
Reverse primer (R-JAKd-1): 5'-ctgaatagtcctacagtgttttcagtttca-3'(SEQ ID NO: 10)
(Reference) Baxter EJ et al., Lancet, 2005, 365, 1054-1061

The method is as follows. Genome sequence PCR products with known concentrations derived from wild-type JAK2 gene and mutant JAK2 gene with allervarden (mutant ratio) of 1%, 10%, 30%, 50%, 70%, 90% or 99 It was mixed so as to be%, and used as a template for a calibration curve. The amount of the mold was ¹⁰⁵ copies. Genomic DNA 100 ng was used as a clone template.

The composition of the reaction solution is as follows: template DNA 10 ⁵ copies / reaction or clone-derived genomic DNA 100 ng / reaction, 100 nM probe (ABQP-JAKd-1), 500 nM forward primer (F-JAKd-1), 150 nM reverse primer ( R-JAKd-1), 0.2mM dNTP mix, 1 X TITANIUM Taq PCR buffer (Takara Bio Inc.), 1 X TITANIUM Taq DNA polymerase (Takara Bio Inc.). The reaction temperature conditions were 94 ° C., 3 minutes (dissociation reaction), then 94 ° C., 30 seconds → 62 ° C., 30 seconds → 72 ° C., 30 seconds for 50 cycles, 72 ° C., 2 minutes (extension reaction). After the PCR reaction, the fluorescence intensities from BODYPY-FL and TAMRA were measured 5 times every 11 seconds at 95 ° C, 2 minutes, and 55 ° C, 2 minutes, and the fluorescence intensities at each temperature were calculated by the following formula. The expressed relative fluorescence intensity was calculated. The fluorescence intensity was measured using Thermal Cycler Dice Real Time System III (Takara Bio Inc.). The relative fluorescence intensity is when the fluorescence intensity of BODYPY-FL of each sample is Bs, the fluorescence intensity of TAMRA is Ts, the fluorescence intensity of BODYPY-FL of the negative control (without template) is Bp, and the fluorescence intensity of TAMRA is Tp. Calculated from (Bs-Bp) / (Tp-Ts).

A calibration curve of allerbaden was prepared from the relative fluorescence intensity obtained from the PCR product having a known concentration, and the allerbaden value was measured from the relative fluorescence intensity of the cloned genomic DNA.

2) -4 Single allele sequence A single allele sequence was performed on clones in which high mutagenesis into the JAK2 gene was observed by ABC-PCR. The PCR product containing the mutagenesis region was TA cloned into the pMD20-T vector, and plasmids were isolated from 24 E. coli colonies per clone. Sequence analysis was performed, and clones in which the G1849T mutation was introduced into all alleles of the JAK2 gene in UT-7 / EPO cells were designated as UT-7 / EPO / JAK2V617F cells.

2) -5 STAT5 phosphorylation signaling analysis of UT-7 / EPO / JAK2V617F cells UT-7 / EPO cells and UT-7 / EPO / JAK2V617F cells were seeded at 4x10 ⁵ cells / mL and 2 unit / mL. The cells were cultured in EPO-containing or EPO-free IMDM / 10% FCS for 3 days. To the cell pellet of 4x10 ⁶ cells, 200 μL of RIPA buffer was added and incubated at 4 ° C. for 20 minutes. The supernatant after centrifugation at 14000 rpm, 4 ° C. and 15 minutes was used as a lysate solution.

30 μg of heat-reduced lysate was subjected to SDS-PAGE under the conditions of 40 mA and 40 minutes. The polyacrylamide gel was transferred to the PVDF membrane under the conditions of 75 mA and 90 minutes. The PVDF membrane was immersed in TBST / 5% BSA solution and blocked at room temperature for 3 hours. The primary antibody (Table 2) reaction was carried out at 4 ° C. for 18 hours, and the cells were washed 3 times with TBST for 10 minutes. The secondary antibody reaction was carried out at room temperature for 1 hour, and the cells were washed 3 times with TBST for 10 minutes.

A chemiluminescent reagent was added to the PVDF film, reacted for 10 minutes, and then photographed with LAS-3000 (Fujifilm Co., Ltd.).

2. 2. Mutant EPO structure simulation Coordinate data (PDB ID: 1EER) (Syed et al. 1998) of the three-dimensional structure of the EPO and EPOR extracellular domain complex is available at Protein Data Bank (http://www.rcsb.org/pdb/). Obtained from. 1EER is composed of EPO (chain A) and EPOR extracellular domain dimer (chain B, C), and is determined by X-ray crystallography. The three-dimensional structure model of the EPOR mutant was created by the software Swiss-Pdbviewer (Guex et al. 2009) using 1EER chain A. Substitution of amino acid residues was performed in the state of the simple substance of the three-dimensional structure of EPO (chain A), and the direction of the side chain (rotorer) that was theoretically the easiest to take was selected. By superimposing the prepared EPO mutant model on the three-dimensional structure of 1EER, a complex model of the EPO mutant and EPOR was prepared. The software Waals (Altif Labs. Inc.) was used to create the complex model, detect neighboring residues, measure the interatomic distance, and display the three-dimensional structure. Hydrogen bonds were determined based on the general parameters of a minimum distance of 2.195 [Å] between atoms, a maximum distance of 3.300 [Å], and a minimum angle of 90.0 ° C. As a hydrophobic interaction, a pair of carbons in contact with each other with an interatomic distance of less than 3.500 [Å] was detected. The electrostatic potential to the surface shape was obtained from PDBj's eF-site (http://service.pdbj.org/eF-site/) and displayed in Waals and CueMol 2.0. The electrostatic potential of the mutant model was displayed on the Swiss-Pdb viewer.

3. 3. Expression of mutant EPO (mEPO1 ~ mEPO10)
1) Expression of mutant EPO A plasmid pcDNA3.4- (each mutant EPO) in which an artificially synthesized mutant EPO was introduced into the plasmid pcDNA3.4-TOPO was purchased. Mutant EPO was expressed using the ExpiCHO Expression System (Thermo Fisher Scientific Fix). ExpiCHO-S cells 1 vial (1 x 10 ⁷ cells) were melted in a water bath and transferred to ExpiCHO Expression Medium 25 mL, which had been preheated to 37 ° C. Shake culture was performed at 125 ± 5 rpm in an incubator at 37 ° C and 8% CO ² . The cells were cultured to 4 to 6 x 10 ⁶ cells / mL, and passage was repeated 2-3 times or more before transfection. The cell number was adjusted to 3-4 x 10 ⁶ cells / mL 1 day before transfection, and then shaken and cultured overnight. Cells were counted and adjusted to 125 mL culture flasks to 6 x 10 ⁶ cells / mL in 25 mL of ExpiCHO Expression Medium. 40 ug of pcDNA3.4- (mEPO) was diluted with 1 mL of OptiPRO medium and added to a mixture of 0.92 mL of OptiPRO medium and 80uL of ExpiFectamine CHO Reagent. After incubating this at room temperature for 5 minutes, it was added to a flask containing cells, and shaking culture was started. The next day, ExpiCHO ^TM Enhancer 150uL and ExpiCHO ^TM Feed 7.5 mL were added and cultured for 12 days. After culturing, centrifugation was performed at 3000 rpm for 10 minutes to obtain a culture supernatant. This culture supernatant was filtered through a 0.45 um filter, and the supernatant further filtered through a 0.22 um filter was used for activity measurement.

