Gene therapy for hemophilia A using viral vectors encoding recombinant FVIII variants with increased expression

文档序号：939419 发布日期：2021-03-05 浏览：45次中文

阅读说明：本技术 使用编码具有增加的表达的重组fviii变体的病毒载体的a型血友病的基因疗法 (Gene therapy for hemophilia A using viral vectors encoding recombinant FVIII variants with increased expression ) 是由 H·罗滕施泰纳 W·赫尔里格尔于 2019-07-15 设计创作，主要内容包括：本公开尤其提供编码在哺乳动物细胞中表达的因子VIII变体的密码子改变的多核苷酸。在一些实施方案中,本公开还提供用于治疗A型血友病的哺乳动物基因疗法载体和方法。在一些实施方案中,本公开提供用于向A型血友病患者给予编码因子VIII多肽的多核苷酸,例如密码子改变的多核苷酸的方法。(The present disclosure provides, inter alia, polynucleotides encoding codon alterations of factor VIII variants expressed in mammalian cells. In some embodiments, the present disclosure also provides mammalian gene therapy vectors and methods for treating hemophilia a. In some embodiments, the disclosure provides methods for administering a polynucleotide, e.g., a codon altered polynucleotide, encoding a factor VIII polypeptide to a hemophilia a patient.)

1. A method for treating hemophilia A, comprising intravenously infusing 2x 10 into a human subject diagnosed with hemophilia A¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of said human subject, wherein said AAV particles comprise a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA).

2. A method for treating hemophilia A, comprising intravenously infusing 6 x 10 into a human subject diagnosed with hemophilia A¹²Adeno-associated virus (AAV) particles/kgA dose of a body weight of a human subject, wherein the AAV particle comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA).

3. The method of claim 1 or 2, further comprising administering prednisolone or a course of prednisone to the human subject diagnosed with hemophilia a.

4. The method of claim 3, wherein prednisolone or the course of prednisone is administered after infusion of the AAV particles.

5. The method of claim 3 or 4, wherein the course of treatment of prednisolone or prednisone administration comprises:

administering to the human subject 60mg prednisolone or prednisone/day during the first week immediately following infusion of the AAV particles;

administering to the human subject 40mg prednisolone or prednisone/day during a second week immediately following infusion of the AAV particles; and

administering to the human subject 30mg prednisolone or prednisone per day during the third week immediately following infusion of the AAV particles.

6. The method of claim 5, further comprising administering a tapered dose of prednisolone or prednisone immediately after a third week following infusion of the AAV particles.

7. The method of claim 6, wherein administering the tapered dose of prednisolone or prednisone comprises:

administering to said human subject 20mg prednisolone or prednisone/day for immediately following the completion of an initial course of prednisolone or prednisone;

administering to the human subject 20mg prednisolone or prednisone for 5 days followed by 15mg prednisolone or prednisone/day for 3 consecutive days;

administering to the human subject 15mg prednisolone or prednisone for 3 days, followed by 10mg prednisolone or prednisone for each of the consecutive 3 days; and

administering to the human subject 10mg prednisolone or prednisone for 3 days, followed by administering to the human subject 5mg prednisolone or prednisone/day for 3 consecutive days.

8. The method of claim 6, wherein administering the tapered dose of prednisolone or prednisone comprises:

administering to said human subject 30mg prednisolone or prednisone/day for immediately following 7 consecutive days after completion of the initial course of prednisolone or prednisone;

administering to the human subject 20mg prednisolone or prednisone/day for 7 consecutive days following administration of 30mg prednisolone or prednisone to the human subject for 7 days;

administering to the human subject 20mg prednisolone or prednisone for 7 days, followed by 15mg prednisolone or prednisone/day for 5 consecutive days;

administering to the human subject 15mg prednisolone or prednisone for 5 days followed by 10mg prednisolone or prednisone for each day for 5 consecutive days; and

administering to the human subject 10mg prednisolone or prednisone for 5 days, followed by 5 consecutive days of administering to the human subject 5mg prednisolone or prednisone per day.

9. A method, the method comprising:

determining a first level of factor VIII activity in a blood sample collected from a human subject diagnosed with hemophilia a following administration of adeno-associated virus (AAV) particles comprising a polynucleotide encoding a factor VIII protein to the human subject, and concurrently with an initial course of glucocorticoid steroid treatment of the human subject;

determining a second level of factor VIII activity in a blood sample collected from the human subject after completion of the initial course of glucocorticoid steroid treatment;

comparing the second level of factor VIII activity to the first level of factor VIII activity; and

administering a gradually decreasing dose of the glucocorticoid steroid, wherein:

administering a first tapering-down dose of the glucocorticoid steroid for a period of time of no more than three weeks when the second level of factor VIII activity is not below the first level of factor VIII activity; and is

Administering a second tapering-down dose of the glucocorticoid steroid for a period of more than three weeks when the second level of the factor VIII activity is lower than the first level of the factor VIII activity.

10. A method, the method comprising:

determining a first level of liver enzyme activity in a blood sample collected from a human subject diagnosed with hemophilia a prior to administering adeno-associated virus (AAV) particles comprising a polynucleotide encoding a factor VIII protein to the human subject;

determining a second level of liver enzyme activity in a blood sample collected from the human subject after administration of AAV particles comprising a polynucleotide encoding a factor VIII protein to the human and after completion of an initial course of glucocorticoid steroid therapy;

comparing the second level of liver enzyme activity to the first level of liver enzyme activity; and

administering a gradually decreasing dose of the glucocorticoid steroid, wherein:

administering a first tapering dose of the glucocorticoid steroid for a period of no more than three weeks when the second level of liver enzyme activity does not exceed the first level of liver enzyme activity; and is

Administering a second tapering dose of the glucocorticoid steroid for a period of more than three weeks when the second level of liver enzyme activity exceeds the first level of factor VIII activity.

11. The method of claim 9 or 10, wherein administering the first tapering dose of the glucocorticoid steroid comprises:

administering to said human subject 20mg prednisolone or prednisone/day for immediately following the completion of said initial course of glucocorticoid steroid therapy for 5 consecutive days;

administering to the human subject 20mg prednisolone or prednisone for 5 days followed by 15mg prednisolone or prednisone/day for 3 consecutive days;

administering to the human subject 15mg prednisolone or prednisone for 3 days, followed by 10mg prednisolone or prednisone for each of the consecutive 3 days; and

administering to the human subject 10mg prednisolone or prednisone for 3 days, followed by administering to the human subject 5mg prednisolone or prednisone/day for 3 consecutive days.

