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  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
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  • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

• the present invention relates to a modified protein C polypeptide, and more particularly to a novel modified protein C polypeptide into which a self-cleavage site has been inserted, and a pharmaceutical composition containing an activated protein C protein produced from the polypeptide. .
• Protein S (PS), protein C (PC), and antithrombin (AT) deficiencies are the three major congenital thrombotic predispositions in Japanese people. Both are autosomal dominant (dominant) inherited diseases, with heterozygous mutation carriers causing deep vein thrombosis, and homozygous and compound heterozygous severe forms causing purpura fulminans in newborns. Decreased activity of these three factors was identified in 65% of Japanese adults who developed deep vein thrombosis, and gene mutations were identified in about half of them. The full scope of the perinatal maternal and infant area is not clear.
• Protein C is a blood protein that acts as a physiological anticoagulant factor. It is activated by thrombin at the site of thrombus formation and exerts an anticoagulant effect by cleaving activated factor V and activated factor VIII, functioning as a so-called "coagulation brake.” Congenital abnormalities in the protein C gene (PROC) disrupt the balance between coagulation and anticoagulation, leading to a tendency toward thrombosis.
• PROC protein C gene
• Heterozygotes 0.16%, homozygotes: approximately 1 per 500,000 people.
• Heterozygous patients have protein C activity in the blood reduced to 30-50% of normal. Although the disease is often asymptomatic until adolescence, infections, trauma, surgery, pregnancy, etc. can lead to the development of deep vein thrombosis, pulmonary thromboembolism, etc.
• Homozygous patients have reduced protein C activity in the blood to less than 5% of normal. It causes a fulminant hemorrhagic condition called purpura fulminans in the neonatal period.
• purpura fulminans in the neonatal period.
• lower extremity deep vein thrombosis, thrombophlebitis, and associated pulmonary thromboembolism occur repeatedly from a young age. Arterial thrombosis is rare.
• Anact C (registered trademark), an activated protein C (APC) preparation, is used to treat neonatal purpura fulminans.
• Anact C® is plasma-derived and requires plasma for production. Therefore, there is a risk of unknown infectious diseases. Furthermore, Anact C has an extremely short half-life and requires continuous administration, and there are no results of long-term administration. Since the drug product has a short half-life and requires continuous administration, anticoagulant therapy such as warfarin is used during the stable phase, but anticoagulant therapy is difficult to control, and thrombotic tendencies or, conversely, drug-induced bleeding tendencies can easily occur. Occasionally, fatal cerebral hemorrhage associated with anticoagulation is experienced.
• the blood concentration of protein C is reported to be 70 nM, and the concentration of the active form is 40 pM, and the blood concentration of the active form remains less than 0.1% of the total. Therefore, the emergence of an epoch-making drug that can cure congenital protein C deficiency is awaited.
• Patent Document 1 describes human protein C derivatives that have increased anticoagulant activity compared to wild-type protein C and that exhibit the biological activity of wild-type human protein C. These derivatives may be administered less frequently and/or in the treatment of acute coronary syndromes, vascular occlusive disorders, hypercoagulable states, thrombotic disorders and thrombosis-prone disease states than wild-type human protein C.
• the amino acid Ser at position 11 of wild-type human protein C is replaced with Gly and the amino acid Ser at position 12 is replaced with Asn, the maximum It has been described to exhibit 4 times higher anticoagulant activity.
• Patent Document 2 describes a human protein C derivative, which has enhanced anticoagulant activity, resistance to serpin inactivation, and resistance to thrombin activation compared to wild type protein C. Having an enhanced susceptibility and retaining the biological activity of wild-type human protein C, the derivative is susceptible to acute coronary syndromes, vascular occlusive disorders, hypercoagulable states, thrombotic disorders and thrombosis. Although the potential for requiring either less frequent administration and/or lower dosages than wild-type human protein C in the treatment of disease states has been described, specific anticoagulant activity relative to wild-type is not shown.
• Patent Document 3 describes a protein C derivative, but does not show any specific activity compared to the wild type.
• U.S. Pat. No. 5,050,200 describes protein C derivatives, which show that these polypeptides retain the biological activity of wild-type human protein C and have a substantially longer half-life in human blood.
• the polypeptide may be administered less frequently and/or less frequently than wild-type human protein C in the treatment of vaso-occlusive diseases, hypercoagulable states, thrombotic diseases, and disease states predisposing to thrombosis.
• the amino acid at position 194 of the wild type protein C sequence was substituted from Leu to Ser, it was reported that the in vivo stability was approximately four times higher than that of the wild type. ing.
• Patent Document 5 describes a transformed cell containing a nucleotide sequence encoding a protein that is cleavable by furin and exhibits an Arg-(Lys/Arg)-Arg motif, and describes protein C as an option for the protein.
• Patent Document 6 describes a composition comprising a modified blood coagulation factor having a normally non-existent proteolytic cleavage site that has been engineered to allow intracellular cleavage and secretion of the active form, and which is used as a blood coagulation factor. , protein C has been described.
• Patent Documents 1 to 4 do not describe the insertion of a self-cleavage site into protein C.
• Patent Documents 5 and 6 neither disclose nor suggest an amino acid sequence in which a self-cleavage site is inserted at a specific position in the protein C polypeptide of the present invention.
• An object of the present invention is to provide an innovative activated protein C that can cure protein C deficiency, a pharmaceutical composition containing the same, and the like.
• the present inventors inserted a self-cleavage site into the site of protein C that is cleaved by thrombin to produce the polypeptide of the present invention. It was found that the polypeptide was expressed in cultured cells, and protein C activity and anticoagulant effect were observed in the supernatant of the culture solution without artificial activation. Furthermore, the present inventors expressed the polypeptide of the present invention in vivo in mice using a viral vector, and found that protein C activity and anticoagulant effect increased in the blood, leading to the completion of the present invention.
• a 1 -A 2 -A 3 (I) (In the formula, A1 is an amino acid sequence that includes the light chain amino acid sequence of protein C or its homologue, A2 is an amino acid sequence that constitutes a self-cleavage site, and A3 is the amino acid sequence of the heavy chain of protein C or its homolog. (indicates the amino acid sequence containing) A polypeptide or a partial polypeptide thereof, which contains an amino acid sequence represented by A polypeptide or a partial polypeptide thereof having protein C activity.
• the polypeptide meets the following conditions: (1) The amino acid sequence (formula: A 1 -A 3 (II)) in which A 1 is connected to the N-terminal side and A 3 is connected to the C-terminal side includes the amino acid sequence of SEQ ID NO: 2; (2) An amino acid sequence in which A1 is connected to the N-terminal side and A3 is connected to the C-terminal side is the deletion, substitution, insertion, or addition of 1 to 45 amino acids in the amino acid sequence represented by SEQ ID NO: 2. or (3) an amino acid sequence in which the amino acid sequence in which A 1 is connected to the N-terminus and A 3 is connected to the C-terminus has 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2.
• A2 is RKRRKR (SEQ ID NO: 3), KRRKR (SEQ ID NO: 4), RKR, KR, RHQR (SEQ ID NO: 5), RSKR (SEQ ID NO: 6), ATNFSLLKQAGDVEENPGP (P2A) (SEQ ID NO: 7), RKRRKRRKR (SEQ ID NO: 8), RKRRKRRKRRKR (SEQ ID NO: 9), or a partial polypeptide thereof according to [1] or [2].
• polypeptide or partial polypeptide thereof according to any one of [1] to [3], wherein A 2 is RKRRKR (SEQ ID NO: 3) or KRRKR (SEQ ID NO: 4).
• a 2 is RKRRKR (SEQ ID NO: 3) or KRRKR (SEQ ID NO: 4).
• polypeptide comprises the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14.
• [6] A dimeric protein consisting of a fragment on the N-terminal side and a fragment on the C-terminal side of the A2 cleavage site of the polypeptide according to any one of [1] to [5] or a partial polypeptide thereof, or A protein or a partial protein thereof, wherein the protein or partial protein has protein C activity.
• a nucleic acid comprising a nucleotide sequence encoding the polypeptide according to any one of [1] to [5] or a partial polypeptide thereof.
• a vector comprising the nucleic acid according to [7].
• the vector according to [8], wherein the vector is an expression vector.
• the vector according to [8], wherein the vector is a donor vector.
• the viral vector is an adeno-associated virus (AAV) vector.
• a host cell population comprising the host cell according to [14].
• polypeptide according to any one of [1] to [5] or a partial polypeptide thereof, the protein according to [6] or a partial protein thereof, the nucleic acid according to [7], [8] to [13] ] A pharmaceutical composition comprising the vector according to any one of [14], the host cell according to [14], or the host cell population according to [15].
• a pharmaceutical composition comprising the vector according to any one of [10] to [13] and a vector comprising a nucleic acid encoding a nucleic acid metabolic enzyme.
• a vector in which the nucleic acid metabolic enzyme is a CRISPR/Cas9-based nucleic acid metabolic enzyme, further comprising a vector containing a nucleic acid encoding a guide RNA, or a vector comprising a nucleic acid encoding a guide RNA together with a nucleic acid encoding a nucleic acid metabolic enzyme.
• the thrombosis is selected from the group consisting of venous thrombosis, disseminated intravascular coagulation, (neonatal) purpura fulminans, deep vein thrombosis pulmonary thromboembolism, and thrombosis associated with novel coronavirus infection. , [20].
• a method for producing protein C-expressing cells which comprises introducing the vector according to any one of [8] to [13] into mammalian cells in vitro.
• a method for producing a recombinant preparation of protein C comprising producing protein C-expressing cells by the method described in [22], isolating and purifying activated protein C from the cells, and formulating it. .
• a method for treating or preventing thrombosis which comprises administering to a subject the vector according to [12] or [13] or the pharmaceutical composition according to any one of [16] to [18].