2) Confirmation of mutant EPO expression The expression of mEPO in the CHO cell culture supernatant was confirmed by Western blot. SDS-PAGE was performed on 500 ng of the heat-reduced CHO cell culture supernatant under the conditions of 40 mA and 40 minutes, and the gel was transferred to the PVDF membrane under the conditions of 75 mA and 90 minutes. The PVDF membrane was immersed in PBST / 5% BSA solution and blocked at room temperature for 2 hours. Rabbit anti-EPO antibody The primary antibody was diluted 1,000-fold with PBST / 5% BSA and allowed to stand at 4 ° C for 18 hours. The PVDF membrane was washed 3 times with PBST for 10 minutes. The HRP-labeled secondary antibody was diluted 10,000-fold with PBST / 5% BSA and soaked at room temperature for 1 hour. The PVDF membrane was washed 3 times with PBST for 10 minutes. The chemiluminescent reagent was added to the PVDF membrane and reacted for 10 minutes. Taken with LAS-3000 (Fuji Film Co., Ltd.).

In addition, the culture supernatant was treated with glycosidase F, a sugar chain-cleaving enzyme, at 37 ° C. for 20 hours, and western blot was performed by the above method.

4. Measurement of activity of mutant EPO (mEPO1 ~ mEPO10)
1) EPO activity of mutant EPO using UT-7 / EPO cells Adjust UT-7 / EPO cells to 4.0x10 ⁵ cells / mL with EPO-free IMDM / 10% FCS, and inoculate 50 μL each on a 96-well plate. did. 50 μL of the mutant EPO culture supernatant diluted 5-fold with EPO-free IMDM / 10% FCS was added to each well, and the cells were cultured for 48 hours under 37 ° C. and 5% CO ₂ conditions. Eventually, the dilution ratio of the mutant EPO will be 10 times.

10 μL of MTT reagent was added to each well and incubated at 37 ° C for 3 hours. Absorbance at a wavelength of 450 nm was measured with a microplate reader, and EPO-dependent cell proliferation activity due to mutant EPO was measured.

2) Competitive activity with wild-type EPO by mutant EPO using UT-7 / EPO cells Prepare UT-7 / EPO cells to 4.0x10 ⁵ cells / mL with EPO-free IMDM / 10% FCS and 96-well plate. Was sown in 50 μL increments. Add ₅₀ μL of mutant EPO culture supernatant diluted 5-fold with IMDM / 10% FCS containing 2 times the concentration of EPO (0.06 unit / mL) to each well under 37 ° C. and 5% CO ₂ conditions. Was cultured for 48 hours. Finally, the dilution ratio of the mutant EPO is 10 times, and the EPO concentration is EC ₅₀ = 0.03 unit / mL.

10 μL of MTT reagent was added to each well and incubated at 37 ° C. for 3 hours. Absorbance at a wavelength of 450 nm was measured with a microplate reader, and EPO-dependent cell proliferation activity due to mutant EPO was measured.
3) Growth inhibitory activity by mutant EPO using PV disease model cells UT-7 / EPO / JAK2V617F UT-7 / EPO / JAK2V617F cells were cultured by the same method as in 4-1).

10 μL of MTT reagent was added to each well and incubated at 37 ° C for 3 hours. The absorbance at a wavelength of 450 nm was measured with a microplate reader, and the EPO-dependent cell proliferation inhibitory activity by the mutant EPO was measured.

5. Improved mutant EPO structure simulation
Structural simulation analysis of the improved mutant EPO with higher cell proliferation inhibitory activity was performed by the same method as in 2.

6. Expression of improved mutant EPO (mEPO4.1 ~ mEPO4.11, mEPO4.4.10), purification
1) We purchased the plasmid pcDNA3.4- (each mutant EPO) in which the mutant EPO synthesized by artificial gene was introduced into the expression plasmid pcDNA3.4-TOPO of the improved mutant EPO. Mutant EPO was expressed using the ExpiCHO Expression System (Thermo Fisher Scientific Fix). ExpiCHO-S cells 1 vial (1 x 10 ⁷ cells) were melted in a water bath and transferred to 30 mL of ExpiCHO Expression Medium preheated to 37 ° C. Shake culture was performed at 125 ± 5 rpm in an incubator at 37 ° C and 8% CO ² . The cells were cultured to 4 to 6 x 10 ⁶ cells / mL, and passage was repeated 2-3 times or more before transfection. The cell number was adjusted to 3-4 x 10 ⁶ cells / mL 1 day before transfection, and then shaken and cultured overnight. The cells were counted and adjusted to a 250 mL culture flask to 6 x 10 ⁶ cells / mL in 50 mL of ExpiCHO Expression Medium. 40 ug of pcDNA3.4- (mEPO) was diluted with 2 mL of OptiPRO medium and added to a mixture of 1.84 mL of OptiPRO medium and 160uL of ExpiFectamine CHO Reagent. After incubating this at room temperature for 5 minutes, it was added to a flask containing cells, and shaking culture was started. The next day, 300uL of ExpiCHO ^TM Enhancer and 12 mL of ExpiCHO ^TM Feed were added, and the culture conditions were changed to 32 ° C. and 5% CO2, and the cells were cultured for 8 days. After culturing, centrifugation was performed at 3000 rpm for 10 minutes to obtain a culture supernatant. The culture supernatant was filtered through a 0.45 um filter and further filtered through a 0.22 um filter.

2) Confirmation of expression of improved mutant EPO The expression of mEPO in the CHO cell culture supernatant was confirmed by the same method as in 3-2).

3) 35 mL of the purified and filtered culture supernatant of the improved mutant EPO was ultrafiltered three times with a molecular weight of Amicon Ultra-15 30 kDa (Merck Millipore) with 15 mL of buffer (20 mM Tris-hydrochloric acid, 0 M sodium chloride). The upper layer was subjected to ultrafiltration 7 times with the above buffer with Amicon Ultra-15 100 kDa (Merck Millipore), and the lower layer was recovered. This lower layer was ultrafiltered with Amicon Ultra-15 10 kDa (Merck Millipore), and the upper layer concentrated to 2 mL was recovered.

7. Activity measurement of improved mutant EPO (mEPO4.1 ~ mEPO4.11, mEPO4.4.10)-2
The EPO activity, EPO competitive activity, and growth inhibitory activity of the improved mutant EPO were measured by the same method as in 4.

8. Amino acid sequences of mutant EPO and improved mutant EPO
> Wild-type EPO
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
* 1 Residual mutation EPO
> mEPO1 (R103A)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL A SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO2 (R103F)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL F SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO3 (R103D)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL D SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4 (R103E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO5 (L108A)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTT A LRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO6 (V11Y)
APPRLICDSR Y LERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO7 (L108Y)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTT Y LRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO8 (L5N)
APPR N ICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO9 (L108S)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTT S LRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO10 (L108N)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTT N LRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
* 2 Residual mutation EPO
> mEPO4.1 (S100D / R103E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAV D GL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.2 (S100E / R103E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAV E GL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.3 (R103E / T107D)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLT D LLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.4 (R103E / T107E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLT E LLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.5 (R14D / R103E)
APPRLICDSRVLE D YLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.6 (R14E / R103E)
APPRLICDSRVLE E YLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.7 (K97D / R103E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVD D AVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.8 (K97E / R103E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVD E AVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.9 (R103E / R110D)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLL D ALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.10 (R103E / R110E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLL E ALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
> mEPO4.11 (R4E / R103E)
APP E LICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
* 3 Residue mutation EPO
> mEPO4.4.10 (R103E / T107E / R110E)
APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGL E SLT E LL E ALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR

9. Measurement of JAK2-STAT5 signaling inhibition by mutant EPO (mEPO4) and improved mutant EPO (mEPO4.4, mEPO4.10, mEPO4.4.10)

1) Acquisition of UT-7 / EPO / JAK2V617F / pSTAT5-luc cells that stably express the pGL4.52 [luc2P / STAT5 RE / Hygro] vector Luciferase gene and hygro having five STAT5 response sequences (STAT5 RE) in the promoter region 600 ng of the pGL4.52 [luc2P / STAT5 RE / Hygro] vector (Promega) incorporating the mycin resistance gene was introduced into UT-7 / EPO / JAK2V617F cells 6x10 ⁵ cells by the electroporation method. Electroporation was performed using the Neon Transfection System (Thermo Fisher) under the conditions of 1115 V, 30 ms, and 2 pulse. UT-7 / EPO / JAK2V617F / pSTAT5-that stably expresses the pGL4.52 [luc2P / STAT5 RE / Hygro] vector by culturing in EPO-free IMDM / 10% FCS containing 200 μg / mL of hygromycin for about 1 month. Obtained luc cells.