12. The method of any one of claims 9-11, wherein administering the second, tapered dose of the glucocorticoid steroid comprises:

administering to said human subject 30mg prednisolone or prednisone/day for immediately following completion of said initial course of glucocorticoid steroid therapy for 7 consecutive days;

administering to the human subject 20mg prednisolone or prednisone/day for 7 consecutive days following administration of 30mg prednisolone or prednisone to the human subject for 7 days;

administering to the human subject 20mg prednisolone or prednisone for 7 days, followed by 15mg prednisolone or prednisone/day for 5 consecutive days;

administering to the human subject 15mg prednisolone or prednisone for 5 days followed by 10mg prednisolone or prednisone for each day for 5 consecutive days; and

administering to the human subject 10mg prednisolone or prednisone for 5 days, followed by 5 consecutive days of administering to the human subject 5mg prednisolone or prednisone per day.

13. A method for monitoring the efficacy of factor VIII gene therapy for hemophilia a using adeno-associated virus (AAV) particles comprising a polynucleotide encoding a factor VIII polypeptide, comprising:

determining whether factor VIII inhibitor antibodies are present in a blood sample collected from a human subject following administration of the AAV particles to the human subject; and

upon detecting the presence of a factor VIII inhibitor in the blood of the human subject, administering to the human subject an alternative agent for treating hemophilia A.

14. The method of claim 13, wherein the surrogate agent comprises a chemically modified human factor VIII protein.

15. The method of claim 13, wherein the replacement agent comprises porcine factor VIII protein.

16. The method of claim 13, wherein the surrogate agent is a factor VIII shunt therapeutic comprising factor II, factor IX, and factor X.

17. A method, the method comprising:

a) administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA); and

b) measuring the level of SEQ ID NO:1 or fragments thereof in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

18. A method, the method comprising:

a) administering 6 x 10 to a hemophilia a patient at a first time point¹²Individual adeno-associated virus (AAV) particles per kilogram of said patientA dosage of a subject's body weight, wherein the AAV particle comprises a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA); and

b) measuring the level of SEQ ID NO:1 or fragments thereof in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

19. A method, the method comprising:

a) administering a dose of adeno-associated virus (AAV) particles to a hemophilia a patient at a first time point, wherein the AAV particles comprise a polynucleotide encoding a factor VIII protein; and

b) measuring the level of a polynucleotide encoding the factor VIII protein or fragment thereof in the patient's blood stream at a later time point, wherein the later time point is 7 days or longer.

20. The method of any one of claims 17-19, wherein the later time point is 7 days old.

21. The method of any one of claims 17-19, wherein the later time point is at 14 days.

22. The method of any one of claims 17-19, wherein the later time point is 21 days old.

23. A method, the method comprising:

a) administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA); and

b) measuring the level of the capsid protein in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

24. A method, the method comprising:

a) administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA); and

b) measuring the level of the capsid protein in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

25. A method, the method comprising:

a) administering a dose of adeno-associated virus (AAV) particles to a hemophilia a patient at a first time point, wherein the AAV particles comprise a capsid protein and a polynucleotide encoding a factor VIII protein; and

b) measuring the level of the capsid protein in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

26. The method of any one of claims 23-25, wherein the later time point is 7 days old.

27. The method of any one of claims 23-25, wherein the later time point is at 14 days.

28. The method of any one of claims 23-25, wherein the later time point is 21 days old.

29. A method, the method comprising:

b) measuring the level of anti-factor VIII antibody in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

30. A method, the method comprising:

a) administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA); and

b) measuring the level of anti-factor VIII antibody in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

31. A method, the method comprising:

b) measuring the level of anti-factor VIII antibody in the patient's blood stream at a later time point, wherein the later time point is 7 days or more.

32. The method of any one of claims 29-31, wherein the later time point is 7 days old.

33. The method of any one of claims 29-31, wherein the later time point is at 14 days.

34. The method of any one of claims 29-31, wherein the later time point is 21 days old.

35. A method, the method comprising:

a) administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA);and

b) measuring the level of anti-capsid protein antibodies in the patient's bloodstream at a later time point, wherein the later time point is 7 days or more.

36. A method, the method comprising:

b) measuring the level of anti-capsid protein antibodies in the patient's bloodstream at a later time point, wherein the later time point is 7 days or more.

37. A method, the method comprising:

b) measuring the level of anti-capsid protein antibodies in the patient's bloodstream at a later time point, wherein the later time point is 7 days or more.

38. The method of any one of claims 35 to 37, further comprising:

c) measuring a baseline level of anti-capsid protein antibodies in the patient's blood stream prior to administering the dose of the adeno-associated virus (AAV) particles to the patient; and

d) optionally, comparing the measured level of anti-capsid protein antibodies in the patient's bloodstream at a later point in time with the baseline level of anti-capsid protein antibodies in the patient's bloodstream.

39. The method of any one of claims 35-38, wherein the later time point is 7 days old.

40. The method of any one of claims 35-38, wherein the later time point is at 14 days.

41. The method of any one of claims 35-38, wherein the later time point is 21 days old.

Background

Blood coagulation proceeds via a complex and dynamic biological pathway of interdependent biochemical reactions called the coagulation cascade (cascade). Coagulation factor viii (fviii) is a key component in the cascade. Factor VIII is recruited to the bleeding site and forms a Xase complex with activated factor ix (fixa) and factor x (fx). The Xase complex activates FX, which in turn activates prothrombin to thrombin, which in turn activates other components in the coagulation cascade to generate stable clots (reviewed in Saenko et al, Trends Cardiovasc. Med.,9: 185-.

Hemophilia a is a congenital X-linked bleeding disorder characterized by a lack of factor VIII activity. Attenuated factor VIII activity inhibits the positive feedback loop in the coagulation cascade. This results in incomplete coagulation, which is manifested as bleeding episodes of increased duration, extensive congestion, spontaneous bleeding in the mouth and nose, joint stiffness and chronic pain, and in severe cases may manifest as internal bleeding and anemia (Zhang et al, clinical. rev. allerg. immunol.,37: 114-.

Conventionally, hemophilia a is treated by factor VIII replacement therapy, which consists of administering a factor VIII protein (e.g., plasma-derived or recombinantly produced factor VIII) to an individual with hemophilia a. Factor VIII is administered prophylactically to prevent bleeding episodes or to reduce the frequency of bleeding episodes in response to acute bleeding episodes, and/or before and after surgery to control bleeding during surgery. However, there are several undesirable characteristics of factor VIII replacement therapy.

First, factor VIII replacement therapy is used to treat or control hemophilia a, but does not cure potential factor VIII deficiency. Because of this, subjects with hemophilia a require factor VIII replacement therapy during their life cycle. Continuous treatment is expensive and requires the subject to maintain strict compliance, as for subjects with severe haemophilia a, the omission of only a small number of prophylactic doses can have serious consequences.

Second, because factor VIII has a relatively short in vivo half-life, conventional prophylactic factor VIII replacement therapy requires administration every two or three days. This places the burden on the subject to maintain compliance throughout its life. While third generation "long-acting" factor VIII drugs can reduce the frequency of administration, prophylactic factor FVIII replacement therapy with these drugs still requires permanent monthly, weekly, or more frequent administration. For example, using elocat^TM[ antihemophilic factor (recombinant), Fc fusion protein]Is administered every three to five days (elocat)^TMPrescription information, Biogen Idec, (2015)). Furthermore, the long-term effects of chemically modified biologies (e.g., pegylated polypeptides) are not fully understood.