• the thrombosis is selected from the group consisting of venous thrombosis, disseminated intravascular coagulation, (neonatal) purpura fulminans, deep vein thrombosis, and acute pulmonary thromboembolism.
• activated protein C can be obtained from the culture supernatant of cultured cells expressing the polypeptide. Furthermore, protein C activity and anticoagulant effect in blood can be increased by expressing the polypeptide of the present invention in vivo using a viral vector.
• FIG. 1 is a diagram showing the structure of protein C and the inserted self-cleavage sequence.
• Figure 5 shows an AAV8-type vector expressing SaCas9 (Cas9) used for genome editing therapy, and a wild-type PC sequence combined with a P2A sequence having homologous recombination sequences (approximately 1 kb) at the gene locus at both ends.
• SaCas9 SaCas9
• FIG. 6 is a diagram showing the results of testing the blood coagulation inhibitory effect of activated mouse protein C.
• AAV type 8 vector expressing wild-type mouse protein C sequence (mPC) or modified mouse protein C sequence (mPC variant) was administered once intravenously, and blood was collected from 4 to 8 weeks after administration to collect plasma. .
• Figure 8 shows active oxygen-dependent pathological thrombosis in mice that received a single intravenous administration of an AAV type 8 vector expressing wild-type mouse protein C sequence (mPC) or modified mouse protein C sequence (mPC variant).
• FIG. 9 is a diagram showing the results of evaluating the phenotype of protein C-deficient mice. Two types of AAV vectors were administered to neonatal mice (Proc-/-) born from crossbreeding of protein C-deficient mice (Proc+/-), and activated protein C was expressed in the neonatal livers through genome editing. Ta.
• the polypeptide of the present invention has the formula: A 1 -A 2 -A 3 (I)
• A1 is an amino acid sequence that includes the light chain amino acid sequence of protein C or its homologue
• A2 is an amino acid sequence that constitutes a self-cleavage site
• A3 is the amino acid sequence of the heavy chain of protein C or its homolog. (indicates the amino acid sequence containing)
• a polypeptide or a partial polypeptide thereof, which contains an amino acid sequence represented by A polypeptide or a partial polypeptide thereof having protein C activity is provided.
• amino acid residues and peptides are written with the N-terminus on the left and the C-terminus on the right, according to conventional methods.
• protein C is one of the blood coagulation control regulators in the blood coagulation system of vertebrates, and is a complex of thrombin generated during the blood coagulation process and thrombomodulin on vascular endothelial cells. refers to the precursor of a vitamin K-dependent serine protease that is activated by V. a. and specifically inactivates blood coagulation factors Va and VIII a through proteolysis. Most protein C is a heterodimer consisting of a heavy chain and a light chain, which are modified with sugar chains, and are linked by disulfide bonds between cysteine residues in the heavy chain and cysteine residues in the light chain. There is.
• human protein C the heavy chain (41 kDa) and light chain (21 kDa) are linked by a disulfide bond between Cys183 and Cys319.
• Protein C is mainly synthesized in the liver.
• human protein C consists of a signal peptide (positions 1-32), a light chain (positions 43-88) from the N-terminal side (the light chain consists of a Gla domain (positions 43-88), a helical aromatic segment (positions 89-96), ), two EGF-like domains (positions 97-132 and 136-176)), an activation peptide (positions 200-211), and a heavy chain (positions 212-461) (the heavy chain is a trypsin-like serine protease domain). (including positions 212-450)), and is translated as a full-length 461 amino acid polypeptide.
• activated protein C refers to a form that has serine protease activity and is generated when the activation peptide bound to the N-terminus of the heavy chain of protein C is excised by thrombin.
• activated protein C refers to a dimeric protein consisting of an N-terminal fragment and a C-terminal fragment formed by self-cleavage of A2 from the polypeptide of the present invention, or its dimeric protein. Refers to a partial protein.
• protein C activity refers to serine protease activity, which specifically inhibits blood coagulation factors Va and VIIIa through proteolysis in the blood coagulation system of the biological species that has the same origin as protein C. Refers to the activity to be activated.
• protein C activity is measured using, for example, the activity measuring reagent Verichrome Protein C (Sysmex, Kobe, Japan) using a fully automatic blood coagulation analyzer CS1600 (Sysmex). can do.
• the composition of the kit is as follows. 1. Protein C activator snake venom 3.0U/vial2. Substrate reagent pyro-glutamic acid-proline-arginine-methoxynitroanilide (p-Glu-Pro-Arg-MNA) 4 mmol/L 3. buffer solution
• the origin of protein C is not particularly limited as long as it is a vertebrate, and examples of the vertebrate include mammals, birds, reptiles, amphibians, fish, etc.
• the fish include tilapia, Thai , flounder, shark, and salmon; amphibians include frogs and newts; reptiles include crocodiles, turtles, and lizards; birds include chickens, quail, , ducks, geese, ostriches, and guinea fowl;
• mammals include rodents such as mice, rats, hamsters, and guinea pigs; experimental animals such as rabbits; pigs, cows, goats, horses, and sheep.
• domestic animals such as mink, pets such as dogs and cats, humans, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees, and such mammals are preferred, with humans being particularly preferred.
• the term "homolog" of protein C refers to either or both of the following (A) or (B).
• (A) Amino acid sequence of Protein C A molecule consisting of an amino acid sequence in which 1 to X amino acids are deleted, substituted, inserted, or added.
• X is 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less of the number of amino acids in the full-length protein C precursor. , 1% or less, or 0.5% or less.
• Identity herein refers to the optimal alignment of two amino acid sequences when using a mathematical algorithm known in the art (preferably, the algorithm refers to the ratio (%) of identical and similar amino acid residues to all overlapping amino acid residues (in which the introduction of a gap in one or both may be considered).
• Similar amino acids means amino acids similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, He, Val), polar amino acids (Gln, Asn ), basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is predicted that such a substitution with a similar amino acid will not result in a change in the protein phenotype (ie, it is a conservative amino acid substitution).
• the polypeptides of the invention meet the following conditions: (1) The amino acid sequence (formula: A 1 -A 3 (II)) in which A 1 is connected to the N-terminal side and A 3 is connected to the C-terminal side includes the amino acid sequence of SEQ ID NO: 2; (2) An amino acid sequence in which A 1 is connected to the N-terminus and A 3 is connected to the C-terminus is the deletion, substitution, insertion, or addition of 1 to 50 amino acids in the amino acid sequence represented by SEQ ID NO: 2. or (3) an amino acid sequence in which the amino acid sequence in which A 1 is connected to the N-terminus and A 3 is connected to the C-terminus has 90% or more identity with the amino acid sequence represented by SEQ ID NO: 2. Contains arrays.
• the amino acid sequence containing A 1 on the N-terminal side and A 3 on the C-terminal side includes 1 to 2, 1 to 3, and 1 in the amino acid sequence represented by SEQ ID NO: 2.
• the amino acid sequence containing A 1 on the N-terminal side and A 3 on the C-terminal side contains 1 to 2, 1 to 3, or 1 to 3 amino acids in the amino acid sequence represented by SEQ ID NO: 2.
• the amino acid sequence that is linked with A1 on the N-terminal side and A3 on the C-terminal side contains an amino acid sequence that is 70% or more, 75% or more, 80% or more of the amino acid sequence represented by SEQ ID NO: 2, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more identical or have 100% identity.
• the polypeptide of the invention is (a-1) A 1 contains the amino acid sequence represented by SEQ ID NO: 15; (a-2) A 1 contains an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 15; or (a-3) A 1 contains an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 15; and (b-1) A 3 contains the amino acid sequence represented by SEQ ID NO: 16; (b-2) A3 contains an amino acid sequence in which 1 to 30 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 16; or (b-3) A 3 is a polypeptide containing an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 16, and the active protein formed by self-cleavage of A2 has protein C activity.
• the polypeptide of the invention is (c-1) A 1 contains the amino acid sequence represented by SEQ ID NO: 15, and A 3 contains the amino acid sequence represented by SEQ ID NO: 16; (c-2) A 1 contains an amino acid sequence in which 1 to 50 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 15, and A 3 contains an amino acid sequence in which 1 to 50 amino acids are deleted, substituted, inserted, or added to the amino acid sequence represented by SEQ ID NO: 15, and A 3 is SEQ ID NO: 16.
• the amino acid sequence represented by SEQ ID NO: 15 contains an amino acid sequence in which 1 to 50 or more amino acids are deleted, substituted, inserted, or added; or (c-3)
• a 1 is the amino acid represented by SEQ ID NO: 15.
• A3 is a polypeptide containing an amino acid sequence having 90% or more identity with the amino acid sequence represented by SEQ ID NO: 16, and A3 is a polypeptide containing an amino acid sequence having 90% or more identity with The active protein formed by cleavage has protein C activity.
• the amino acid sequence contained in A1 is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 2 in the amino acid sequence represented by SEQ ID NO: 15. 7 pieces, 1-8 pieces, 1-9 pieces, 1-10 pieces, 1-11 pieces, 1-12 pieces, 1-13 pieces, 1-14 pieces, 1-15 pieces, 1-16 pieces, 1- This is an amino acid sequence in which 17, 1 to 18, 1 to 19, or 1 to 20 amino acids are deleted, substituted, inserted, or added.
• the amino acid sequence contained in A1 is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 in the amino acid sequence represented by SEQ ID NO: 15. pieces, 1 to 8 pieces, 1 to 9 pieces, 1 to 10 pieces, 1 to 11 pieces, 1 to 12 pieces, 1 to 13 pieces, 1 to 14 pieces, 1 to 15 pieces, 1 to 16 pieces, 1 to 17 pieces.