2) Purification of mutant EPO (mEPO4) and improved mutant EPO (mEPO4.4, mEPO4.10, mEPO4.4.10) Mutant EPO (mEPO4) and improved mutant EPO (mEPO4.4, mEPO4.10, mEPO4.4.10) 30 mL of the culture supernatant of the CHO cells expressing the above was filtered, placed in 2 bottles of 15 mL each in Amicon 30 kDa, centrifuged at 3000 rpm for 30 minutes, concentrated to 5 mL, and subjected to ultrafiltration. 10 mL of 20 mM phosphate buffer / 50 mM sodium chloride (pH 7.4) (buffer A) was added to the concentrate, and the mixture was centrifuged and concentrated to 5 mL. This was repeated two more times.
Next, the above concentrate was added to 10 mL of Blue Sepharose 6 Fast Flow (Cytiva) equilibrated with Buffer A, and the mixture was rotated at 4 ° C. for 4 hours. Centrifuge at 3000 rpm for 5 minutes at 4 ° C. to separate the supernatant from Blue Sepharose and discard the supernatant. Washed 3 times with 20 mL buffer A. 15 mL of 20 mM phosphate buffer / 2M NaCl (pH 7.4) (buffer B) was added to Blue Sepharose and rotated at 4 ° C. for 30 minutes to elute the mutant EPO and the improved mutant EPO. Centrifugation was performed at 3000 rpm for 5 minutes at 4 ° C., and the supernatant containing the mutant EPO and the improved mutant EPO was collected. This was repeated once to obtain an elution fraction.
30 mL of the eluted fraction was placed in 2 bottles of 15 mL each in Amicon 10 kDa, centrifuged at 3000 rpm for 30 minutes, and concentrated to 3 mL. 12 mL of PBS was added to the concentrate, and the mixture was centrifuged and concentrated to 3 mL. This was repeated two more times. Centrifugal concentration was performed until the final volume was 3 mL.

3) Measurement of JAK2-STAT5 signaling inhibition by mutant EPO and improved mutant EPO using UT-7 / EPO / JAK2V617F / pSTAT5-luc cells
UT-7 / EPO / JAK2V617F / pSTAT5-luc cells were adjusted to 2.2x10 ⁵ cells / mL with EPO-free IMDM / 10% FCS and seeded in 90 μL increments on 96-well plates. A sample obtained by diluting the purified mutant EPO and the improved mutant EPO 20-fold with PBS was added to each well in an amount of 10 μL, and the cells were cultured under 37 ° C. and 5% CO2 conditions for 6 hours. Eventually, the dilution ratio of the mutant EPO will be 200 times.
100 μL of ONE-Glo ^TM Reagent (Promega) was added to each well and incubated for 3 minutes to lyse the cells. Chemiluminescence of this plate by luciferase was measured with a luminometer and by dimerization of JAK2V617F via EPOR with the addition of mutant EPO (mEPO4) and improved mutant EPO (mEPO4.4, mEPO4.10, mEPO4.4.10). Inhibition of phosphorylation followed by STAT5 phosphorylation inhibition was evaluated.

Result 1. Preparation of UT-7 / EPO / JAK2V617F cells
A complex consisting of Cas9 protein, sgRNA, and ssODN was introduced into UT-7 / EPO cells by electroporation, and a V617F mutation was introduced into the JAK2 gene by genome editing. By repeating this three times in total, a cell line capable of growing in the absence of EPO was obtained.

In the cloning after the first genome editing, 65 clones proliferated in the culture for 2 weeks. As a result of sequence analysis of the region around the introduction of the JAK2 gene mutation, all clones were heterozygous mutations. Based on the waveform data, select clones (No.11, 16, 25, 35, 54, 55, 58) with strong thymidine fluorescence signal at the mutation introduction site (G1849T), and use the ABC-PCR method to select allelic Baden values. As a result of quantifying, No. 11 and 54 showed 56.70% and 56.41%, respectively, which are higher allele-baden values than other clones.

It is known that mutations in the JAK2 gene are homozygous in PV patients. Therefore, mutations in the JAK2 gene need to be homozygous for the construction of PV disease model cells. In order to obtain clones with higher allele-baden values, genome editing was performed for No. 11 and No. 54 with high allele-badden values for the second time. Cloning yielded 299 clones. 11 clones (No. 11B-78, 11R-11, 11R-22, 11R-59, 11R-61, 54-8, 54-15, 54-29, The allelic Baden values of 54-36, 54-39, 54-75) were quantified.

The allele-baden value of No.54-15 was the highest at 88%, but since it did not reach 100%, the allele-baden value was high and cell proliferation was good. On the other hand, the genome was edited for the third time. Cloning yielded 88 clones. 6 clones (No.11R-61-21, 11R-61-44, 11R-61-52, 54-15-17, 54-15-21, 54- As a result of quantifying the allele-baden value of 15-29), it was about 90% in all 6 clones, suggesting that it is a homozygous mutant.

No. 54-15-17, which has a high allele Baden value, was found to be a homozygous mutant by single allele sequence analysis, and was named UT-7 / EPO / JAK2V617F cells that mimic PV disease.

In the signal transduction analysis of UT-7 / EPO / JAK2V617F cells, in the absence of EPO, phosphorylation of STAT5 and ERK1 / 2 disappeared in UT-7 / EPO cells, whereas phosphorylation of UT-7 / EPO / JAK2V617F cells disappeared. Was constantly phosphorylated.

2. 2. Mutant EPO structure simulation Prior to the study of mutation introduction, in order to clarify the factors of binding between wild-type EPO and EPOR, we used the three-dimensional structure data of the complex of EPO and EPOR dimer, and used EPO (chain A). We investigated the interaction of EPOR (chain C). Amino acid residues related to the interaction between the low affinity binding surface of EPO (site2) and EPOR were detected, and the amino acid residues involved in the binding between EPO and EPOR were inferred.

First, in the EPO and EPOR complex, all the interatomic distances of EPO (chain A) and EPOR (chain C) are measured, and the amino acid residues in which the interatomic distance between the two chains is within 4.5 Å are determined. Detected. The results of color-coding the amino acid residues according to the interatomic distance are shown on the three-dimensional structure and amino acid sequence (Fig. 3).

From the amino acid residues detected between EPO and EPOR, the residues directly involved in the interaction were inferred. As amino acid residues forming hydrogen bonds, EPO Asp8, Arg14, Lys97, Arg103, and Ser104 were inferred (Fig. 4). The inferred hydrogen bond was shown in. Among them, it was speculated that the side chain of Arg103 forms a total of four hydrogen bonds with the side chain of Glu62, Asp89, Ser91 and the main chain of Ala88 of EPOR.

Two hydrophobic interactions were speculated: between Arg103 and EPOR of EPO and between Phe93 and EPO of EPOR. Figure 5 shows the amino acid residues inferred to have hydrophobic interactions. The side chain of Arg103 contributes to both EPOR and hydrogen bonds and hydrophobic interactions, and is thought to play an important role in binding to EPOR.