Third, between 15% and 30% of all subjects receiving factor VIII replacement therapy develop anti-factor VIII inhibitor antibodies, making the therapy ineffective. Factor VIII bypass therapy (e.g., administration of a complex concentrate of prothrombin either plasma-derived or recombinantly produced) can be used to treat hemophilia in individuals who form inhibitor antibodies. However, factor VIII bypass therapy is not as effective as factor VIII replacement therapy (Mannucci p.m., J thramb haemans, 1(7):1349-55(2003)) and may be associated with increased risk of cardiovascular complications (Luu and Ewenstein, haempolia, supplement 102: 10-16 (2004)).

Somatic gene therapy has great promise for treating hemophilia a because it offsets the potential for under-expression of functional factor VIII activity (e.g., due to missense or nonsense mutations) rather than providing a single dose of factor VIII activity to an individual. Due to this difference in mechanism of action, a single administration of the factor VIII gene therapy vector can provide the individual with factor VIII for several years, compared to factor VIII replacement therapy, thereby reducing treatment costs and eliminating the need for continued patient compliance.

Coagulation factor IX (fix) gene therapy has been effectively used to treat subjects with hemophilia B (an associated blood coagulation disorder characterized by diminished factor IX activity) (mann c.s., et al, Nat med.,12(3):342-47 (2006)). However, factor VIII gene therapy presents several unique challenges. For example, a full length wild-type factor VIII polypeptide (2351 amino acids; UniProt accession P00451) is five times larger than a full length wild-type factor IX polypeptide (461 amino acids; UniProt accession P00740). Thus, the coding sequence for wild-type factor VIII is 7053 base pairs, which is too large to be packaged in conventional AAV gene therapy vectors. Furthermore, reported recombinant expression of the B domain deletion variant of factor VIII (BDD-FVIII) was poor. Thus, several groups have attempted to alter the codon usage of BDD-FVIII constructs with limited success.

Disclosure of Invention

Thus, there is a need for factor VIII variants whose coding sequences are more efficiently encapsulated in and delivered via gene therapy vectors. There is also a need for nucleic acids that more efficiently express factor VIII synthetic codon changes. Such factor VIII variants and codon altered nucleic acids may improve the treatment of factor VIII deficiencies (e.g., hemophilia a). The above deficiencies and other problems associated with the treatment of factor VIII deficiencies (e.g., hemophilia a) are reduced or eliminated by the disclosed codon-altered factor VIII variants.

According to some embodiments, the present disclosure provides nucleic acids encoding factor VIII variants having high sequence identity to the disclosed sequences of codon changes of the factor VIII heavy chain (e.g., CS04-HC-NA) and light chain (e.g., CS 04-LC-NA). In some embodiments, the nucleic acids further comprise, between the sequences encoding the factor VIII heavy and light chains, a sequence encoding a linker sequence (e.g., a linker sequence comprising a furin cleavage site) in place of the native factor VIII B domain.

In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence encoding a factor VIII polypeptide. The factor VIII polypeptide comprises a light chain, a heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence having at least 95% identity to CS04-HC-NA (SEQ ID NO: 3). The light chain of the factor FVIII polypeptide is encoded by a second nucleotide sequence having at least 95% identity to CS04-LC-NA (SEQ ID NO: 4). The polypeptide linker comprises a furin cleavage site.

In one embodiment of the polynucleotides described above, the polypeptide linker is encoded by a third nucleotide sequence having at least 95% identity to BDLO04(SEQ ID NO: 5).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide has at least 96% identity to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide has at least 96% identity to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide has at least 97% identity to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide has at least 97% identity to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide has at least 98% identity to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide has at least 98% identity to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is at least 99% identical to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is at least 99% identical to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is at least 99.5% identical to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is at least 99.5% identical to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is at least 99.9% identical to the corresponding heavy chain sequence (e.g., CS04-HC-NA (SEQ ID NO:3)), and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is at least 99.9% identical to the corresponding light chain sequence (e.g., CS04-LC-NA (SEQ ID NO: 4)).

In one embodiment of the polynucleotides described above, the first nucleotide sequence encoding the heavy chain of the factor VIII polypeptide is CS04-HC-NA (SEQ ID NO:3) and the second nucleotide sequence encoding the light chain of the factor FVIII polypeptide is CS04-LC-NA (SEQ ID NO: 4).

In one aspect, the present disclosure provides a polynucleotide comprising a nucleotide sequence having at least 95% identity to CS04-FL-NA, wherein the polynucleotide encodes a factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 96% identical to the corresponding full-length polynucleotide sequence, e.g., CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 97% identical to the corresponding full-length polynucleotide sequence, e.g., CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 98% identical to the corresponding full-length polynucleotide sequence (e.g., CS04-FL-NA (SEQ ID NO: 1)).

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 99% identical to a corresponding full-length polynucleotide sequence, e.g., CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 99.5% identical to the corresponding full-length polynucleotide sequence, e.g., CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is at least 99.9% identical to the corresponding full-length polynucleotide sequence, e.g., CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the nucleotide sequence is CS04-FL-NA (SEQ ID NO: 1).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 95% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 96% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 97% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 98% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 99% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 99.5% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising an amino acid sequence having at least 99.9% identity to CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotides described above, the polynucleotides encode a factor VIII polypeptide comprising the amino acid sequence of CS04-FL-AA (SEQ ID NO: 2).

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 95% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 96% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 97% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 98% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 99% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence has at least 99.5% identity to a sequence selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotide described above, the nucleotide sequence is selected from the group consisting of CS04-FL-NA, CS04-HC-NA and CS 04-LC-NA.

In one embodiment of the polynucleotides described above, the polynucleotide further comprises a promoter element operably linked to the polynucleotide encoding the factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide further comprises an enhancer element operably linked to the polynucleotide encoding the factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide further comprises a polyadenylation element operably linked to the polynucleotide encoding the factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the polynucleotide further comprises an intron operably linked to the nucleotide sequence encoding the factor VIII polypeptide.

In one embodiment of the polynucleotides described above, the intron is located between the promoter element of the nucleotide sequence encoding the factor VIII polypeptide and the translation start site (e.g., the first encoding ATG).

In another aspect, the present disclosure provides a mammalian gene therapy vector comprising a polynucleotide as described above.

In one embodiment of the mammalian gene therapy vector described above, the mammalian gene therapy vector is an adeno-associated virus (AAV) vector.

In one embodiment of the mammalian gene therapy vector described above, the AAV vector is an AAV-8 vector.

In another aspect, the present disclosure provides a method for treating hemophilia a comprising administering to a patient in need thereof a mammalian gene therapy vector as described above.

In another aspect, the present disclosure provides a mammalian gene therapy vector as described above for use in treating hemophilia a.