• SEQ ID NO: 15 is an amino acid sequence in which five amino acids are deleted, substituted, inserted, or added.
• the amino acid sequence included in A3 is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 2 in the amino acid sequence represented by SEQ ID NO: 16. 7 pieces, 1-8 pieces, 1-9 pieces, 1-10 pieces, 1-11 pieces, 1-12 pieces, 1-13 pieces, 1-14 pieces, 1-15 pieces, 1-16 pieces, 1- 17 pieces, 1-18 pieces, 1-19 pieces, 1-20 pieces, 1-21 pieces, 1-22 pieces, 1-23 pieces, 1-24 pieces, 1-25 pieces, 1-26 pieces, 1- This is an amino acid sequence in which 27, 1 to 28, 1 to 29, or 1 to 30 amino acids are deleted, substituted, inserted, or added.
• the amino acid sequence contained in A3 is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, or 1 to 7 in the amino acid sequence represented by SEQ ID NO: 16. pieces, 1 to 8 pieces, 1 to 9 pieces, 1 to 10 pieces, 1 to 11 pieces, 1 to 12 pieces, 1 to 13 pieces, 1 to 14 pieces, 1 to 15 pieces, 1 to 16 pieces, 1 to 17 pieces Deletion, substitution, or insertion of 1 to 18, 1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, or 1 to 25 amino acids , or an added amino acid sequence.
• the amino acid sequence contained in A1 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more of the amino acid sequence represented by SEQ ID NO: 15, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% identity.
• the amino acid sequence contained in A1 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the amino acid sequence represented by SEQ ID NO: 15. % or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7 % or more, 99.8% or more, or 99.9% or more, or 100% identity.
• the amino acid sequence contained in A3 is 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more of the amino acid sequence represented by SEQ ID NO: 16, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more, or 100% identity.
• the amino acid sequence contained in A3 is 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more of the amino acid sequence represented by SEQ ID NO: 16. % or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7 % or more, 99.8% or more, or 99.9% or more, or 100% identity.
• amino acid sequence represented by SEQ ID NO: 15 includes the sequence of the light chain of human wild-type protein C
• amino acid sequence represented by SEQ ID NO: 16 includes the sequence of the heavy chain of human wild-type protein C. including.
• partial polypeptide refers to a portion of a polypeptide that includes a continuous amino acid sequence of the polypeptide.
• partial polypeptide when referring to a “partial polypeptide” of the polypeptide of the present invention, the “partial polypeptide” includes a part of the continuous amino acid sequence of the polypeptide of the present invention, and includes the entire sequence of A2 .
• amino acid sequence constituting a self-cleavage site refers to a polypeptide or peptide containing a site where self-cleavage of a peptide bond in the main chain occurs, a length of 50 amino acids or less, a length of 45 amino acids or less, a length of 40 amino acids 35 amino acids or less, 30 amino acids or less, 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, 14 amino acids or less, 13 amino acids or less, 12 amino acids or less, 11 amino acids or less, 10 amino acids refers to an amino acid sequence having a length of 9 amino acids or less, 8 amino acids or less, 7 amino acids or less, 6 amino acids or less, or 5 amino acids or less.
• self-cleavage refers to cleavage due to the autolytic activity of a polypeptide or peptide consisting of an amino acid sequence constituting a self-cleavage site, as well as the polypeptide of the present invention or a partial polypeptide thereof, the protein or It also refers to cleavage of the cleavage site by another molecule (eg, protease) present in the system for producing the partial protein.
• the self-cleavage site includes an amino acid sequence that is specifically recognized by another molecule (eg, a protease) present in the system for producing the protein of the invention or a partial protein thereof. It also refers to cleavage caused by ribosome skipping of peptide bonds between amino acids within the amino acid sequence that constitute the self-cleavage site during polypeptide synthesis.
• A2 is, for example, RKRRKR (SEQ ID NO: 3), KRRKR (SEQ ID NO: 4), RKR, KR, RHQR (SEQ ID NO: 5), RSKR (SEQ ID NO: 6), ATNFSLLKQAGDVEEN PGP (P2A).
• RSKR SEQ ID NO: 6
• ATNFSLLKQAGDVEEN PGP P2A
• P2A ATNFSLLKQAGDVEEN PGP
• RKRRKR SEQ ID NO: 3
• KRRKR SEQ ID NO: 4
• polypeptide of the present invention comprises or consists of the amino acid sequence represented by SEQ ID NO: 13 or SEQ ID NO: 14.
• Protein in the polypeptide of the present invention the amino acid sequence constituting the self-cleavage site is cleaved at the cleavage site, resulting in activated protein C (hereinafter also referred to as the protein of the present invention).
• partial protein refers to a part of a protein that includes part of a continuous amino acid sequence of the protein.
• the "partial protein” includes the entire fragment on the N-terminal side of the A2 cleavage site of the polypeptide of the present invention and a part of the fragment on the C-terminal side, It includes a part of the N-terminal fragment and all of the C-terminal fragment, or a part of the N-terminal fragment and a part of the C-terminal fragment.
• a "partial protein” of the protein of the present invention is produced by processing the protein of the present invention in vivo or within cells.
• the amino acid sequence of the fragment on the N-terminal side of the A2 cleavage site of the polypeptide of the present invention is the same as the amino acid sequence of the light chain of wild-type protein C.
• the polypeptide or partial polypeptide thereof, protein or partial protein thereof of the present invention is preferably isolated.
• isolated means that an operation is performed to remove factors other than the target component, and the component is removed from its naturally existing state.
• the purity of the "isolated protein or its partial protein” (the percentage of the weight of the target protein or its partial protein in the total weight of the evaluation target) is usually 70% or more, preferably 80% or more, more preferably is 90% or more, more preferably 99% or more.
• the polypeptide or partial polypeptide thereof, protein or partial protein thereof of the present invention may be in the form of a salt.
• physiologically acceptable acids eg, inorganic acids, organic acids
• bases eg, alkali metals
• physiologically acceptable acid addition salts are particularly preferred.
• Such salts include, for example, salts with inorganic acids (e.g. hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or with organic acids (e.g. acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Salts with tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, etc. are used.
• nucleic Acids The present invention provides nucleic acids comprising nucleotide sequences encoding the polypeptides of the present invention or partial polypeptides thereof.
• the nucleic acid encoding the polypeptide of the present invention or a partial polypeptide thereof may be DNA or RNA, or may be a DNA/RNA chimera. Furthermore, the nucleic acid may be double-stranded or single-stranded. If it is double-stranded, it may be double-stranded DNA, double-stranded RNA, or a DNA:RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).
• DNA nucleic acid encoding the polypeptide of the present invention or a partial polypeptide thereof examples include synthetic DNA.
• full-length protein C cDNA for example, in the case of human, SEQ ID NO: 1
• the base sequence represented by can be amplified and obtained.
• it can also be obtained by cloning from a cDNA library prepared by inserting (a fragment of) the above cDNA into an appropriate vector, by colony or plaque hybridization, PCR, or the like.
• Vectors used in the library may be bacteriophages, plasmids, cosmids, phagemids, or the like.
• the nucleic acid of the present invention has an enhancer, a promoter, a transcription initiation signal, a splicing signal, a transcription termination signal, a polyA addition signal, a cap structure, a 5' untranslated region, a Kozak sequence, and an internal ribosome introduction that can exhibit activity in host cells. (IRES), 3' untranslated region, etc. in some cases.
• IRS internal ribosome introduction that can exhibit activity in host cells.
• the nucleic acid of the present invention is more stably transcribed and translated within a host cell.
• the nucleic acid of the present invention may be linked to sequences (homology arms) homologous to sequences before and after a site in the genome sequence of an organism. With such a configuration, the nucleic acid of the present invention can be integrated into the genome sequence of an organism by homologous recombination.
• the nucleic acid comprises a codon-optimized nucleotide sequence encoding a polypeptide described herein or a partial polypeptide thereof. Codon optimization of nucleotide sequences is believed to increase translation efficiency of mRNA transcripts. Codon optimization of a nucleotide sequence may involve replacing a native codon with another codon that encodes the same amino acid but can be translated by tRNAs that are more readily available in the cell, thus reducing translation efficiency. rises. Nucleotide sequence optimization may also reduce mRNA secondary structure that interferes with translation, thus increasing translation efficiency.
• the invention also provides nucleotide sequences that are complementary to, or hybridize under stringent conditions to, the nucleotide sequences of any of the nucleic acids described herein.
• a nucleic acid comprising a nucleotide sequence is provided.
• High stringency conditions mean that the nucleotide sequences hybridize specifically to the nucleotide sequences of the nucleic acids described herein in an amount detectably greater than non-specific hybridization. means.
• High stringency conditions include low salt conditions and/or high temperature conditions, such as, for example, a temperature of about 50-70° C., about 0.02-0.1 M NaCl. It is generally understood that conditions can be made more stringent by increasing the amount of formamide added.
• the present invention also provides for at least about 70% or more, e.g., about 80%, about 90%, about 91 %, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% identical.
• the nucleic acid may consist of the nucleotide sequences described herein.
• the present invention provides a vector containing the above-mentioned nucleic acid of the present invention.
• the vector may be a cloning vector, such as a bacteriophage, plasmid, cosmid, phagemid, etc.
• the vector is an expression vector.
• the above-described nucleic acid of the present invention or the nucleic acid encoding the same is functionally linked to a promoter that can exhibit promoter activity in the cells of the organism to which it is administered (also referred to as host cells, for example, human cells). is connected to.
• Those skilled in the art can select an appropriate promoter depending on the type of host cell.
• the host cell is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, ⁇ PL promoter, lpp promoter, T7 promoter, etc. are used, but the promoter is not limited to these.
• the SPO1 promoter, SPO2 promoter, penP promoter, etc. are used, but the promoter is not limited to these.