EPOR's Phe93 binds into the hydrophobic pocket formed by EPO's Leu5, Val11, Tyr15, and Leu108, and all carbons in the aromatic ring of Phe93 form a hydrophobic interaction with the carbon of the four residues of EPO. Was.

Next, in order to confirm the electrostatic interaction between EPO and EPOR, the surface shape was created, the electrostatic potential was mapped, and the areas involved in the interaction were circled (Fig. 6). It was confirmed that the surface of EPO has a positive electrostatic potential (blue) and the surface of EPOR has a negative electrostatic potential (red) on the bonding surface of site2. The region showing each positive or negative electrostatic potential includes the region where the previously detected interaction was inferred (circled in FIG. 6). From this, it was speculated that the interaction between EPO (positive) and EPOR (negative) due to the electrostatic potential also contributed to the binding between EPO and EPOR. Arg103, which has a positively charged side chain of EPO inferred to have hydrogen bonds, was present in the positive region, and Glu62 and Asp89, which had negatively charged side chains of EPOR, were present in the negative region. It is speculated that Arg103 of EPO and Glu62 and Asp89 of EPOR not only form hydrogen bonds but also cause positive or negative electrostatic potentials on their respective surfaces and contribute to electrostatic interaction.

From the above, it was inferred that site 2 of EPO is bound to EPOR by three factors: hydrogen bond, hydrophobic interaction, and interaction between EPO (positive) and EPOR (negative) due to electrostatic potential. Regarding hydrogen bonds and hydrophobic interactions, the hydrogen bonds between Arg103 of EPO and EPOR and the hydrophobic interaction between Phe93 of EPOR and EPO were considered to be particularly important interactions.

2-1. Examination of introduction of mutation into EPO to inhibit dimer formation of EPOR
From the analysis results of the interaction between EPO and EPOR, three factors were inferred: hydrogen bonding, hydrophobic interaction, and interaction between EPO (positive) and EPOR (negative) due to electrostatic potential. Eliminating these interactions was considered effective in inhibiting the binding of EPO site 2 to EPOR.

We focused on the two most important positions for introducing mutations. One is EPO's Arg103, which is called site2-A here. At site2-A, the EPO Arg103 binds to the EPOR Glu62, Ala88, Asp89, Ser91 so as to enter the recess, and the Arg side chain forms a hydrogen bond with the EPOR Glu62, Ala88, Asp89, Ser91. It was (Fig. 7). The surface area of this EPOR depression had a negative electrostatic potential and was suitable for the binding of positive Arg103.

It was also considered that the nitrogen of the guanidyl group having a positive charge on the side chain of Arg103 of EPO may be electrostatically bonded to the oxygen of the carbonyl group of the side chains of EPOR Glu62 and Asp89. Introducing a mutation into site2-A eliminates these interactions of Arg103, resulting in a mutation that eliminates hydrogen bonds, a mutation that reduces or reverses positive electrostatic potential, and a mutation that causes steric hindrance with EPOR. (Fig. 8, left).

The position where the other mutation is introduced is Leu5, Val11, Tyr15, Leu108, which are inferred to have a hydrophobic interaction with Phe93 of EPOR, and these are called site2-B. At site2-B, these residues formed hydrophobic pockets, which are hydrophobic depressions, on the surface of site 2 of the EPO (Fig. 7). EPOR's Phe93 bound to fit in this hydrophobic pocket and formed a hydrophobic interaction with EPO's Leu5, Val11, Tyr15, Leu108. Therefore, we investigated mutations that eliminate the hydrophobic interaction of EPOR with Phe93 and mutations that prevent Phe93 from binding to the hydrophobic pocket by disrupting steric hindrance and hydrophobic environment in the hydrophobic pocket (Fig. 8). ,right).
By eliminating the interaction required for EPO and EPOR to form the original complex at site2, EPOR cannot bind to site2 of the mutant EPO and form the original dimer structure. Conceivable. Furthermore, it was expected to have the effect of inhibiting EPOR from approaching its original position with respect to EPO due to the repulsion of electrostatic potential and mutation that causes steric hindrance with EPOR.

2-2. Preparation and evaluation of a three-dimensional structural model of mutant EPO and selection of mutation candidates Amino acid residues for each residue of site2-A (Arg103) and site2-B (Leu5, Val11, Tyr15, Leu108) A mutant model was prepared in which the above was replaced, and the effect of the mutation was investigated by comparing it with the wild-type EPO. We also confirmed that the mutation does not affect the three-dimensional structure of the EPO itself.

2-2-1. Mutation introduction into site2-A In site2-A, we examined a mutant model in which a mutation was introduced into R103 of EPO. Arg103 of EPO formed a hydrogen bond with Glu62, Ala88, Asp89, Ser91 of EPOR. Furthermore, the side chain with a positive charge of Arg is considered to be a factor that makes the electrostatic potential of the EPO surface positive, and as a result, contributes to the binding to the negative EPOR surface.

In order to eliminate these interactions, mutations were examined for Arg103 according to the following three policies (Fig. 8, left).
(1) Eliminate the hydrogen bond with EPOR.
(2) Causes steric hindrance with EPOR.
(3) Decrease and invert the positive electrostatic potential of the EPO.

Based on these policies, a mutant model in which Arg103 was replaced was prepared.
(1) Eliminate hydrogen bonds with EPOR
It was speculated that the substitution of Arg with hydrophobic residues Ala, Val, Ile, Leu and the substitution with Ser, Thr, Asn with small side chains all eliminated the hydrogen bond with EPOR by the side chain of Arg. .. However, in the complex model substituted with polar residues (Ser, Thr, Asn), a space where water molecules can hydrate was confirmed with EPOR, and it is possible that hydrogen bonds may be formed via water molecules. rice field. Substitution with Ala, whose side chain is a methyl group, was considered to be the most direct and effective in the direction of eliminating hydrogen bonds with EPOR. Substitution from Arg to Ala also has the effect of reducing the positive electrostatic potential of the EPO surface because it eliminates the guanidyl group in the positively charged Arg side chain. In addition, the hydrophobic interaction between Arg103 and EPOR with Leu59, Ser91, and Val94 (Fig. 5, right) was also considered to disappear.

(2) Causes steric hindrance with EPOR As the direction of causing steric hindrance, we created a mutant model for Phe, Tyr, and Trp, which has a side chain that is bulkier than the Arg residue. It was speculated that Phe, Tyr, and Trp may cause the side chain of Val94 of EPOR and steric clash. However, considering that this region of EPOR has a loop structure and that tyrosine clashes are side chain atoms, steric hindrance with EPOR may occur depending on the side chain orientation (rotorer) of Phe, Tyr, and Trp. It may be possible to avoid it, and it may not be effective enough to actually inhibit the binding with EPOR. In addition, in the mutation to Tyr and Trp, it is possible that the side chain forms a hydrogen bond with the side chain of EPOR and binds in a different conformation from the original.

Of these three, mutation to Phe was considered preferable in terms of directly confirming the effect of steric hindrance with EPOR. The

(3) Decrease and invert the positive electrostatic potential of EPO In order to reduce and invert the positive electrostatic potential of EPO, the side chain of Arg residues with positive charge is replaced with amino acid residues with negative charge. Substitution with Glu was performed. It was confirmed that it has the effect of making the electrostatic potential on the surface of the EPO negative (Fig. 9, right). This was also the case when replaced with Asp, which is also negatively charged. Glu, which has a longer side chain than Asp, is considered to be more effective in inducing repulsion with EPOR because the negatively charged carbonyl group protrudes more to the surface. Substitution from Arg to Ala also eliminates the guanidyl group in the positively charged Arg side chain, thus reducing the positive electrostatic potential on the EPO surface (center of Figure 9), but from Arg to Asp, Glu. Since the electrostatic potential on the surface of EPO can be reversed to negative by the substitution of, the effect of causing repulsion with EPOR and separating EPOR can be expected.