In another aspect, the present disclosure provides the use of a mammalian gene therapy vector as described above for the manufacture of a medicament for the treatment of hemophilia a.

Drawings

Figure 1 shows a schematic illustration of wild-type and refecto-type human factor VIII protein constructs.

FIGS. 2A and 2B show a nucleotide sequence (SEQ ID NO:1) encoding a CS04 codon alteration of a factor VIII variant (for the full-length coding sequence, "CS 04-FL-NA"), according to some embodiments.

Figure 3 shows a factor VIII variant amino acid sequence (SEQ ID NO:2) encoded by a nucleotide sequence altered by codon CS04 (for the full-length amino acid sequence, "CS 04-FL-AA"), according to some embodiments.

Figure 4 shows a portion of a nucleotide sequence encoding a CS04 codon alteration of a heavy chain of a factor VIII variant (SEQ ID NO:3) ("CS 04-HC-NA"), according to some embodiments.

Figure 5 shows a portion of a nucleotide sequence encoding a CS04 codon alteration of a light chain of a factor VIII variant (SEQ ID NO:4) ("CS 04-LC-NA"), according to some embodiments.

FIG. 6 shows an exemplary coding sequence for a B domain substituted linker according to some embodiments (SEQ ID NO: 5). BDLO04(SEQ ID NO:5) is the corresponding part of the nucleotide sequence encoding the B domain altered by the CS04 codon of the substituted linker.

FIGS. 7A, 7B, and 7C show an AAV vector sequence (SEQ ID NO:8) ("CS 04-AV-NA") comprising a nucleotide sequence having a change in CS04 codon, according to some embodiments.

FIGS. 8A and 8B show a nucleotide sequence encoding a CS08 codon alteration of a factor VIII variant (SEQ ID NO:7) ("CS 08-FL-NA") according to some embodiments.

FIGS. 9A and 9B show a nucleotide sequence encoding a CS10 codon alteration of a factor VIII variant (SEQ ID NO:8) ("CS 10-FL-NA") according to some embodiments.

FIGS. 10A and 10B show a nucleotide sequence encoding a CS11 codon alteration of a factor VIII variant (SEQ ID NO:9) ("CS 11-FL-NA") according to some embodiments.

FIGS. 11A and 11B show a CS40 wild-type ReFacto coding sequence (SEQ ID NO:10) ("CS 40-FL-NA") according to some embodiments.

FIGS. 12A and 12B show a nucleotide sequence encoding a codon change of CH25 of a factor VIII variant (SEQ ID NO:11) ("CH 25-FL-NA") according to some embodiments.

FIG. 13 shows a wild-type human factor VIII amino acid sequence (SEQ ID NO:12) ("FVIII-FL-AA"), according to some embodiments.

FIG. 14 illustrates the procedure for cloning the pCS40, pCS04, pCS08, pCS1O, pCS11 and pCh25 constructs by inserting synthetic Refacto type BDD-FVIII DNA sequences into the vector backbone pCh-BBO1 via Ascl and Notl restriction sites.

Figure 15 shows the integrity of AAV vector genome formulations as analyzed by agarose gel electrophoresis. Lane 1, DNA marker; lane 2, vCS 40; lane 4, vCS 04. The AAV vectors all have the same size genome, migrating at approximately 5kb (right arrow). The scale on the left indicates the size of the DNA fragment in kilobases (kb).

Figure 16 shows protein analysis of AAV vector preparations by PAGE and silver staining. Lane 1, protein marker (M); lane 2, vCS 40; and lanes 4, vCS 04. The constructs all have the same AAV8 capsid consisting of VP1, VP2, and VP3 (right arrow). The scale on the left indicates the size of the protein tag in kilodaltons (kDa).

Figure 17 shows FVIII activity following systemic administration of an (r) AAV 8-based gene therapy vector containing a CS04 factor VIII codon optimized construct as described in example 3. cp, vector capsid particles; FVIII, factor VIII; LLOQ, lower limit of quantitation. 14. Time points of 28, 42 and 56 days are shown from left to right in the illustration.

Figure 18 shows a reduction in blood loss following systemic administration of an (r) AAV 8-based gene therapy vector containing a CS04 factor VIII codon optimized construct as described in example 3 in a tail tip bleeding assay. cp, vector capsid particles.

Figures 19A, 19B and 19C show biodistribution of (r) AAV 8-based gene therapy vectors containing CS04 factor VIII codon-optimized construct DNA after systemic administration. 1902-liver; 1904 as lymph nodes; 1906 skeletal muscle; 1908 heart; 1910 ═ kidney; 1912, spleen; 1914 pulmonary; 1916 testis; 1918 the brain.

Detailed Description

I. Introduction to the design reside in

AAV-based gene therapy holds great promise for treating hemophilia patients. For hemophilia B, the first clinical data is encouraging to maintain FIX levels of about 10% for more than 1 year in at least some patients. However, for hemophilia a, it remains challenging to obtain 5% -10% therapeutic expression levels of AAV vectors for a variety of reasons. First, the factor VIII coding sequence is too large for conventional AAV-based vectors. Second, the effect of poor expression in the engineered B domain deleted or truncated factor VIII construct receptor, even when codon optimized. Third, these B domain deleted or truncated factor VIII variant constructs have a short half-life in vivo, exacerbating the effects of poor expression. Fourth, even when expressed, FVIII is not secreted from the cell as efficiently as other coagulation factors (such as factor IX). Therefore, strategies to improve FVIII expression are needed to make FVIII gene therapy a viable treatment option for hemophilia a patients.

The present disclosure relates to the discovery of codon-altered factor VIII variant coding sequences that address these and other problems associated with factor VIII gene therapy. For example, the polynucleotides disclosed herein provide significantly improved expression in mammalian cells and show improved viral particle encapsulation due to stabilized fill interactions. In some embodiments, these advantages are achieved by using coding sequences for the heavy and light chains of factor VIII that have high sequence identity to the codon altered CS04 construct (e.g., high sequence identity to the CS04-HC heavy chain coding sequence and high sequence identity to the CS04-LC light chain coding sequence).

In some embodiments, the factor VIII molecule encoded by the polynucleotides described herein has been shortened by truncation, deletion, or substitution of the wild-type B domain. Thus, polynucleotides are more suitable for expressing factor VIII via conventional gene therapy vectors that inefficiently express larger polypeptides, such as wild-type factor VIII.

Advantageously, the factor VIII variant coding sequence with the CS04 codon alteration shown herein provides superior expression in vivo of B domain deleted factor VIII constructs. For example, it was demonstrated in example 2 and Table 4 that intravenous administration of an AAV-based gene therapy vector having the CS04(SEQ ID NO:1) coding sequence provided a 74-fold increase in factor VIII expression relative to the corresponding CS40 construct encoded with the wild-type polynucleotide sequence (SEQ ID NO:10) in factor VIII knockout mice (Table 4).