• the host cell is yeast, Gal1 promoter, Gal1/10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are used, but are not limited to these.
• polyhedrin promoter, P10 promoter, etc. are used, but are not limited to these.
• CaMV35S promoter, CaMV19S promoter, NOS promoter, etc. are used, but are not limited to these.
• SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus) Viral thymidine kinase) promoters can be used, but are not limited to these.
• a polI promoter when the host cell is a human cell, a polI promoter, a polII promoter, a polIII promoter, etc. can be used.
• SV40-derived early promoters viral promoters such as cytomegalovirus LTR, mammalian constituent protein gene promoters such as ⁇ -actin gene promoters, and RNA promoters such as tRNA promoters are used.
• RNA promoters such as tRNA promoters
• polIII promoters include U6 promoter, H1 promoter, tRNA promoter, and the like.
• an expression vector in addition to the above, one containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication (hereinafter sometimes abbreviated as SV40 ori), etc. may be used as desired. I can do it.
• selectable markers include dihydrofolate reductase gene (hereinafter sometimes abbreviated as dhfr, methotrexate (MTX) resistance), neomycin resistance gene (hereinafter sometimes abbreviated as neor, G418 resistance), etc. It will be done.
• dhfr gene-deficient Chinese hamster cells and using the dhfr gene as a selection marker the target gene can also be selected using a thymidine-free medium.
• Expression vectors of the present invention include the pUC series (Fermentas Life Sciences), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), and the pGEX series (Pharmacia Biosciences). otech, Uppsala, Sweden), and pEX series (Clontech, Palo Alto, Calif.) may be used.
• Bacteriophage vectors such as ⁇ GT10, ⁇ GT11, ⁇ ZapII (Stratagene), ⁇ EMBL4 and ⁇ NM1149 can also be used.
• the recombinant expression vector is a viral vector, eg an AAV vector.
• the recombinant expression vector is an AAV vector carrying an ITR.
• the present invention provides a donor vector that can provide the above-mentioned nucleic acid of the present invention as donor DNA in gene homologous recombination.
• the donor vector contains a site in the genome sequence of a cell of an organism to be administered (also referred to as a host cell, for example, a human cell) into which the above-described nucleic acid of the present invention is integrated by homologous recombination.
• the preceding and succeeding sequences contain homologous sequences.
• the site in the genome sequence is not particularly limited, and is a sequence that exists in the form of being inserted between genes, and that even if the sequence is changed, it does not affect the survival of the cell. It is preferable that there be.
• the nucleic acid of the present invention in the donor vector, is located in the sequence before and after the site in the genome sequence of the cell of the organism to which the above-mentioned nucleic acid of the present invention is integrated by homologous recombination. Linked to homologous sequences (homology arms).
• the site in the genome sequence may be a site present in a gene on the genome, and such a gene includes, for example, a site in the Alb (ALB) gene locus.
• the homologous sequence comprises a selected target nucleotide sequence (described below) within the sequence, and within the target nucleotide sequence, DNA (donor DNA (e.g., donor vector) and/or genomic duplex This is a sequence that has sufficient sequence identity and length to cause homologous recombination with genomic DNA containing the sequence when the DNA) is cut.
• the homologous sequence comprises a sequence homologous to (part of) a sequence upstream and/or downstream of a selected target nucleotide sequence in the sequence, which is attached to the nucleic acid of the invention as a homology arm. and have sufficient sequence identity and length to cause homologous recombination to the genomic DNA containing the target nucleotide sequence when the genomic double-stranded DNA is cleaved within the target nucleotide sequence. It is an array.
• the degree of identity of the homologous sequence to the sequence is not particularly limited as long as homologous recombination is possible.
• the degree of identity that enables homologous recombination varies depending on the length of the polynucleotide, but is, for example, at least about 80% or more, preferably at least about 85% or more, more preferably at least about 90% or more, and most preferably It can be about 95-100%.
• the length of the homologous sequence is not particularly limited as long as it is long enough to allow homologous recombination with genomic DNA. However, in general terms, the longer the homologous region, the better for homologous recombination with genomic DNA to occur efficiently. On the other hand, the length of DNA that can be inserted is limited by the efficiency of introducing donor DNA (eg, donor vector) into cells. Therefore, considering these, the length of the homologous sequence is, for example, 0.15 kb to 20 kb, 0.18 kb to 10 kb, 0.2 kb to 8 kb, 0.3 kb to 5 kb, 0.5 kb to 2 kb, 0.7 kb to It can be 1kb.
• the homologous sequence includes a nucleotide sequence homologous to the sequence or a partial sequence thereof existing in the genome.
• the homologous sequence is prepared from a host cell by, for example, synthesizing an oligo DNA primer to cover a region encoding a desired portion (a portion containing a target nucleotide sequence described below) based on the DNA sequence information of the sequence. Cloning can be performed by amplifying by PCR using the obtained genomic DNA as a template. It is also possible to use the sequence cloned from a species other than the host cell, as long as the degree of identity that allows the above-mentioned homologous recombination is maintained.
• the donor vector may further contain a selection marker gene for selecting transformants in which the nucleic acid of the present invention has been inserted into the genome.
• selectable marker genes include, but are not limited to, genes that confer resistance to drugs such as tetracycline, ampicillin, and kanamycin, and genes that complement auxotrophic mutations. Genes that complement auxotrophic mutations are used in combination with host cells that have the corresponding auxotrophic mutations.
• Donor vectors used in the method of the present invention include double-stranded DNA, single-stranded DNA (circular double-stranded DNA, linear double-stranded DNA, circular single-stranded DNA, linear single-stranded DNA). , circular double-stranded DNA containing single-stranded DNA.
• “bp" shall be read as "b”.
• the donor vector will be explained mainly by exemplifying circular double-stranded DNA as a representative example, but the explanation is equally applicable to other donor DNAs other than circular double-stranded DNA. can be easily understood by those skilled in the art.
• the circular double-stranded DNA is a circular double-stranded DNA plasmid.
• Circular double-stranded DNA plasmids include Escherichia coli-derived plasmids (e.g., pBR322, pBR325, pUC12, pUC13); Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, pC194); yeast-derived plasmids (e.g., YCplac33, pRS403, YIplac128); insect cell expression plasmids (e.g. pFast-Bac); plant cell expression plasmids (e.g.
• animal cell expression plasmids e.g. pCAGGS, pSR ⁇ , pA1- 11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo
• animal cell expression plasmids e.g. pCAGGS, pSR ⁇ , pA1- 11, pXT1, pRc/CMV, pRc/RSV, pcDNAI/Neo
• the donor vector of the present invention can be an adeno-associated virus (AAV) vector, an adenovirus vector, a lentivirus vector, a Sendai virus vector, a retrovirus vector.
• AAV adeno-associated virus
• the vector is an AAV vector.
• the AAV vector may be a vector of serotype AAV2, AAV3, AAV6, AAV7, AAV8, AAV9, which is directed against hepatocytes.
• Adeno-associated viruses are members of the parvoviridae family and contain linear, single-stranded DNA genomes of less than about 5 kb of nucleotides.
• AAV requires co-infection with a helper virus (ie, adenovirus or herpesvirus) or expression of helper genes for efficient replication.
• helper virus ie, adenovirus or herpesvirus
• helper genes for efficient replication.
• AAV vectors used for administration of therapeutic nucleic acids contain approximately 50% of the parental genome so that only the inverted terminal repeats (ITRs), which contain the recognition signals for DNA replication and packaging, remain. 96% are deleted. This eliminates immunological or toxic side effects due to viral gene expression.
• AAV vectors containing AAV ITRs into specific regions of the cell's genome is possible as needed (e.g., in the US See Patent Nos. 6,342,390 and 6,821,511).
• Host cells containing integrated AAV genomes do not exhibit changes in cell proliferation or morphology (see, eg, US Pat. No. 4,797,368).
• the invention provides host cells containing the vectors of the invention.
• the term "host cell” refers to any type of cell that can contain a vector of the invention.
• the host cell may be a eukaryotic cell, eg a plant, animal, fungus or algae, or a prokaryotic cell, eg a bacterium.
• the host cell may be a cultured cell or a primary cell, ie, one isolated directly from an organism, eg, a human.
• Host cells can be adherent cells or cells in suspension. Suitable host cells are known in the art and include, for example, DH5 ⁇ E.
• the host cell is, for example, a prokaryotic cell, such as a DH5 ⁇ cell.
• the host cell is, for example, a vertebrate cell.
• the host cell is a mammalian cell, more preferably a human cell.
• the type of host cell, tissue of origin, and developmental stage are not limited.
• the host cell is not a transformed cell that contains a nucleotide sequence encoding a proteolytic enzyme (eg, furin).
• compositions comprising the polypeptides or partial polypeptides thereof, proteins or partial proteins thereof, nucleic acids, vectors, host cells, or host cell populations of the present invention.
• the pharmaceutical composition of the present invention comprises, in addition to the polypeptide of the present invention or a partial polypeptide thereof, a protein or a partial protein thereof, a nucleic acid, a vector, a host cell, or a population of host cells, an arbitrary carrier such as a pharmaceutically acceptable carrier. can include.
• Pharmaceutically acceptable carriers include, for example, excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone. , binders such as gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch, carboxymethyl cellulose, hydroxypropyl starch, sodium glycol starch, disintegrants such as sodium bicarbonate, calcium phosphate, calcium citrate, magnesium stearate.
• excipients such as sucrose, starch, mannitol, sorbitol, lactose, glucose, cellulose, talc, calcium phosphate, calcium carbonate, cellulose, methylcellulose, hydroxypropylcellulose, polypropylpyrrolidone.