Based on the above, regarding the introduction of the mutation into site2-A, the mutation from Arg to Ala (R103A) as a mutation that eliminates the hydrogen bond with EPOR, and the mutation from Arg to Asp, Glu as a mutation that reverses the positive electrostatic potential of EPO. Mutations (R103D, R103E) were considered as candidates (Fig. 10). R103D and R103E not only eliminate the binding ability of EPOR from site 2 of EPO, but also cause charge repulsion between EPO and EPOR, so they seemed to be effective mutations in separating the binding surface of EPOR. The mutation that causes steric hindrance includes the mutation from Arg to Phe (R103F), but the effect seems to be limited from the mutant model. It was speculated that neither mutation affected the three-dimensional structure of EPO.

2-2-2. Introduction of mutation to site2-B
As a mutation introduction to site2-B, a mutant model in which amino acid residues were substituted was prepared for Leu5, Val11, Tyr15, and Leu108 of EPO. The amino acid residues of site2-B of EPO form a hydrophobic pocket, which is a hydrophobic depression, on the surface of site2. EPOR's Phe93 binds to fit in this hydrophobic pocket, forming a hydrophobic interaction.

In order to eliminate these hydrophobic interactions, we investigated mutations in site2-B using the following three policies (Fig. 8, right).
(1) Expand the hydrophobic pocket of the EPO to create a gap.
(2) Causes steric hindrance with EPOR and inhibits Phe93 binding.
(3) Break the hydrophobic environment of the pocket to which the EPO Phe93 binds.

Based on these policies, we created a mutant model in which each amino acid residue of site2-B was replaced. The

(1) Expand the hydrophobic pocket of EPO to create a gap
It eliminates the direct hydrophobic interaction with Phe93 by widening the hydrophobic pocket of the EPO to which Phe93 of EPOR binds and creating an extra gap with Phe93. As the direction of this mutation, it was considered that the mutation was made to a smaller amino acid residue.

In the prepared mutant model, EPOR Phe93 was used for the substitution of Leu5 to Ala, Val, the substitution of Val11 to Ala, the substitution of Tyr15 to Ala, Val, Ile, Leu, and the substitution of Leu108 to Ala, Val. The disappearance of the hydrophobic interaction with was inferred. These mutations not only eliminate the direct hydrophobic interaction with Phe93, but also have the effect of destabilizing the binding of Phe93 by opening a gap in the tight binding between the hydrophobic pocket and Phe93. Thought. In order to leave as much space as possible between Phe93 and Phe93, it is preferable that the amino acid residue to be substituted is small, and Ala is considered to be the most suitable type of amino acid residue. The

Based on the above, the policy of widening the hydrophobic pocket to create a gap is to mutate Leu5 to Ala (L5A), mutation from Val11 to Ala (V11A), mutation from Tyr to Ala (Y15A), and mutation from Leu108 to Ala. Mutation (L108A) was considered as a candidate (Fig. 11). In single-residue substitution, the greater the number of hydrophobic interactions between each amino acid residue and Phe93, the greater the effect. Considering the number of hydrophobic interactions by each residue, L108A (5 hydrophobic interactions), L5A (4), V11A in terms of effectively eliminating hydrophobic interactions with one residue. (2 locations), Y15A (1 location) can be considered in that order. In addition, since Phe93 interacts with four amino acid residues, it is considered effective to introduce multiple mutations when the effect of one-residue substitution is observed. It was speculated that neither mutation affected the three-dimensional structure of EPO.

(2) Causes steric hindrance with EPOR
As a direction that causes steric hindrance to the hydrophobic pocket of EPO and inhibits the binding of Phe93 of EPOR, it is considered that the amino acid residues Phe, Tyr, Trp, etc., which have a bulkier side chain, are mutated. It is expected that EPOR's Phe93 will not be able to bind by filling the space of the hydrophobic pocket with the bulky side chain and causing steric hindrance with Phe93.

Replacement with Met, Trp in Leu5, replacement with Phe, Tyr, Trp in Val11, replacement with Phe, Tyr, Lys, Trp in Leu108 causes steric hlash with Phe93 of EPOR, causing steric hindrance with EPOR. It was speculated that it could cause it. On the other hand, in the substitution from Leu5 to Ile, Phe, Lys, the substitution from Val11 to Ile, Leu, Met, and the substitution from Leu108 to Ile, Met, a hydrophobic interaction with Phe93 in a different form was observed. Therefore, it was considered inappropriate. Tyr15 produced steric clash only by substitution with Trp, which has a bulky side chain, but substitution with Trp was excluded from the candidates because of steric hindrance within the structure of EPO itself. Among these mutant models, the Phe residue of the introduced EPO is a hydrophobic pocket, especially in the Val11 to Phe, Tyr mutation (V11F, V11Y) and the Leu108 to Phe, Tyr mutation (L108F, L108Y). And because tyrosine clash with multiple carbon atoms in the side chain of Phe93 of EPOR was inferred, it was speculated that it has the effect of inhibiting the binding of Phe93 to the pocket. The

From the above, the mutation from Val11 to Phe, Tyr (V11F, V11Y) and the mutation from Leu108 to Phe, Tyr (L108F, L108Y) were considered to be candidates for mutations that cause steric hindrance with EPOR (Fig. 12). Mutation to Tyr not only causes steric hindrance, but also brings hydrophilic OH groups to the contact surface of EPOR with Phe93, so it can be expected to have the effect of destroying the hydrophobic environment. It was speculated that none of the mutations affected the three-dimensional structure of EPO.

(3) Break the hydrophobic environment of the hydrophobic pocket of the EPO
The hydrophobic pocket of EPO exerts a great binding force by forming clusters of hydrophobic amino acid residues. Therefore, by introducing a hydrophilic amino acid residue into the hydrophobic pocket, the effect of eliminating the hydrophobic interaction with Phe93 and the effect of attracting water molecules into the pocket were expected.

In the mutant model in which Leu5 is replaced with Ser, Thr, hydrophilic OH groups are arranged, but the effect of disrupting the hydrophobic environment is limited because the OH groups in the side chains are displaced from the hydrophobic pocket. So I thought. It was speculated that the substitution with Asn could cause hydrophilic side chains to enter the hydrophobic pocket and disrupt the hydrophobic environment. In the variant model in which Val11 was replaced with Ser and Thr, the OH group in the side chain formed a hydrogen bond with the main chain of Leu5 and Asp8 of EPO itself and was not exposed on the surface of the hydrophobic pocket. Therefore, it was considered that the effect of destroying the hydrophobic environment was low. In addition, substitution with Asn and Lys is not preferable because it forms a hydrogen bond with EPOR. Substitution of Tyr15 to hydrophilic residues did not reach the hydrophobic clusters of any of the amino acid residues, and it seemed difficult to obtain an effect enough to destroy the hydrophobic environment. In the substitution from Leu108 to Ser, Thr, the OH group of the side chain of Ser, Thr was introduced inside the hydrophobic pocket, and it was speculated that it was effective in destroying the hydrophobic environment. In addition, the replacement of Leu108 with Asn is considered to be effective because the hydrophilic side chains of Asn are arranged so as to fill the hydrophobic pocket.