In addition, factor VIII variant coding sequences with codon changes to CS04 are also shown herein to provide excellent virion encapsulation and viral production. For example, it was demonstrated in example 1 that AAV vector constructs containing the CS04 construct provide 5 to 7 fold greater viral yields relative to the corresponding CS40 construct encoded with the wild-type polynucleotide sequence when isolated from the same amount of cell pellets.

Definition of

As used herein, the following terms have the meanings ascribed thereto unless otherwise specified.

As used herein, the terms "factor VIII" and "FVIII" are used interchangeably and refer to any protein having factor VIII activity (e.g. active FVIII, commonly referred to as FVIIIa) or a protein precursor (e.g. a proprotein or preproprotein) of a protein having factor VIII activity, in particular factor IXa cofactor activity. In an exemplary embodiment, a factor VIII polypeptide refers to a polypeptide having a sequence with high sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or more) to the heavy and light chains of a wild-type factor VIII polypeptide. In some embodiments, the B domain of the factor VIII polypeptide is deleted, truncated, or replaced with a linker polypeptide to reduce the size of the polynucleotide encoding the factor VIII polypeptide. In an exemplary embodiment, amino acids 20-1457 of CS04-FL-AA constitute a factor VIII polypeptide.

Non-limiting examples of wild-type factor VIII polypeptides include human prophase causal factor VIII (e.g., GenBank accession nos. AAA52485, CAA25619, AAA58466, AAA52484, AAA52420, AAV85964, BAF82636, BAG36452, CAI41660, CAI41666, CAI41672, CAI43241, CA003404, EAW72645, AAH22513, AAH64380, AAH98389, AAI11968, AAI11970, or AAB61261), the corresponding causal factor VIII and native variants thereof; porcine pro-causal factor VIII (e.g. UniProt accession number F1RZ36 or K7GSZ5), corresponding causal factor VIII and natural variants thereof; mouse pre-causal factor VIII (e.g. GenBank accession No. AAA37385, CAM15581, CAM26492 or EDL29229), the corresponding causal factor VIII and native variants thereof; rat pro-causal factor VIII (e.g., GenBank accession No. AAQ21580), counterpart causal factor VIII and natural variants thereof; rat procause VIII; and other mammalian factor VIII homologs (e.g., monkey, ape, hamster, guinea pig, etc.).

As used herein, factor VIII polypeptides include natural variants and artificial constructs having factor IX cofactor activity. As used in this disclosure, factor VIII encompasses any natural variant, alternative sequence, isoform or mutein that retains some essential factor IX cofactor activity (e.g., at least 5%, 10%, 25%, 50%, 75% or greater corresponding to wild-type activity). Examples of factor VIII amino acid variants present in the human population (relative to FVIII-FL-AA (SEQ ID NO:12)) include, but are not limited to: s19, R22, Y24, Y25, L26/30, W33, Y35/41, R48/67/69, E72, D75/V/83, G89/92/97, E98, V99, D101/H/104, K108, M110, A111/113/117/121, E129, G130, E132, Y133, D135/137/138, E141, D145, V147, Y155, V159, N163, G164/165, C172, S176, S179, V181/185, D186/N/189, L191, G193, L195, C198, S202/214, L217, A219/220, D222, E223, G224, T252, V253, N254, G255, L261, P262, G263, G266, C267, W274, H275, G278, G280, E284, V295, V285, V297, R294, R285/297, R294, R181/297, R102, V297, R, A303E/P, I307S, S308L, F312S, T314A/I, A315V, G323E, L326P, L327P/P329P, 133IV, M339P, E340P, V345P/P348P/S/P365P, R391P/H/P392P/36394P, W401P, I405P/P409P, W412/P427P, L431P/P437 544/P, G439P/S/P442P, K444P, Y450P/P, F455P, G466P, P470P/R474/R/P/36553, G477 36553, D P, P/P, 36563672/P, P/P, 36563672/P, 36563672, 3656365636563672, 36563672/P, 36563672, 365636563672, P/P, 3656365636563672, 36563672/P, 3656365636563656369/P, P/P, 36563672/P, P, I585/586, D588/594, S596, N601/602, S603/604, Y605/609, R612, N631/633, S635, N637/I/639, L644, L650, V653/659, A663, Q664, F677, M681, V682, Y683/686, F698, M699/701, G705, G710, N713, R717/720/721/727, L725, V727, E739, Y742, R795, P947, V1012, E1057, H1066, D1260, K1289, Q1331800, N1460, L1481761, A1610, I1698, Y16999/1701, Q1705, R8/1711720 4, R1715, A1713, D1727, Y1728, R1760, K1761, F1751, I16998, Y16999/1701, R178/178, R178/1715, R178, R171845, R178/1746, D173, D1727, Y1728, R1746, R1788, R178/18479, R178, R171, R178/1745, R178, R171, R178, R1763, R1745, R171, R178/1782, R178, D178, S1782, S178, A1853/1858, K1864, D1865/1867/1869/1872, P1873, L1875, V1876, C1877/1882, R1888, E1894, I1901, E1904/1907/1908, Y1909, A1939/1941/1942, M1945, L1951, R1960/1963, S1965, M1966/1967, S1968, N1971, H1973, G1979, H1980/1982, R1985, L1994, Y1998, G2000, T2004, M2013, G2013, W2015, R2016/2018, G2022, G2028, S2030, V2035, Y2036, N2038, 2040/2045, I2056, A2178, W2065, P2067, A2080, S2082, S2083/1865, S2083/S2105, S2106, R2108/S2147, R2103/S2147, R2105, S2108, S2147, S2149/S2147, R2105/S2147, S2105, S2108, S2149/S2147, S2149/S2145, and S2147, H2174D, R2178C/H/C2182C/H/C2183C/C2185C/C2192C, C2193C, P2196C, G2198C, E2200C, I2204C, I2209C, A2211C, A2220C, P2224C, R2228C/L/P/C2229C, V2242C, W2248C/3622572 1C/3622572C, T2264C, Q226365, F223672/C2272C, D2286C, W2290C, G2304C, D2303672, P2329C/G C/H/C/L23272/C/W2336/C, P2336/C, W2336/C, and S233362336. Factor VIII proteins also include polypeptides containing post-translational modifications.

In general, a factor VIII-encoding polynucleotide encodes an inactive single chain polypeptide (e.g., a preproprotein) that undergoes post-translational processing to form an active factor VIII protein (e.g., FVIIIa). For example, referring to fig. 1, a wild-type human factor VIII preproprotein is first cleaved to release the encoded signal peptide (not shown) to form a first single chain proprotein (shown as "human wild-type FVIII"). The proprotein is then cleaved between the B and A3 domains to form a first polypeptide comprising a factor VIII heavy chain (e.g., a1 and a2 domains) and a B domain, and a second polypeptide comprising a factor VIII light chain (e.g., including A3, C1, and C3 domains). The first polypeptide is further cleaved to remove the B domain, and a1 is also isolated from the a2 domain, which remains associated with the factor VIII light chain in the mature factor Villa protein. For an overview of the factor VIII maturation method, see Graw et al, Nat Rev Genet, 6(6):488-501(2005), the contents of which are incorporated herein by reference in their entirety for all purposes.