• binders such as gelatin, gum arabic, polyethylene glycol, sucrose, starch, starch,
• the pharmaceutical composition of the present invention can further contain a reagent for nucleic acid introduction.
• nucleic acid introduction reagents cationic lipids such as lipofectin, lipofectamine, DOGS (transfectam), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly(ethyleneimine) (PEI) are used.
• polysaccharides such as schizophyllan (SPG) can be used.
• retrovirus when a retrovirus is used as an expression vector, retronectin, fibronectin, polybrene, etc. can be used as an introduction reagent.
• the dosage unit form of the pharmaceutical composition of the present invention includes solutions, tablets, pills, drinking solutions, powders, suspensions, emulsions, granules, extracts, fine granules, syrups, infusions, decoctions, and eye drops. , troches, poultices, liniments, lotions, eye ointments, plasters, capsules, suppositories, enemas, injections (solutions, suspensions, etc.), patches, ointments, jellies, pasta Examples include agents, inhalants, creams, sprays, nasal drops, and aerosols.
• the content of the polypeptide of the present invention or its partial polypeptide, protein or its partial protein, nucleic acid, or vector in the pharmaceutical composition is not particularly limited and can be appropriately selected within a wide range, but for example, It is 0.01 to 100% by weight.
• the concentration of the polypeptide of the present invention or its partial polypeptide, protein or its partial protein, nucleic acid, vector of the present invention in the pharmaceutical composition is not particularly limited and can be appropriately selected within a wide range, but for example, It is 0.01 nM to 1M, preferably 0.1 nM to 10 mM, and more preferably 1 nM to 100 nM.
• the pharmaceutical composition of the present invention is administered in various ways depending on its use. For example, in the case of an injection, it is administered intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly, or intraperitoneally.
• the pharmaceutical composition of the present invention has low toxicity and is administered parenterally (e.g., to humans or other vertebrates (e.g., mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, birds, etc.)). , intravascular administration, subcutaneous administration, etc.).
• parenterally e.g., to humans or other vertebrates (e.g., mice, rats, rabbits, sheep, pigs, cows, cats, dogs, monkeys, birds, etc.).
• the dosage of the pharmaceutical composition of the present invention varies depending on the activity and type of the active ingredient, the mode of administration, the severity of the disease, the species of animal to be administered, the drug acceptability of the subject, body weight, age, etc.
• the amount of active ingredient per day is usually about 0.001 mg/kg to about 2.0 g/kg.
• the present invention also provides pharmaceutical compositions containing host cells or populations of host cells of the present invention.
• the pharmaceutical composition of the present invention containing the host cell or host cell population of the present invention can be used, but is not limited to, the host cell or host cell population of the present invention obtained by introducing the nucleic acid or vector of the present invention. It can be obtained by suspending it in saline or an appropriate buffer (eg, phosphate buffered saline). In this case, if the number of obtained host cells or host cell populations is small, they may be cultured and grown until a predetermined number of cells is obtained.
• an appropriate buffer eg, phosphate buffered saline
• the obtained host cells or host cell population may be cultured in a conventional growth medium, such as DMEM, EMEM, RPMI-1640, F-12, ⁇ -MEM, or MSC growing medium (Bio Whittaker), but is not particularly limited. be able to.
• the culture temperature is usually in the range of about 30-40°C, preferably about 37°C.
• the CO 2 concentration usually ranges from about 1 to 10%, preferably about 5%.
• Humidity is usually in the range of about 70-100%, preferably about 95-100%.
• DMSO dimethyl sulfoxide
• serum albumin etc.
• Antibiotics and the like may be included in the pharmaceutical composition to prevent contamination and proliferation.
• other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
• Those skilled in the art can add these factors and agents to pharmaceutical compositions at appropriate concentrations.
• the number of host cells of the present invention contained in the pharmaceutical composition prepared above is determined based on the gender, age, weight, condition of the affected area, and the number of cells used in order to obtain the desired effect in treating the disease. It can be adjusted as appropriate, taking into consideration the situation.
• target individuals include, but are not limited to, mammals such as humans.
• the pharmaceutical composition of the present invention may be administered multiple times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day, once a week) until the desired therapeutic effect is obtained.
• the drug may be administered twice every day, once a week, once every two weeks, once a month, once every two months, once every three months, or once every six months).
• a therapeutically effective amount includes, for example, 1 to 10 doses of 1 ⁇ 10 3 to 1 ⁇ 10 10 cells per individual per administration.
• the total amount administered in one individual is not limited, but is 1 ⁇ 10 3 cells to 1 ⁇ 10 11 cells, preferably 1 ⁇ 10 4 cells to 1 ⁇ 10 10 cells, and more preferably 1 ⁇ 10 5 cells to 1 ⁇ 10 cells. Examples include 9 cells.
• Methods for administering the pharmaceutical composition containing the host cells or host cell populations of the present invention are not particularly limited, but include intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, and administration to the affected area.
• intravascular administration preferably intravenous administration
• intraperitoneal administration preferably intraperitoneal administration
• intraintestinal administration preferably intraintestinal administration
• subcutaneous administration preferably intracutaneous administration
• administration to the affected area preferably intravascular administration
• Preferred examples include local administration, and more preferred examples include intravascular administration and local administration.
• the host cells may be autologous or allogeneic (allogeneic, xenogeneic) to the subject to be administered, and the source of the cells is not particularly limited, but for example, When producing the drug of the present invention for administration to animals, etc. that require prevention or treatment of thrombosis, these cells must be prepared to the extent that host cells derived from donor cells can engraft into recipients. It may also be tissue compatible.
• these cells may be the subject's own cells or may be obtained from another individual having an HLA type that is the same or substantially the same as the subject's HLA type.
• substantially the same HLA type means that when the donor's HLA type is transplanted into a subject with the use of immunosuppressants, host cells derived from the donor's cells This means that it matches that of the subject to the extent that engraftment is possible. Examples include HLA types in which the main HLA (eg, the three main loci of HLA-A, HLA-B, and HLA-DR in humans, or the four loci including HLA-Cw) are the same.
• the pharmaceutical composition of the invention is for inhibiting blood coagulation.
• Suppression is a concept that includes stopping the progress of blood coagulation to the point where the progression of the disease is halted, and further includes reducing blood coagulation to the point where the disease is cured.
• the pharmaceutical composition of the invention is for the treatment or prevention of thrombosis.
• Thrombosis to be treated or prevented by the pharmaceutical compositions of the present invention includes venous thrombosis (congenital or acquired), disseminated intravascular coagulation, (neonatal) purpura fulminans, deep vein thrombosis and (acute or chronic) pulmonary thromboembolism, thrombosis associated with the new coronavirus infection.
• treatment and “prevention” do not necessarily mean complete treatment or prevention, but include various degrees of treatment or prevention.
• Treatment or prevention with a pharmaceutical composition of the invention may include treatment or prevention of symptoms of a disease.
• Prevention can also include delaying the onset of a disease or the onset of symptoms thereof, reducing the likelihood of onset of a disease.
• Viral vectors containing nucleic acids encoding the polypeptides of the present invention or partial polypeptides thereof can be prepared by known methods. Briefly, a plasmid vector for viral expression is prepared into which a nucleic acid encoding the polypeptide of the present invention or a partial polypeptide thereof and, if necessary, a nucleic acid having a desired function (e.g., an organ-specific promoter, etc.) are inserted. This may be transfected into appropriate host cells to transiently produce a viral vector containing the nucleic acid of the present invention, and then recovered.
• a plasmid vector for viral expression is prepared into which a nucleic acid encoding the polypeptide of the present invention or a partial polypeptide thereof and, if necessary, a nucleic acid having a desired function (e.g., an organ-specific promoter, etc.) are inserted. This may be transfected into appropriate host cells to transiently produce a viral vector containing the nucleic acid
• an AAV vector when preparing an AAV vector, first, leave the ITRs at both ends of the wild-type AAV genome sequence, and use the polypeptide of the present invention or a partial polypeptide thereof in place of the DNA encoding the other Rep protein and capsid protein.
• a vector plasmid into which a nucleic acid encoding a peptide is inserted is created.
• DNA encoding the Rep protein and capsid protein required for virus particle formation is inserted into a separate plasmid.
• a plasmid containing genes (E1A, E1B, E2A, VA, and E4orf6) responsible for adenovirus helper functions necessary for AAV proliferation is prepared as an adenovirus helper plasmid.
• recombinant AAV ie, AAV vector
• AAV vector recombinant AAV
• the host cell it is preferable to use a cell (for example, 293 cell, etc.) that can supply a part of the gene product (protein) of the gene responsible for the helper action, and when such a cell is used, There is no need for the adenovirus helper plasmid to carry a gene encoding a protein that can be supplied from the host cell. Since the produced AAV vector exists in the nucleus, the desired AAV vector is recovered by freezing and thawing host cells, and is separated and purified by density gradient ultracentrifugation using cesium chloride, column method, etc. A vector is prepared.
• the route of administration is not particularly limited as long as the protein of the present invention or a partial protein thereof, which is an active ingredient, can be delivered to the blood.
• a composition of the invention comprising a viral vector carrying a nucleic acid encoding a polypeptide of the invention or a partial polypeptide thereof is administered via intramuscular injection.
• the compositions may also be administered by infusion, transdermal absorption (e.g., via a transdermal patch), inhalation, topical administration to tissues, or, for example, intravenously, intraperitoneally, intrabuccally, intradermally, subcutaneously or intraarterially. administered to the patient.
• the dosage of viral vectors in the compositions of the invention is from about 1 x 109 to about 6 x 1014 vector genomes (vg)/kg, from about 1 x 1010 to about 4 x 1014 vg/kg, about 1 ⁇ 10 11 to about 2 ⁇ 10 14 vg/kg, about 1 ⁇ 10 12 to about 1 ⁇ 10 14 vg/kg, or about 5 ⁇ 10 12 to about 1 ⁇ 10 14 vg/kg. obtain.