Based on the above, mutations from Leu5 to Ser, Asn (L5S, L5N) and mutations from Leu108 to Ser, Asn (L108S, L108N) were considered as candidates for mutations that disrupt the hydrophobic environment of the hydrophobic pocket (Fig.). 13). It was speculated that neither mutation affected the three-dimensional structure of EPO. In general, in a hydrophobic interaction between proteins, even if a mutation occurs in a hydrophobic residue exposed on the surface, if the mutated residue is a hydrophobic residue, it takes a different conformation from the original and is hydrophobic. Often retains the interaction. The introduction of hydrophilic amino acid residues was considered to be effective in eliminating the hydrophobic interaction of Phe93 of EPOR. The

2-2-3. Selection of mutation candidates As a result of creating a mutant model and simulating in silico, a total of 10 EPO mutation introduction sites were selected for site2-A and site2-B (Table 3).

So far, there have been multiple reports of mutations in the EPO to identify the interaction between the EPO and EPOR before the conformation was determined (Syed RS et al. (1998) Nature 395: 511; Lorenzini. T et al. (1997) Blood 89: 473; Matthews DJ et al. (1996) Proc Natl Acad Sci 93: 9471). Among them, R103A has been shown to suppress the activity of wild-type JAK2. This analysis confirms that Arg103 is extremely important for binding to EPOR. It was speculated that the mutation in R103A caused the deletion of Arg103 to eliminate the binding ability of site2 and inhibit EPOR from forming the original active dimer. The effect of EPO R103A on mutant JAK2 is unknown, but the introduction of site2-A into Arg103 is presumed to have a large effect in the mutant model, so the introduction of mutation into Arg103 is considered to be a candidate for consideration. rice field.

At Site2-A, (1) the hydrogen bond with EPOR is eliminated and the hydrophobic pocket is expanded, R103A, (2) steric hindrance with EPOR is caused, and the positive electrostatic potential of R103F and (3) EPO is exhibited. R103D and R103E, which decrease and reverse and cause repulsion, were selected as candidates.

In addition, among the amino acid residues of site2-B, Leu108 has the largest hydrophobic interaction with Phe93, and it was inferred that mutations are effective in all three directions. I thought that the priority was high. The

EPOR is thought to form a dimer on the cell surface even in the absence of EPO. It has also been reported that when EPO binds to EPOR, it first binds at site1 and then at site2 (Matthews DJ et al. (1996) Proc Natl Acad Sci. 93: 9471). In order to separate the EPOR dimer structure bound to site 1 of the EPO and bind to each EPOR at the site 1 with high affinity, it is necessary to have a mutation that not only eliminates the binding ability of site 2 but also further separates the binding surface of EPOR. I thought it was.

At site2-B, candidates for (1) expand the hydrophobic pocket, (2) cause steric hindrance with EPOR, V11Y, L108Y, (3) destroy the hydrophobic environment of EPO, L5N, L108S, L108N. Was selected for.

Figure 14 shows the direction of EPO mutation introduction at site2-A and site2-B, and candidates for mutant amino acids. Table 3 summarizes the candidates for mutant EPO.

3. 3. Expression of mutant EPO (mEPO1 ~ mEPO10)
As a result of Western blotting of the culture supernatant of CHO cells, mEPO1-10 was expressed as a 27 kDa protein. Of these, when mEPO2, standard EPO (Std), and wild-type EPO (hEPO) were treated with Glycosidase F, which is a sugar chain-cleaving enzyme, the band shifted to 20 kDa. It was found that 10 was expressed in full length.

4. Evaluation of mutant EPO activity (mEPO1 ~ mEPO10)
As a result of measuring the EPO activity of the mutant EPO (mEPO1-10) by the MTT assay, UT-7 / EPO cells could not proliferate in the mEPO 1, 3, 4, 7 addition group, and the other mutant EPOs proliferated equivalent to EPO. It was possible (Fig. 15a).

As a result of adding EPO of EC50 = 0.03 U / mL and measuring the competitive activity of EPO and mutant EPO, the proliferation of UT-7 / EPO cells was competitively inhibited in the mEPO1, 3, 4, 7 added group ( Figure 15b).

As a result of measuring the growth inhibitory activity of mutant EPO on UT-7 / EPO / JAK2V617F cells, the growth was inhibited in the mEPO3,4 addition group compared to the medium addition group (Fig. 15c).

5. Improved mutant EPO structure simulation The cell proliferation inhibitory activity of UT-7 / EPO / JAK2V617F cells was observed only in mEPO3 (R103D) and mEPO4 (R103E). The factor of the inhibitory activity is considered to be the introduction of a negative charge to site2. From the evaluation of competitive activity with wild-type EPO, mEPO1, 3, 4, 7 (R103A, R103D, R103E, L108Y) retained site1 with high affinity among the two binding surfaces (site1, site2) of EPO. As it is, it loses the binding ability of site2, which has a low affinity. From this, it was confirmed that it has a high affinity that can compete with wild-type EPO and can inhibit the formation of the active dimer of EPOR.

In addition, from the results of mEPO1 (R103A), in order to inhibit cell proliferation due to the activity of mutant JAK2, it is not enough to simply inhibit EPOR from forming the original active dimer, and there are additional factors. It was inferred that it was necessary. On the bonding surface of site2, the surface of the wild-type EPO shows a positive region, but in mEPO3 (R103D) and mEPO4 (R103E), it is partially changed to negative due to the introduction of side chains with negative charges (Fig. 16). ). In R103D and R103E, the negative charge of the side chain repels the negative region of EPOR, which not only eliminates the binding ability of EPOR from site 2 of EPO, but also causes charge repulsion between EPO and EPOR. Was also predicted to have the effect of pulling away the bonding surface of the EPOR.

As an improved EPO with a high cell proliferation inhibitory effect, by introducing a new mutation focusing on charge repulsion, the EPOR dimer formation is more effectively inhibited, and the improved EPO with a high proliferation inhibitory effect Study was carried out.

5-1. Examination of improved mutant EPO-Design of mutant EPO with enhanced negative electrostatic potential-
It was speculated that the factor of cell proliferation inhibitory activity was the negative charge introduced into site2-A. In order to increase the charge repulsion with the EPOR to the EPO and to have a greater effect on the arrangement of the EPOR, it is considered effective to make the negative region of the charge on the surface of the site 2 of the EPO larger. Therefore, for R103D and R103E, which had the effect of inhibiting cell proliferation, we investigated mutations that introduce a more negative electrostatic potential near site2-A (Fig. 17).

As for the region to introduce the mutation, (1) the introduction of the mutation that enhances the area around Arg103 to the negative electrostatic potential, and (2) the introduction of the mutation that changes the electrostatic potential of the entire site2 from positive to negative were examined. Both Asp and Glu were considered as the types of amino acid residues to be introduced. The

5-1-1. Mutation introduction that enhances the area around EPO Arg103 to a negative electrostatic potential We investigated a mutation that enhances the area around EPO Arg103 to a negative electrostatic potential, which seems to be effective for charge repulsion with EPOR. As candidates, Ser100, Thr107, and Arg110, which are on the same helix as Arg103, were considered. In addition to R103E, the electrostatic potential map of the surface shape of the two-residue substitution EPO S100E / R103E and R103E / T107E in which these amino acid residues are replaced with Glu, and the three-residue substitution R103E / T107E / R110E. Shown (Fig. 18). In the mutant EPO S100E / R103E, R103E / T107E, R103E / T107E / R110E, the vicinity of R103 is shown in red compared to R103E, and it is inferred that the mutation can enhance the area around Arg103 to a negative electrostatic potential.

Figure 19 shows the peripheral structure of the mutation introduction site. In the complex of EPO and EPOR, there is a loop consisting of amino acid residues Glu60-Asp61-Glu62 having a negative charge of EPOR in the vicinity of Arg103. Therefore, the introduction of a negative charge around Arg103 is considered to have the effect of keeping EPOR away by inducing charge repulsion with this loop of EPOR. In particular, since Thr107 is located near this loop, the introduction of negatively charged amino acid residues was considered to be effective.