However, in some embodiments, the factor VIII polypeptide is a single chain factor VIII polypeptide. Single chain factor VIII polypeptides are engineered to remove the native cleavage site, and optionally to remove, truncate, or replace the B domain of factor VIII. Thus, it is immature due to cleavage (except for optional cleavage of the signal and/or leader peptide) and is active as a single chain. Non-limiting examples of single chain factor VIII polypeptides are described in Zollner et al (Thromb Res,134(1):125-31(2014)) and Donath et al (Biochem I.,312(1):49-55(1995)), the disclosures of which are incorporated herein by reference in their entirety for all purposes.

As used herein, the term "factor VIII heavy chain" or simply "heavy chain" refers to an aggregate of the a1 and a2 domains of a factor VIII polypeptide. In an exemplary embodiment, amino acids 20-759 of CS04-FL-AA (SEQ ID NO:2) comprise the factor VIII heavy chain.

As used herein, the term "factor VIII light chain" or simply "light chain" refers to an aggregate of the a3, C1, and C2 domains of a factor VIII polypeptide. In an exemplary embodiment, amino acids 774 and 1457 of CS04-FL-AA (SEQ ID NO:2) comprise the factor VIII light chain. In some embodiments, the factor VIII light chain does not include the acidic a3 peptide that is released in vivo during maturation.

In general, the factor VIII heavy and light chains are expressed as a single polypeptide chain, e.g., along with an optional B domain or substituted linker for the B domain. However, in some embodiments, the factor VIII heavy chain and the factor VIII light chain are expressed as separate polypeptide chains (e.g., co-expressed) and reconstituted to form the factor VIII protein (e.g., in vivo or in vitro).

As used herein, the terms "B domain substituted linker" and "factor VIII linker" are used interchangeably and refer to truncated versions of the wild-type factor VIII B domain (e.g., amino acids 760-1667 of FVIII-FL-AA (SEQ ID NO:12)), or peptides engineered to replace the B domain of a factor VIII polypeptide. As used herein, in a factor VIII variant polypeptide according to some embodiments, the factor VIII linker is located between the C-terminus of the factor VIII heavy chain and the N-terminus of the factor VIII light chain. Non-limiting examples of substituted linkers for the B domain are disclosed in: U.S. Pat. nos. 4,868,112, 5,112,950, 5,171,844, 5,543,502, 5,595,886, 5,610,278, 5,789,203, 5,972,885, 6,048,720, 6,060,447, 6,114,148, 6,228,620, 6,316,226, 6,346,513, 6,458,563, 6,924,365, 7,041,635 and 7,943,374; U.S. patent application publication nos. 2013/024960, 2015/0071883, and 2015/0158930; and PCT publication nos. WO 2014/064277 and WO 2014/127215, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

Unless otherwise specified herein, the numbering of factor VIII amino acids refers to the corresponding amino acids in the full length wild-type human factor VIII sequence (FVIII-FL-AA) (presented as SEQ ID NO:12 in FIG. 13). Thus, when referring to amino acid substitutions in the factor VIII variant proteins disclosed herein, the recited amino acid numbering refers to analogous (e.g., structural or functional equivalents) and/or homologous (e.g., evolutionarily conserved in the primary amino acid sequence) amino acids in the full-length wild-type factor VIII sequence. For example, a T2105N amino acid substitution refers to the T to N substituent at position 2105 of the full length wild type human factor VIII sequence (FVIII-FL-AA; SEQ ID NO:12) and the T to N substituent at position 1211 of the factor VIII variant protein encoded by CS04(CS 04-FL-AA; SEQ ID NO: 2).

As described herein, the factor VIII amino acid numbering system depends on whether a factor VIII signal peptide (e.g., amino acids 1 through 19 of the full length wild-type human factor VIII sequence) is included. Where a signal peptide is included, the numbering is referred to as "signal peptide includes" or "SPI". Where no signal peptide is included, the numbering is referred to as "signal peptide exclusion" or "SPE". For example, for the same amino acid as F309S in SPE numbering, F328S is SPI numbering. Unless otherwise indicated, all amino acid numbering refers to the corresponding amino acids in the full length wild-type human factor VIII sequence (FVIII-FL-AA) (presented as SEQ ID NO:12 in FIG. 13).

As described herein, the codon altered polynucleotide provides for increased expression of transgenic factor VIII in vivo (e.g., when administered as part of a gene therapy vector) as compared to the level of factor VIII expression provided by a naturally encoded factor VIII construct (e.g., a polynucleotide encoding the same factor VIII construct using wild-type human codons). As used herein, the term "increased expression" refers to an increased level of transgenic factor VIII activity in the blood of an animal administered a factor VIII encoding codon altered polynucleotide compared to the level of transgenic factor VIII activity in the blood of an animal administered a naturally encoded factor VIII construct. The level of activity can be measured using any factor VIII activity known in the art. An exemplary assay for determining factor VIII activity is the Technochrome FVIII assay (Technoclone, Vienna, Austria).

In some embodiments, increased expression refers to at least 25% greater transgenic factor VIII activity in the blood of an animal administered the codon altered factor VIII polynucleotide compared to the level of transgenic factor VIII activity in the blood of an animal administered the naturally encoded factor VIII polynucleotide. In some embodiments, increased expression refers to a transgenic factor VIII activity that is at least 50% greater, at least 75% greater, at least 100% greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 1O-fold greater, at least 15-fold greater, at least 20-fold greater, at least 25-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 1 OO-fold greater, at least 125-fold greater, at least 150-fold greater, at least 175-fold greater, at least 200-fold greater, at least 225-fold greater, or at least 250-fold greater in the blood of an animal administered the codon altered factor VIII polynucleotide compared to the level of transgenic factor VIII activity in the blood of the animal.

As described herein, a codon altered polynucleotide provides increased vector production compared to the level of vector production provided by a naturally encoded factor VIII construct (e.g., a polynucleotide encoding the same factor VIII construct using wild-type human codons). As used herein, the term "increased viral production" refers to increased vector production (e.g., titer/liter culture) in a cell culture inoculated with a codon altered polynucleotide encoding factor VIII as compared to the vector production in a cell culture inoculated with a naturally encoded factor VIII construct. Vector yield can be measured using any vector titer assay known in the art. An exemplary assay for determining vector yield (e.g., of AAV vectors) is qPCR targeting the AAV2 inverted terminal repeat (Aurnhammer, Human Gene Therapy Methods: Part B23: 18-28 (2012)).

In some embodiments, increased viral production refers to a vector yield at least 25% greater in codon changes compared to the yield of the naturally encoded factor VIII construct in the same type of culture. In some embodiments, increased vector production refers to a vector yield of codon alterations that is at least 50% greater, at least 75% greater, at least 100% greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 1O-fold greater, at least 15-fold greater, or at least 20-fold greater than the yield of a naturally encoded factor VIII construct in the same type of culture.