• the present invention provides a pharmaceutical composition comprising a donor vector of the present invention and a vector comprising a nucleic acid encoding a nucleic acid metabolic enzyme.
• nucleic acid metabolic enzyme refers to a molecular complex that has DNA cleaving activity and is endowed with the ability to recognize a specific nucleotide sequence.
• the complex comprises either a nucleic acid sequence recognition module with DNA cleaving activity, or a nucleic acid sequence recognition module and a DNA cleaving domain without DNA cleaving activity.
• the “complex” includes not only one composed of multiple molecules, but also one having a nucleic acid sequence recognition module and a DNA cleavage domain in a single molecule, such as a fusion protein.
• the nucleic acid metabolic enzyme of the invention is a nuclease and comprises or consists of a protein as a constituent.
• the nucleases of the invention include components other than proteins (eg, nucleic acids).
• nucleic acid sequence recognition module refers to a molecule or molecular complex that has the ability to specifically recognize and bind to a specific nucleotide sequence (ie, target nucleotide sequence) on a DNA strand. Binding of a nucleic acid sequence recognition module to a target nucleotide sequence allows the module or a DNA cleavage domain linked to the module to act specifically on a targeted site of DNA.
• the "nucleic acid sequence recognition module” itself has DNA cleaving activity. In other embodiments, the "nucleic acid sequence recognition module” itself does not have DNA cleaving activity.
• DNA cleavage domain refers to a polypeptide that catalyzes a reaction that cleaves one or both strands of a double helix that constitutes DNA.
• polypeptide include a polypeptide of the restriction enzyme FokI.
• the target nucleotide sequence in DNA that is recognized by the nucleic acid sequence recognition module is not particularly limited as long as the module can specifically bind to it, and it may be any sequence in DNA.
• the length of the target nucleotide sequence is sufficient as long as it is sufficient for specific binding by the nucleic acid sequence recognition module, and is, for example, 12 nucleotides or more, preferably 15 nucleotides or more, more preferably 17 nucleotides or more.
• the upper limit of the length is not particularly limited, but is preferably 25 nucleotides or less, more preferably 22 nucleotides or less.
• the nucleic acid sequence recognition module includes the CRISPR-Cas system.
• the nucleic acid sequence recognition module since the nucleic acid sequence recognition module itself has DNA cleavage activity, it is not necessarily necessary to form a complex between the nucleic acid sequence recognition module and the DNA cleavage domain.
• the above CRISPR-Cas system recognizes the sequence of the target donor DNA (double-stranded DNA or single-stranded DNA) using a guide RNA having a target nucleotide sequence (however, an RNA sequence). Any sequence can be targeted simply by synthesizing an oligo DNA that can hybridize to it.
• the CRISPR/Cas system also recognizes single-stranded DNA as a substrate and has the activity of cleaving it (Ma, E., Mol. Cell, (2015) 60(3), 398-407).
• donor DNA can be double-stranded DNA, including single-stranded DNA.
• the cut end of the donor DNA is trimmed to expose the single-stranded DNA, and that single strand binds to a homologous sequence site on the chromosome, so that the donor DNA can be more efficiently integrated into the chromosome by homologous recombination.
• a nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of a Cas protein and an RNA molecule (guide RNA) consisting of a target nucleotide sequence (RNA sequence) and tracrRNA necessary for recruiting the Cas protein.
• a nucleic acid sequence recognition module using CRISPR-Cas is provided as a complex of crRNA containing RNA having the same sequence as the target nucleotide sequence, tracrRNA, and Cas.
• the Cas protein used in the present invention is not particularly limited as long as it belongs to the CRISPR system, and examples include Cas9 and Cpf1, but Cas9 is preferred.
• Cas9 include Cas9 derived from Streptococcus pyogenes (SpCas9), Cas9 derived from Streptococcus thermophilus (StCas9), and Staphylococcus pyogenes.
• the nucleic acid sequence recognition module is preferably introduced into cells in the form of a nucleic acid (expression vector) encoding the module. That is, by introducing an expression vector encoding guide RNA and Cas protein into cells and expressing the guide RNA and Cas protein, a complex of guide RNA and Cas protein is formed within the cell.
• the guide RNA and Cas protein may be encoded on the same expression vector, or may be encoded on different expression vectors.
• DNA encoding Cas can be cloned from cells that produce Cas by methods well known in the art. The obtained Cas-encoding DNA can be inserted downstream of the promoter of an expression vector depending on the host.
• DNA encoding guide RNA can be chemically synthesized by designing an oligo DNA sequence that connects a target nucleotide sequence (RNA sequence) and a known tracrRNA sequence, and using a DNA/RNA synthesizer. can.
• DNA encoding guide RNA can also be inserted into an expression vector depending on the host.
• the guide RNA and Cas may be encoded on the same expression vector, or may be encoded on different expression vectors.
• the DNA encoding Cas and the DNA encoding guide RNA and tracrRNA are inserted into the same expression vector downstream of separate promoters.
• the target nucleotide sequence in the present invention a sequence adjacent to the PAM sequence (on the 5' side or 3' side) within the sequence contained in the host cell genome is selected.
• the Cas protein is SpCas9 and the sequence is selected that is immediately adjacent to the 5' side of the PAM sequence (5'-NGG) within the sequence contained in the host cell genome.
• the Cas protein is SaCas9 and the sequence immediately adjacent to the 5' side of the PAM sequence (NNGRR(T)) within the sequence contained in the host cell genome is selected.
• the PAM sequence includes 5'-NG or 5'-NNG.
• the target nucleotide sequences of other Cas are sequences 3' to the PAM sequence.
• Sequences in the donor DNA (eg, donor vector) used in the present invention that are homologous to the sequences before and after the site in the genome sequence into which the nucleic acid of the present invention is integrated by homologous recombination include the target nucleotide sequence.
• RNA encoding Cas can be prepared, for example, by using the above-described DNA encoding Cas as a template and transcribing it into mRNA using a known in vitro transcription system.
• Guide RNA can be chemically synthesized using a DNA/RNA synthesizer by designing an oligo RNA sequence that connects a target nucleotide sequence (RNA sequence) and a known tracrRNA sequence.
• nucleotide sequences are described as DNA sequences unless otherwise specified, but if the polynucleotide is RNA, thymine (T) shall be read as uracil (U) as appropriate.
• the nucleic acid sequence recognition module includes a zinc finger motif (Japanese Patent No. 4968498), a TAL effector (Japanese Patent Application Publication No. 2013-513389), and a PPR motif (Japanese Patent Application Laid-open No. 2013-128413).
• a zinc finger motif Japanese Patent No. 4968498
• a TAL effector Japanese Patent Application Publication No. 2013-513389
• a PPR motif Japanese Patent Application Laid-open No. 2013-128413
• the nucleic acid sequence recognition module can be provided as a fusion protein with the DNA cleavage domain, or can recognize protein binding domains such as SH3 domain, PDZ domain, GK domain, GB domain and their binding partners by recognizing the nucleic acid sequence.
• the module may be fused to a DNA cleavage domain, respectively, and provided as a protein complex through interaction between the protein binding domain and its binding partner.
• inteins can be fused to the nucleic acid sequence recognition module and the DNA cleavage domain, respectively, and the two can be linked by ligation after each protein is synthesized.
• the nucleic acid sequence recognition module or the nucleic acid sequence recognition module and the DNA cleavage domain, may each be present as a nucleic acid encoding a fusion protein thereof, or in a form capable of forming a complex within a host cell after translation into a protein.
• it is prepared as a nucleic acid encoding a constituent factor.
• the nucleic acid may be DNA or RNA.
• DNA it is preferably double-stranded DNA, and is provided in the form of an expression vector capable of expressing each component under the control of a functional promoter in the host cell.
• RNA it is preferably single-stranded RNA.
• DNA encoding a nucleic acid sequence recognition module such as a zinc finger motif, TAL effector, or PPR motif can be obtained by any of the methods described in the above-mentioned literature for each module.
• DNA encoding a sequence recognition module such as a restriction enzyme, a transcription factor, or an RNA polymerase covers a region encoding a desired portion of the protein (a portion containing a DNA binding domain), for example, based on the cDNA sequence information thereof.
• Cloning can be carried out by synthesizing oligo-DNA primers as described above, and amplifying by RT-PCR using total RNA or mRNA fractions prepared from cells producing the protein as a template.
• oligo DNA primers are synthesized based on the cDNA sequence information of the domain to be used, and total RNA or mRNA fractions prepared from cells producing the domain are used as templates.
• Cloning can be performed by amplifying by RT-PCR method.
• DNA encoding FokI can be cloned from Flavobacterium okeanokoites (IFO 12536)-derived mRNA by RT-PCR by designing appropriate primers upstream and downstream of CDS based on the cDNA sequence.
• the cloned DNA can be used as is or after digestion with restriction enzymes or addition of appropriate linkers and/or nuclear/organelle export signals, the cloned DNA can be ligated with DNA encoding a nucleic acid sequence recognition module to produce a fusion protein.
• DNA encoding can be prepared.
• DNA encoding a nucleic acid sequence recognition module and DNA encoding a DNA cleavage domain are each fused with DNA encoding a binding domain or its binding partner, or both DNAs are fused with DNA encoding a separated intein. may allow the nucleic acid sequence recognition module and the DNA cleavage domain to form a complex after translation within the host cell.
• a linker and/or nuclear localization signal can be ligated to an appropriate position of one or both of the DNAs, if desired.
• An expression vector containing a DNA encoding a nucleic acid sequence recognition module and/or a DNA cleavage domain can be produced, for example, by ligating the DNA downstream of a promoter in an appropriate expression vector.