In addition, Thr107 and Arg110 with EPO triad substitution were considered to be effective because they invert the binding surface with EPOR to a wide negative electrostatic potential.

5-1-2. Mutation introduction that changes the electrostatic potential of the entire EPO site2 from positive to negative There are multiple amino acid residues with positive charges in addition to Arg103 in site2 of the EPO, and these are the surface of the entire site2. The electrostatic potential is set to positive. In order to reverse the electrostatic potential of the bonding surface with EPOR on the site2 surface from positive to negative, it was examined to replace these amino acid residues with Asp and Glu having negatively charged side chains.

Arg4, Arg14, Lys97, Arg110 were considered as candidates. Fig. 20 shows the electrostatic potential map of R4E / R103E, R14E / R103E, K97E / R103E, R103E / R110E in which these amino acid residues are replaced with Glu, and Fig. 21 shows the electrostatic potential maps of R14E / R103E, K97E / R103E, R103E / R110E. The peripheral structure is shown. It was confirmed that the electrostatic potential on the surface was negative compared to R103E. The

In the vicinity of Arg110, there is a loop of Glu60-Asp61-Glu62 with the above-mentioned negative charge of EPOR (Fig. 21c). In the wild-type EPO-EPOR complex, Glu62 forms hydrogen bonds with Arg103. Although Arg110 itself is not coupled to this loop, the positive charge of Arg110 contributes to the positive electrostatic potential on the surface of site2, and it seems that it plays a role in attracting the structure of this loop with negative charge of EPOR. .. Due to the mutation of R103E / R110E, the electrostatic potential on the surface on the EPO side to which this loop approaches is reversed negatively (Fig. 20b), so charge repulsion is similar to R103E / T107E described in 5-1-1. Is expected to keep the EPOR loop away.

R4 is located in the helix below R103, and its side chain is not very close to EPOR (Fig. 22a). However, by substituting this amino acid with Glu, the side chain becomes closer to EPOR (Fig. 22b), and the negative charge of EPO site2 is also widely strengthened, so charge repulsion is expected.

5-1-3. Candidates for improved mutation EPO by two-residue substitution and three-residue substitution As mutations that make the electrostatic potential of the site2 surface of EPO negative, (1) Strengthen the periphery of Arg103 to negative electrostatic potential. Mutations (S100E, T107E, R110E) and (2) mutations (R14E, K97E, R110E, R4E) that make positively charged amino acids on the surface of site2 negatively charged were investigated. Glu mutations were investigated for R4. Two-residue substitution mutations in which Asp or Glu mutations are introduced at the positions shown in (1) and (2) in addition to R103E, that is, S100D / R104E, S100E / R104E, R103E / T107D, R103E / T107E, R103E / T107E / Eleven types of R110E, R14D / R103E, R14E / R103E, K97D / R103E, K97E / R103E, R103E / R110D, R103E / R110E, and R4E / R103E were considered as candidates.

FIG. 23 shows the positions of mutation introduction candidates on the three-dimensional structure of EPO and on the amino acid sequence.
Table 4 shows the candidates for the improved mutant EPO.

6. Expression of improved mutant EPO (mEPO4.1 ~ mEPO4.11, mEPO4.4.10)
As a result of Western blotting of the culture supernatant of CHO cells, mEPO4.1-4.11 and mEPO4.4.10 were expressed as 27 kDa proteins.

7. Evaluation of the activity of improved mutant EPO (mEPO4.1 ~ mEPO4.11, mEPO4.4.10)
The activity of the improved mutant EPO (mEPO4.1 ~ 4.11), which was further mutated based on mEPO4, was evaluated by the MTT assay. UT-7 / EPO cells were unable to proliferate in all improved mutant EPO-added groups (Fig. 24a).

As a result of adding EPO of EC50 = 0.03 U / mL and measuring the competitive activity of EPO and improved mutant EPO, proliferation of UT-7 / EPO cells in the group to which mEPO4.1, 4.2, 4.3, 4.4, 4.9, 4.10 was added. Was competitively inhibited (Fig. 24b).

As a result of measuring the growth inhibitory activity of the improved mutant EPO on UT-7 / EPO / JAK2V617F cells, the growth was inhibited in the mEPO4.3, 4.4, 4.9, 4.10 addition group. Of these, mEPO 4.4 and 4.10 had higher inhibitory activity than the base mEPO 4 (Fig. 24c).

EPO activity disappeared and EPO competitive activity was observed in mEPO 4.4.10 into which all the mutation sites of mEPO 4.4, 4.10 were introduced, and the growth inhibitory activity was higher than that of mEPO4, mEPO4.4, and mEPO4.10 (Fig. 25a-c). ).

8. Stable expression of the pGL4.52 [luc2P / STAT5 RE / Hygro] vector to measure JAK2-STAT5 signaling inhibition by mutant EPOs and improved mutant EPOs. UT-7 / EPO / JAK2V617F / pSTAT5-luc cells were generated. The pGL4.52 [luc2P / STAT5-RE / Hygro] vector is a vector in which a STAT5 response sequence (STAT5 RE) is bound to a luciferase gene (luc2P) as a promoter sequence, and STAT5 phosphorylated by JAK2 translocates to the nucleus. Since luc2P is expressed by binding to STAT5-RE, it is possible to measure STAT5 signaling by measuring luciferase activity. STAT5 is located downstream of EPOR, where EPO binds to two EPORs, resulting in dimerization and phosphorylation of JAK2, which binds to the intracellular domain of EPOR. Then, STAT5 is phosphorylated by phosphorylated JAK2, nuclear translocation occurs, and EPO-related gene expression occurs. On the other hand, the reporter cell line UT-7 / EPO / JAK2V617F / pSTAT5-luc prepared from disease model cells of polycythemia vera is EPO-independent and constitutively dimerized and phosphorylated by JAK2V617F mutation. ing. As a result, STAT5 phosphorylation and nuclear translocation occur, and luc2P having a STAT5 response sequence as a promoter is expressed. By adding mutant EPO and improved mutant EPO to this cell line, luc2P can inhibit JAK2 dimerization and phosphorylation by changing the positional relationship between the two EPORs, and subsequently inhibit STAT5 phosphorylation and nuclear translocation. It can be measured by a decrease in luciferase activity.

When the luciferase activity of the medium-added group is 100%, mEPO4 is 84%, mEPO4.4 is 78%, mEPO4.10 is 87%, mEPO4.4.10 is 84%, and mutant EPO (mEPO4) and improved mutants. It was revealed that the phosphorylation of STAT5 was inhibited in all the groups to which EPO (mEPO4.4, mEPO4.10, mEPO4.4.10) was added (Fig. 26). That is, the mutant EPO (mEPO4) in which the 103rd arginine of the EPO was converted to glutamic acid, the improved mutant EPO (mEPO4.4) in which the 103rd arginine and the 107th threonine were converted to glutamic acid, and the 103rd and 110th arginines were used. Positions of two EPORs by adding improved mutant EPO (mEPO4.10) converted to glutamic acid, improved mutant EPO (mEPO4.4.10) converted to arginine 103 and 110 and threonine 107 Abnormal proliferation of erythropoiec precursors in true erythropoiegia by inhibiting the phosphorylation of JAK2V617F, which is constantly phosphorylated and activated, and the phosphorylation of STAT5 by inactivation due to changes in the relationship. Was shown to be suppressed.

All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as is.

INDUSTRIAL APPLICABILITY The present invention can be used as a drug that suppresses the growth of tumor cells.