As used herein, the term "hemophilia" refers to a group of disease states generally characterized by reduced blood clotting or coagulation. Hemophilia may refer to hemophilia a, B or C, or to the complex of all three disease types. Hemophilia a (hemophilia a) is caused by a reduction or loss of factor viii (fviii) activity and is the most important hemophilia subtype. Hemophilia B (hemophilia B) is caused by a loss or reduction in coagulation function of factor ix (fix). Haemophilia C (hemophilia C) is a consequence of a loss or reduction in coagulation activity of factor xi (fxi). Haemophilia a and B are X-linked diseases, whereas haemophilia C is autosomal. Conventional treatments for hemophilia include prophylactic and on-demand administration of coagulation factors such as FVIII, FIX (including-VH) and FXI, as well as FEIBA-VH, desmopressin and plasma infusion.

As used herein, the term "FVIII gene therapy" includes any treatment method that provides a nucleic acid encoding factor VIII to a patient to alleviate, attenuate or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. The term encompasses the administration of any compound, drug, procedure or regimen comprising a nucleic acid encoding a factor VIII molecule, including any modified form of factor VIII (e.g., factor VIII variant), to maintain or improve the health of an individual with hemophilia. One skilled in the art will appreciate that the course of FVIII therapy or dose of FVIII therapeutic agent can be varied, for example, based on results obtained in accordance with the present disclosure.

As used herein, the term "bypass therapy" includes any treatment that provides a non-factor VIII hemostatic agent, compound, or coagulation factor to a patient to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. Non-factor VIII compounds and coagulation factors include, but are not limited to, factor VIII inhibitor bypass activity (FEIBA), recombinant activated factor vii (fviia), prothrombin complex concentrates, and activated prothrombin complex concentrates. These non-factor VIII compounds and coagulation factors may be recombinant or plasma derived. One skilled in the art will appreciate that the course of bypass therapy or the dose of bypass therapy may be varied, for example, based on the results obtained according to the present invention.

As used herein, "combination therapy" comprising administration of a nucleic acid encoding a factor VIII molecule and a conventional type a hemophilia therapeutic agent includes any method of treatment that provides a nucleic acid encoding a factor VIII molecule to a patient with a factor VIII molecule and/or a non-factor VIII hemostatic agent (e.g., a bypass therapeutic agent) to alleviate, reduce, or prevent the recurrence of one or more symptoms (e.g., clinical factors) associated with hemophilia. The term encompasses the administration of any compound, drug, procedure or regimen comprising a nucleic acid encoding a factor VIII molecule (including any modified form of factor VIII), which is suitable for maintaining or improving the health state of a subject suffering from hemophilia and includes any of the therapeutic agents described herein.

The term "therapeutically effective amount or dose" or "therapeutically sufficient amount or dose" or "effective or sufficient amount or dose" refers to a dose that produces the therapeutic effect for which it is intended to be administered. For example, a therapeutically effective amount of a drug suitable for treating hemophilia may be an amount capable of preventing or alleviating one or more symptoms associated with hemophilia. The precise Dosage will depend on The purpose of The treatment and will be determined by one of skill in The Art using known techniques (see, e.g., Lieberman, Pharmaceutical delivery Forms (Vol.1 to Vol.3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, delivery calls (1999); and Remington, The Science and Practice of Pharmacy, 20 th edition, 2003, Gennaro eds., Lippincott, Williams & Wilkins).

As used herein, the term "gene" refers to a segment of a DNA molecule (e.g., a coding region) that encodes a polypeptide chain. In some embodiments, a gene is located by a region immediately preceding, after, and/or inserted into the coding region that is involved in the production of a polypeptide chain (e.g., a regulatory element such as a promoter, enhancer, polyadenylation sequence, 5 'untranslated region, 3' untranslated region, or intron).

As used herein, the term "regulatory element" refers to a nucleotide sequence that provides for expression of a coding sequence in a cell, such as a promoter, enhancer, terminator, polyadenylation sequence, intron, and the like.

As used herein, the term "promoter element" refers to a nucleotide sequence that helps control the expression of a coding sequence. Generally, promoter elements are located 5' to the translation start site of a gene. However, in certain embodiments, the promoter element may be located within an intron sequence or 3' of the coding sequence. In some embodiments, a promoter suitable for use in a gene therapy vector is derived from an endogenous gene of a protein of interest (e.g., a factor VIII promoter). In some embodiments, promoters suitable for use in gene therapy vectors are specific for expression in a particular cell or tissue of the target organism (e.g., liver-specific promoters). In other embodiments, one of a plurality of well-characterized promoter elements is used in a gene therapy vector described herein. Non-limiting examples of well characterized promoter elements include the CMV early promoter, the β -actin promoter, and the methyl CpG binding protein 2(MeCP2) promoter. In some embodiments, the promoter is a constitutive promoter that drives substantially constant expression of the protein of interest. In other embodiments, the promoter is an inducible promoter that drives expression of the protein of interest in response to a particular stimulus (e.g., exposure to a particular treatment or agent). For a review of designing promoters for AAV-mediated Gene Therapy, see Gray et al (Human Gene Therapy 22:1143-53(2011)), the contents of which are expressly incorporated by reference in their entirety for all purposes.

As used herein, the term "vector" refers to any vehicle used to transfer a nucleic acid (e.g., encoding a factor VIII gene therapy construct) into a host cell. In some embodiments, the vector includes a replicon for replicating the vector along with the target nucleic acid. Non-limiting examples of vectors suitable for gene therapy include plasmids, phages, cosmids, artificial chromosomes and viruses, which serve as autonomous units of replication in vivo. In some embodiments, the vector is a viral vehicle for introducing a nucleic acid of interest (e.g., a codon-altered polynucleotide encoding a factor VIII variant). Many modified eukaryotic viruses suitable for use in gene therapy are known in the art. For example, adeno-associated viruses (AAV) are particularly useful for human gene therapy because the natural host for the virus, to which the virus is not known to contribute to any disease, and which elicits a mild immune response.

As used herein, the term "CpG island" refers to a region within a polynucleotide having a statistically higher density of CpG dinucleotides. As used herein, if within a 200 base pair window: (i) a region having a GC content of greater than 50%, and (ii) a region of a polynucleotide (e.g., a polynucleotide encoding a codon altered factor VIII protein) is a CpG island if the observed CpG dinucleotide/expected CpG dinucleotide ratio is at least 0.6, as defined by the relationship:

for additional information on methods for identifying CpG islands, see Gardiner-Garden m. et al, J Mol biol.,196(2):261-82(1987), the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

As used herein, the term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form, as well as their complementary sequences. The term encompasses nucleic acids containing synthetic, naturally occurring and non-naturally occurring known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and which are metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methyl ribonucleotide, and Peptide Nucleic Acid (PNA).