• an expression vector encoding guide RNA and Cas protein is introduced into a host cell, and the guide RNA and Cas protein are expressed in the host cell. Forms a complex with Cas protein.
• the guide RNA and Cas protein may be encoded on the same expression vector, or may be encoded on different expression vectors.
• the expression vector may optionally contain an enhancer, a splicing signal, a terminator, a polyA addition signal, a selection marker such as a drug resistance gene, an auxotrophic complementary gene, an origin of replication, and the like.
• RNA encoding the nucleic acid sequence recognition module and/or the DNA cleavage domain can be prepared, for example, in a known in vitro transcription system using the above-described vector encoding the DNA encoding the nucleic acid sequence recognition module and/or the DNA cleavage domain as a template. It can be prepared by transcription into mRNA.
• a pharmaceutical composition comprising a donor vector of the invention and a vector comprising a nucleic acid encoding a nucleic acid metabolic enzyme is administered to a subject, and a genome comprising a selected target nucleotide sequence of a cell of the subject is administered to a subject.
• the double-stranded DNA and the donor vector are contacted with a nuclease that cleaves the DNA within the target nucleotide sequence.
• the nuclease is formed in the host cell by introducing the donor DNA (e.g., donor vector) and the expression vector encoding the constituent elements (nucleic acid sequence recognition module and/or DNA cleavage domain) of the nuclease into cells.
• the nuclease can be brought into contact with the genomic double-stranded DNA and the donor vector.
• a pharmaceutical composition comprising an RNA encoding a nucleic acid sequence recognition module and/or a DNA cleavage domain and a guide RNA is administered to a subject, and the genome of a cell of the subject comprising a selected target nucleotide sequence is The double-stranded DNA and the donor vector are contacted with a nuclease that cleaves the DNA within the target nucleotide sequence.
• the pharmaceutical composition can be embedded in lipid nanoparticles (LNPs) and introduced into cells.
• the nuclease is formed in the host cell by introducing the donor DNA (e.g., donor vector) and the RNA encoding the component of the nuclease (nucleic acid sequence recognition module and/or DNA cleavage domain) into the cell. , the nuclease can be brought into contact with the genomic double-stranded DNA and the donor vector.
• the donor DNA e.g., donor vector
• the RNA encoding the component of the nuclease nucleic acid sequence recognition module and/or DNA cleavage domain
• Donor DNA e.g. donor vector
• Donor DNA can be introduced into cells by known methods (e.g. lysozyme method, competent method, PEG method, CaCl co-precipitation method, electroporation method, microinjection method) depending on the cell type. method, particle gun method, lipofection method, Agrobacterium method, etc.).
• the expression vector containing the donor vector, the nucleic acid sequence recognition module and/or the DNA encoding the DNA cleavage domain is a viral vector. In this case, it can be administered directly to a subject (eg, intravenously) and introduced into cells within the subject.
• the number of molecules of the donor DNA (e.g., donor vector) used in the introduction operation is, for example, 1 x 10 2 molecules to 1 x 10 8 molecules, preferably 4 x 10 3 molecules, when converted as the number of copies of homologous nucleotide sequences per host cell.
• the number of molecules is ⁇ 4 ⁇ 10 4 molecules.
• the dosage of the viral vector in the composition of the invention is about 1 x 10 9 to about 6 x 10 14 vector genomes (vg)/kg, about 1 ⁇ 10 10 to approximately 4 ⁇ 10 14 vg/kg, approximately 1 ⁇ 10 11 to approximately 2 ⁇ 10 14 vg/kg, approximately 1 ⁇ 10 12 to approximately 1 ⁇ 10 14 vg/kg, or approximately 5 ⁇ 10 12 to approximately It can be 1 ⁇ 10 14 vg/kg.
• the number of molecules of the expression vector encoding the component of the nuclease (nucleic acid sequence recognition module and/or DNA cleavage domain) used in the introduction operation is, for example, 1 x 10 2 to 1 x 10 9 molecules per host cell, preferably 1 x 10 2 to 1 x 10 9 molecules.
• the number is 4 ⁇ 10 4 molecules to 4 ⁇ 10 5 molecules.
• the ratio of the number of molecules of those expression vectors to be introduced is, for example, 1:0.
• the ratio is 4 to 1:1.6, preferably 1:0.5 to 1:1.5.
• the dose of the viral vector in the composition of the invention is about 1 ⁇ 10 9 to approximately 6 ⁇ 10 14 vector genome (vg)/kg, approximately 1 ⁇ 10 10 to approximately 4 ⁇ 10 14 vg/kg, approximately 1 ⁇ 10 11 to approximately 2 ⁇ 10 14 vg/kg, approximately 1 x10 12 to about 1 x 10 14 vg/kg or about 5 x 10 12 to about 1 x 10 14 vg/kg.
• nucleic acid sequence recognition module When a nucleic acid sequence recognition module or a complex (nuclease) of a nucleic acid sequence recognition module and a DNA cleavage domain is expressed from a nucleic acid or an expression vector introduced into a cell, the nucleic acid sequence recognition module becomes a donor DNA (e.g. donor vector). and/or specifically recognizes and binds to a target nucleotide sequence within genomic double-stranded DNA, and is targeted by the action of the nucleic acid sequence recognition module itself or a DNA cleavage domain linked to the nucleic acid sequence recognition module.
• the DNA is cleaved at a site (which can be adjusted as appropriate within a range of several hundred bases including all or part of the target nucleotide sequence or the vicinity thereof).
• the nuclease preferentially cleaves a target nucleotide sequence in a nucleotide sequence homologous to a sequence on the genome contained in the donor DNA (eg, donor vector).
• a repair mechanism known as homologous recombination (orientated) repair (HDR), which exists in almost all cell types and biological species, combines the sequences on the genomic double-stranded DNA with the donor DNA (e.g. donor vector).
• homologous recombination occurs between the homologous nucleotide sequences contained in the donor DNA (e.g., donor vector), and the DNA sequence encoding the polypeptide of the present invention or a partial polypeptide thereof contained in the donor DNA (e.g., donor vector) becomes a genomic double-stranded DNA.
• the above sequence is inserted into the targeted site. Thereafter, the polypeptide of the present invention or its partial polypeptide is expressed to form the protein or partial protein of the present invention.
• the protein or partial protein of the present invention exhibits a blood anticoagulant effect and can be used to suppress blood coagulation and treat or prevent diseases (thrombosis, etc.).
• the linear double-stranded DNA when linear double-stranded DNA is used as the donor DNA, the linear double-stranded DNA is obtained by cleaving the circular double-stranded DNA at the target nucleotide sequence to obtain linear DNA. It can be. After the linear donor DNA is introduced into the host cell, it is converted into genomic double-stranded DNA by a repair mechanism known as homologous recombination (orientated) repair (HDR), which exists in almost all cell types and biological species.
• HDR homologous recombination
• Homologous recombination occurs between the above sequence and a homologous nucleotide sequence contained in the donor DNA of linear double-stranded DNA, and the present invention contained in the donor DNA (e.g., circular double-stranded DNA)
• the donor DNA e.g., circular double-stranded DNA
• a DNA sequence encoding a polypeptide or a partial polypeptide thereof is inserted into a targeted site on genomic double-stranded DNA.
• the polypeptide of the present invention may further have a signal peptide added thereto.
• Wild-type protein C is translated within cells as a wild-type human protein C prepropolypeptide in which a signal peptide is linked to the N-terminus of the amino acid sequence represented by SEQ ID NO: 2, and when secreted outside the cell, the signal peptide is The peptide is cleaved and converted to the pro-form protein. Due to the addition of a signal peptide, when the polypeptide of the present invention or a partial polypeptide thereof is expressed in cells to produce a recombinant protein, it is secreted outside the cells, making it easy to recover.
• the polypeptide of the present invention or a partial polypeptide thereof can be produced according to known peptide synthesis methods.
• the peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method.
• the target polypeptide or its partial polypeptide is obtained.
• Partial polypeptides can be produced.
• the condensation and removal of the protecting group are carried out according to methods known per se, for example, the methods described in (1) and (2) below.
• polypeptide of the present invention or a partial polypeptide thereof can be purified and isolated using known purification methods.
• purification methods include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof.
• the polypeptide of the present invention or its partial polypeptide obtained by the above method is a free form
• the free form can be converted into an appropriate salt by a known method or a method analogous thereto
• the salt can be converted into a free form or other salts by a known method or a method analogous thereto.
• the polypeptide of the present invention can be produced using a cell-free protein synthesis system.
• RNA transcribed from the DNA containing the nucleic acid of the present invention is used as a translation template, or DNA containing the nucleic acid of the present invention is used to prepare a translation template in vitro. It can be used as a transcription template.
• the translation template may contain an RNA polymerase recognition sequence (eg, SP6, T3 or T7 promoter), a sequence that enhances translation activity in the synthesis system (eg, ⁇ sequence or E01 sequence).
• an RNA polymerase recognition sequence eg, SP6, T3 or T7 promoter
• a sequence that enhances translation activity in the synthesis system eg, ⁇ sequence or E01 sequence.
• the cell-free protein synthesis system methods well known to those skilled in the art can be used as appropriate, such as the method described in WO 05/030954 using wheat germ extract.
• the protein of the present invention or a partial protein thereof can be obtained by culturing a host cell containing an expression vector containing a nucleic acid encoding the polypeptide of the present invention or a partial polypeptide thereof. It can also be produced by separating and purifying.
• the polypeptide of the present invention or a partial polypeptide thereof, or a protein or a partial protein thereof is separated and purified from the culture obtained by culturing the above-mentioned cell-free protein synthesis system or gene-introduced host cells according to a method known per se. be able to.
• a method known per se be able to.