<SEQ ID NO: 1> The amino acid sequence of wild-type EPO is shown.
<SEQ ID NO: 2> The JAK2 gene sequence around the mutation introduction site is shown.
<SEQ ID NO: 3> The ssODN sequence is shown.
<SEQ ID NO: 4> The sgRNA (# 1) sequence is shown.
<SEQ ID NO: 5> The sgRNA (# 2) sequence is shown.
<SEQ ID NO: 6> The sgRNA (# 3) sequence is shown.
<SEQ ID NO: 7> The sgRNA (# 4) sequence is shown.
<SEQ ID NO: 8> The sequence of the fluorescent probe (ABQP-JAKd-1) is shown.
<SEQ ID NO: 9> The sequence of the forward primer (F-JAKd-1) is shown.
<SEQ ID NO: 10> The sequence of the reverse primer (R-JAKd-1) is shown.
<SEQ ID NO: 11> The amino acid sequence of mEPO1 (R103A) is shown.
<SEQ ID NO: 12> The amino acid sequence of mEPO2 (R103F) is shown.
<SEQ ID NO: 13> The amino acid sequence of mEPO3 (R103D) is shown.
<SEQ ID NO: 14> The amino acid sequence of mEPO4 (R103E) is shown.
<SEQ ID NO: 15> The amino acid sequence of mEPO5 (L108A) is shown.
<SEQ ID NO: 16> The amino acid sequence of mEPO6 (V11Y) is shown.
<SEQ ID NO: 17> The amino acid sequence of mEPO7 (L108Y) is shown.
<SEQ ID NO: 18> The amino acid sequence of mEPO8 (L5N) is shown.
<SEQ ID NO: 19> The amino acid sequence of mEPO9 (L108S) is shown.
<SEQ ID NO: 20> The amino acid sequence of mEPO10 (L108N) is shown.
<SEQ ID NO: 21> The amino acid sequence of mEPO4.1 (S100D / R103E) is shown.
<SEQ ID NO: 22> The amino acid sequence of mEPO4.2 (S100E / R103E) is shown.
<SEQ ID NO: 23> The amino acid sequence of mEPO4.3 (R103E / T107D) is shown.
<SEQ ID NO: 24> The amino acid sequence of mEPO4.4 (R103E / T107E) is shown.
<SEQ ID NO: 25> The amino acid sequence of mEPO4.5 (R14D / R103E) is shown.
<SEQ ID NO: 26> The amino acid sequence of mEPO4.6 (R14E / R103E) is shown.
<SEQ ID NO: 27> The amino acid sequence of mEPO 4.7 (K97D / R103E) is shown.
<SEQ ID NO: 28> The amino acid sequence of mEPO4.8 (K97E / R103E) is shown.
<SEQ ID NO: 29> The amino acid sequence of mEPO4.9 (R103E / R110D) is shown.
<SEQ ID NO: 30> The amino acid sequence of mEPO4.10 (R103E / R110E) is shown.
<SEQ ID NO: 31> The amino acid sequence of mEPO4.11 (R4E / R103E) is shown.
<SEQ ID NO: 32> The amino acid sequence of mEPO 4.4.10 (R103E / T107E / R110E) is shown.

Claims (20)

1. The following erythropoietin variant of (a) or (b).
   (a) From the amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, which is an erythropoetin variant in which a mutation is introduced into human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. And another amino acid substituted with arginine at position 103 is the variant that reduces or reverses the positive charge of arginine.
   (b) In the amino acid sequence of the variant of (a), the erythropoetin variant consists of an amino acid sequence in which one or several amino acids other than the 103rd amino acid are deleted, substituted or added, and inhibits erythropoetin signaling.
2. The erythropoietin variant according to claim 1, wherein the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid.
3. Furthermore, in the amino acid sequence of SEQ ID NO: 1, a mutation that enhances the vicinity of the 103rd arginine to a negative electrostatic potential and / or a positively charged amino acid residue on the site 2 surface of human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1 The erythropoetin variant according to claim 1 or 2, wherein a negatively charged mutation has been introduced.
4. The mutation that enhances the negative electrostatic potential around the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is selected from at least the group consisting of the 107th threonine and the 110th arginine in the amino acid sequence of SEQ ID NO: 1. The erythropoetin variant according to claim 3, wherein it is a substitution of one amino acid, and another amino acid substituted with the amino acid at the site reduces or reverses the positive charge of the amino acid at the site.
5. The erythropoietin variant according to claim 4, wherein at least one amino acid selected from the group consisting of threonine at position 107 and arginine at position 110 in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid.
6. The mutation that makes the positively charged amino acid residue on the surface of the site 2 surface of human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1 negatively charged is the substitution of the 110th arginine in the amino acid sequence of SEQ ID NO: 1, and the 110th arginine. The erythropoetin variant according to claim 3, wherein another amino acid substituted with is one that reduces or reverses the positive charge of arginine at position 110.
7. The erythropoietin variant according to claim 6, wherein the 110th arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid.
8. One of the following erythropoietin variants.
   (1) An erythropoietin variant (mEPO3: R103D) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid.
   (2) An erythropoietin variant (mEPO4: R103E) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with glutamic acid.
   (3) An erythropoetin variant (mEPO 4.4:) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 107th threonine is replaced with aspartic acid or glutamic acid. R103E / T107E)
   (4) In the amino acid sequence of SEQ ID NO: 1, the 103rd arginine is replaced with aspartic acid or glutamic acid, the 107th threonine is replaced with aspartic acid or glutamic acid, and the 110th arginine is replaced with aspartic acid or glutamic acid. Erythropoetin variant consisting of the amino acid sequence (mEPO4.4.10: R103E / T107E / R110E)
   (5) An erythropoetin variant (mEPO 4.10:) consisting of an amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with aspartic acid or glutamic acid and the 110th arginine is replaced with aspartic acid or glutamic acid. R103E / R110E)
9. A polynucleotide comprising a nucleotide sequence encoding the erythropoietin variant according to any one of claims 1 to 8 or a sequence complementary thereto.
10. A vector containing the polynucleotide according to claim 9.
11. A cell comprising the vector according to claim 10.
12. The method for producing the erythropoietin mutant according to (a) or (b) below, which comprises culturing the cells according to claim 11.
    (a) From the amino acid sequence in which the 103rd arginine in the amino acid sequence of SEQ ID NO: 1 is replaced with another amino acid, which is an erythropoetin variant in which a mutation is introduced into human erythropoetin consisting of the amino acid sequence of SEQ ID NO: 1. And another amino acid substituted with arginine at position 103 is the variant that reduces or reverses the positive charge of arginine.
    (b) In the amino acid sequence of the variant of (a), the erythropoetin variant consists of an amino acid sequence in which one or several amino acids other than the 103rd amino acid are deleted, substituted or added, and inhibits erythropoetin signaling.
13. Erythropoietin comprising at least one selected from the group consisting of the erythropoietin variant according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, and the cell according to claim 11. A composition for inhibiting signal transduction.
14. JAK2 comprising at least one selected from the group consisting of the erythropoietin variant according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, and the cell according to claim 11. A composition for inhibiting activity.
15. JAK2 comprising at least one selected from the group consisting of the erythropoietin variant according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10 and the cell according to claim 11. Composition for inhibiting cell proliferation by.
16. JAK2 comprising at least one selected from the group consisting of the erythropoietin mutant according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, and the cell according to claim 11. A composition for preventing and / or treating a disease involving a mutation.
17. The composition according to claim 16, wherein the disease in which the JAK2 mutation is involved is a tumor.
18. 17. The composition of claim 17, wherein the tumor is a myeloproliferative neoplasm or solid tumor.
19. The composition according to claim 18, wherein the myeloproliferative neoplasm is polycythemia vera, essential platelet disease, or primary myelofibrosis.
20. A pharmaceutical agent comprising at least one selected from the group consisting of the erythropoietin mutant according to any one of claims 1 to 8, the polynucleotide according to claim 9, the vector according to claim 10, and the cell according to claim 11. ..

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