• host cells collected from a culture by a known method are diluted with a suitable buffer solution.
• the buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100 TM .
• a protein denaturant such as urea or guanidine hydrochloride
• a surfactant such as Triton X-100 TM .
• methods such as separating the culture supernatant from the culture by centrifugation or filtration can be used. used.
• the soluble fraction thus obtained and the mutant AIM of the present invention contained in the culture supernatant can be isolated and purified according to methods known per se.
• Such methods include methods that utilize solubility such as salting out and solvent precipitation methods; methods that mainly utilize differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis.
• methods that utilize differences in charge such as ion exchange chromatography; methods that utilize specific affinity such as affinity chromatography; methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography; Methods that utilize differences in isoelectric points, such as point electrophoresis; methods that use antibodies, etc., are used. These methods can also be combined as appropriate.
• a tag sequence for purification is added to the propeptide or between the signal peptide and the propeptide in order to facilitate the purification of the polypeptide of the present invention, a partial polypeptide thereof, or a protein or a partial protein thereof.
• tag sequences include, but are not limited to, for example, histidine tags, maltose binding protein (MBP) tags, glutathione S-transferase (GST) tags, and the like.
• the polypeptide of the present invention or a partial polypeptide thereof, or a protein or a partial protein thereof, into which a tag sequence for purification has been inserted is prepared in a column packed with a ligand that interacts with the tag sequence (e.g., histidine), depending on the type of the tag sequence.
• tags they can be easily separated and purified by passing the culture supernatant of transfectant mammalian cells through a column on which divalent metal ions such as nickel or cobalt are immobilized.
• the polypeptide of the present invention or its partial polypeptide, or protein or its partial protein adsorbed on the column can be purified by passing an eluent having an appropriate salt concentration through the column.
• the tag sequence for purification is obtained by chemically synthesizing the DNA encoding it based on the known amino acid sequence information, and then combining it with the DNA encoding the signal codon and propeptide by treating it with restriction enzymes or using an appropriate linker. Can be connected.
• DNA encoding a chimeric protein consisting of tag sequence-propeptide or signal peptide-tag sequence-propeptide can also be constructed by combining chemical synthesis and PCR method or Gibson Assembly method in the same manner as above. .
• polypeptide of the present invention a partial polypeptide thereof, or a protein or a partial protein thereof
• polypeptide of the present invention or its partial polypeptide can be made into the protein of the present invention or its partial protein by cleavage at the self-cleavage site and optionally further processing.
• the polypeptide of the present invention or a partial polypeptide thereof can be converted into the protein of the present invention or a partial protein thereof in vitro by treatment with an appropriate protease.
• the present invention provides activated protein C isolated and purified from protein C-expressing cells produced by the method for producing protein C-expressing cells of the present invention. Furthermore, the present invention provides a recombinant protein C preparation prepared by formulating the activated protein C.
• Example 1 Wild-type PROC cDNA was obtained by reverse transcription PCR (RT-PCR) of human liver RNA. Codon-optimized PROC cDNA was synthesized using GenScript or GeneArt TM artificial gene synthesis algorithm. Gene insertion was performed using InFusion Cloning (Takara) after artificially synthesizing the insertion sequence. The codon-optimized sequence was used in cell experiments, and the wild type was used in mouse experiments. A schematic diagram of the polypeptide encoded by the cDNA is shown in FIG.
• Example 2 Various PROC cDNAs were inserted into the pCDNA3 plasmid, and the genes were introduced into HEK293 cells derived from human fetal kidney cells using Lipofectamine® 3000 (Thermo Fisher Scientific, Waltham, MA, USA). Gene expressing cells were selected by G418. Vitamin K (Keito N 5 ⁇ g/ml) was added to the supernatant, and the cell supernatant was collected 24 hours later. Human PC activity was measured using Verichrome Protein C (Sysmex, Kobe, Japan) with a fully automatic blood coagulation analyzer CS1600 (Sysmex).
• PC activity was evaluated by cleavage of the substrate under conditions that did not contain the snake venom (activator) included in the kit. The results are shown in Figure 2. An increase in PC activity was observed when the self-cleavage site was KRRKR, 2RKR (RKRRKR), 3RKR (RKRRKRRKR), and 4RKR (RKRRKRRKRRKR).
• Example 3 Various PROC cDNAs were inserted into pCDNA3 plasmid, and genes were introduced into HEK293 cells using Lipofectamine (registered trademark) 3000. Gene expressing cells were selected by G418. Vitamin K (Keito N 5 ⁇ g/ml) was added to the supernatant, and 24 hours later, the cell supernatant was collected and PC activity was measured under conditions containing the activator. Activated partial is the clotting time after diluting the cell supernatant so that the PC activity is approximately 0%, 2.6, 8%, 26%, and 80% and mixing it with the same amount of human standard plasma (Sysmex).
• Thromboplastin time (Thrombocheck APTT, Sysmex) (A) and prothrombin time (Thrombocheck PT, Sysmex) (B) were measured using CS510 (Sysmex). The results are shown in Figure 3. When the self-cleavage site was 2RKR (RKRRKR), a concentration-dependent prolongation of the clotting time was observed.
• RKRRKR 2RKR
• PROC cDNA (2RKR) was inserted between the liver-specific HCRhAAT promoter and the SV40 polyA sequence.
• the HCRhAAT promoter is composed of the Apo E/C1 hepatic control region and the human ⁇ 1 antitrypsin promoter. This sequence was inserted into a plasmid containing the AAV ITR.
• the AAV type 8 vector was constructed using a plasmid transfection method using a helper-free system in a previous study (Ohmori T, Nagao Y, Mizukami H, Sakata A. Muramatsu SI, Ozawa K, et al.
• the inverted terminal repeat (ITR) of the AAV vector used a sequence derived from AAV type 2.
• the titer of the AAV vector after purification was measured by quantitative PCR (qPCR).
• AAV8 vector was intravenously administered to wild-type C57BL/6 mice (7-8 weeks old, male) through the jugular vein under isoflurane anesthesia (4x10 10 , 4x10 11 , 1.2x10 12 vg/mouse). Blood was collected 2 and 4 weeks after vector administration, and PC activity and APTT in plasma were measured. The results are shown in Figure 4. Increased human PC activity and prolongation of APTT were observed in mouse blood upon administration of the vector.
• Example 5 Previously reported (De Caneva A, Porro F, Bortolussi G, Sola R, Lisjak M, Barzel A, et al. Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.JCI Insight.2019;4(15) :e128863.https://doi.org/10.1172/jci.insight.128863.), a guide RNA was designed at the mouse Alb gene locus, and an AAV8-type vector (Cas9) expressing SaCas9 was added to both ends.
• An AAV8 type vector (Donor) having a wild type PC sequence linked to a P2A sequence having a homologous recombination sequence (approximately 1 kb) at the gene locus was administered to wild type C57BL/6 newborn mice. The results are shown in Figure 5. In neonatal mice administered with an AAV vector containing the Donor sequence and Cas9, human PC activity is persistently increased. Donor sequence alone does not increase PC activity.
• Wild-type mouse protein was administered to wild-type C57BL/6 mice (7 weeks old, male) at three doses: Low (4x10 10 vg/mouse), Medium (1.2x10 11 vg/mouse), and High (4x10 11 vg/mouse).
• AAV type 8 vector expressing C sequence (mPC) or modified mouse protein C sequence (mPC variant) was administered once intravenously, and blood was collected from 4 to 8 weeks after administration to collect plasma.
• the increase in protein C antigen level in plasma Figure 6A
• clotting time [activated partial thromboplastin time (APTT)]
• Figure 6C factor V activity
• factor VIII activity Figure 6D
• SV40 polyadenylation signal contained within the vector was evaluated by quantitative PCR.
• the primer and probe sequences used are as follows. 5'-AGCAATAGCATCACAAATTTCACAA-3' (sense) (SEQ ID NO: 19) 5'-CCAGACATGATAAGATACATTGATGAGTT-3' (antisense) (SEQ ID NO: 20) 5'-AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC-3' (FAM probe) (SEQ ID NO: 21) - Pathological thrombus formation dependent on active oxygen: Anti-platelet GPIb ⁇ antibody (DyLight488 conjugated) (Emfret Analytics GmbH & Co) and rhodamine B (Sigma Aldrich) or Texas Red-conjugated dextran (Thermo Fisher Scientific) were administered to mice under anesthesia.
• hematoporphyrin Sigma Aldrich
• laser-induced thrombus formation in the testicular vein was observed using a confocal microscope (Leica TCS SP8).
• Example 7 Two types of AAV vectors (a vector expressing SaCas9 and a vector carrying an mPC variant) were administered to neonatal mice born from heterozygous crossbreeding of protein C-deficient mice created by genome editing targeting exon 9. Activated PC was expressed in the liver of a child by genome editing. Similar to crossbreeding of hemophilia A mice (F8 -/- ) (kindly provided by Dr. Kazazian, University of Pennsylvania, available from Jackson), survival of protein C-deficient mice was obtained by expression of activated protein C through genome editing. (Figure 9A). Mouse protein C antigen amount (FIG. 9B), factor V activity (FIG. 9C), factor VIII activity (FIG. 9D), and clotting time [activated partial thromboplastin time (APTT)] (FIG. 9E) were determined as in Example 6. It was measured as follows.
• active protein C is produced as a recombinant preparation; 2) It becomes possible to link this to effective gene therapy for protein C deficiency. If activated protein C is produced recombinantly, a safe protein preparation without the risk of infectious diseases can be obtained.
• the blood molarity of protein C is at the same level as factor IX, which is deficient in hemophilia B, and considering the results of previous human clinical trials, administration of AAV vectors is sufficient to achieve therapeutic blood molarity. of protein C can be obtained.

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