The term "amino acid" refers to both naturally occurring and non-natural amino acids, including amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, y-carboxyglutamic acid, and O-phosphoserine. Naturally occurring amino acids can include, for example, D-amino acids and L-amino acids. Amino acids as used herein may also include unnatural amino acids. Amino acid analogs refer to compounds having the same basic chemical structure as a naturally occurring amino acid (i.e., any carbon bound to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, or methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. Amino acids may be referred to herein by their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission (Biochemical Nomenclature Commission). Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

With respect to amino acid sequences, those of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a nucleic acid or peptide sequence that alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are additionally and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.

Conservative amino acid substitutions that provide functionally similar amino acids are well known in the art. Depending on the functionality of a particular amino acid (e.g., a catalytically, structurally, or sterically important amino acid), different groupings of amino acids may be considered conservatively substituted for one another. Table 1 groups of amino acids considered conservative substitutions are provided based on the charge and polarity of the amino acid, the hydrophobicity of the amino acid, the surface exposure/structural properties of the amino acid, and the secondary structure propensity of the amino acid.

TABLE 1. grouping of conservative amino acid substitutions based on the functionality of residues in proteins.

The term "identical" or percent "identity," in the context of two or more nucleic acid or peptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window or specified region), as measured using, for example, the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection.

As is known in the art, a variety of different procedures can be used to identify whether a protein (or nucleic acid as discussed below) has sequence identity or similarity to a known sequence. Sequence identity and/or similarity is determined using standard techniques known in the art, including but not limited to the local sequence identity algorithm of Smith and Waterman, adv.appl.math, 2:482(1981), preferably using default settings or by inspection; sequence identity alignment algorithms of Needleman and Wunsch, J.mol.biol.,48:443 (1970); search for similarity methods of Pearson and Lipman, proc.natl.acad.sci.u.s.a.,85:2444 (1988); computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group,575Science Drive, Madison, Wis.); the Best Fit sequence program described by Devereux et al, Nucl. acid Res.,12:387-395 (1984). Preferably, the percent identity is calculated by FastDB based on the following parameters: a mismatch penalty of 1; gap penalty 1; gap size penalty 0.33; and a connection penalty of 30, "Current Methods in Sequence company and Analysis", macromolecular Sequencing and Synthesis, Selected Methods and Applications, pp.127-149 (1988), Alan R.Liss, Inc., all of which are incorporated by reference.

An example of a suitable algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a set of related sequences using progressive pairwise alignments one by one. It can also plot a tree graph showing the clustering relationships used to generate the alignment. Simplification of PILEEIP using the progressive alignment method of Feng and Doolittle, J.mol.Evol.35:351-360 (1987); the method is similar to that described by Higgins and Sharp CABIOS 5: 151-. Suitable pileip parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a suitable algorithm is the BLAST algorithm, which is described in: altschul et al, J.mol.biol.215,403-410, (1990); altschul et al, Nucleic Acids Res.25:3389-3402 (1997); and Karlin et al, Proc. Natl. Acad. Sci. U.S.A.90:5873-5787(1993), both of which are incorporated by reference. A particularly useful BLAST program is the WU-BLAST-2 program available from Altschul et al, Methods in Enzymology,266: 460-; http:// blast.wustl/edu/blast/README. html. WU-BLAST-2 uses several search parameters, most of which are set to default values. The adjustable parameters were set using the following values: the overlap interval is 1, the overlap score is 0.125, and the word threshold (T) is 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself based on the composition of the particular sequence and the composition of the particular database against which the relevant sequences are searched; however the value can be adjusted to increase sensitivity.

Other suitable algorithms are gapped BLAST, as reported by Altschul et al, Nucl. acids Res.,25: 3389-. Gap BLAST uses the BLOSUM-62 substitution score; the threshold T parameter is set to 9; the two-shot method is used for triggering the vacancy-free extension part, and the vacancy length k is added, so that the cost is 1O + k; xu is set to 16 and Xg is set to 40 for the database retrieval phase and 67 for the output phase of the algorithm. A null alignment is triggered by a score corresponding to about 22 bits (bit).

The% amino acid sequence identity value is determined by dividing the number of matching identical residues by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is one that has the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize alignment score are ignored). In a similar manner, with respect to the coding sequence of the identified polypeptide, "percent (%) nucleic acid sequence identity" is defined as the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the cell cycle protein. The preferred method utilizes the BLASTN module of WU-BLAST-2 set to default parameters, with overlap intervals and overlap scores set to 1 and 0.125, respectively.

Alignment may include introducing gaps into the sequences to be aligned. Furthermore, for sequences containing more or fewer amino acids than the protein encoded by the sequence of FIG. 2 (SEQ ID NO:1), it will be understood that in one embodiment, the percentage of sequence identity will be determined based on the number of identical amino acids or nucleotides relative to the total number of amino acids or nucleotides. Thus, for example, in one embodiment, the sequence identity of a sequence that is shorter than the sequence shown in FIG. 2 (SEQ ID NO:1) as discussed below will be determined using the number of nucleotides in the shorter sequence. In the percent identity calculation, relative weights are not given to various expressions of sequence variants, such as insertions, deletions, substitutions, and the like.

In one embodiment, only the identity score (+1) and all forms of sequence variants including gaps are assigned a value of "0", which avoids the need for weighted scores or parameters as described below for sequence similarity calculations. Percentage sequence identity can be calculated, for example, by dividing the number of identical residues that are matched by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. A "longer" sequence is one that has the most actual residues in the aligned regions.

The term "allelic variant" refers to a polymorphic form of a gene at a particular locus as well as to cDNA derived from the mRNA transcript of the gene and the polypeptides encoded thereby. The term "preferred mammalian codons" refers to a codon subset from among the set of codons encoding amino acids most commonly used for proteins expressed in mammalian cells, such as selected from the following list: gly (GGC, GGG); glu (GAG); asp (GAC); val (GTG, GTC); ala (GCC, GCT); ser (AGC, TCC); lys (aag); asn (AAC); met (atg); ile (atc); thr (acc); tip (tgg); cys (TGC); tyr (TAT, TAC); leu (CTG); phe (TTC); arg (CGC, AGG, AGA); gln (cag); his (cac); and Pro (CCC).

As used herein, the term codon alteration refers to a polynucleotide sequence encoding a polypeptide (e.g., a factor VIII variant protein) in which at least one codon of the native polynucleotide encoding the polypeptide has been altered to improve a property of the polynucleotide sequence. In some embodiments, the improved property facilitates increased transcription of mRNA encoding the polypeptide, increased stability of the mRNA (e.g., increased mRNA half-life), increased translation of the polypeptide, and/or increased polynucleotide encapsulation within the vector. Non-limiting examples of changes that can be used to achieve improved properties include changing the usage and/or distribution of codons for a particular amino acid, adjusting overall and/or local GC content, removing AT-rich sequences, removing repetitive sequence elements, adjusting overall and/or local CpG dinucleotide content, removing recessive regulatory elements (e.g., TATA box and CCAAT box elements), removing intron/exon splice sites, increasing regulatory sequences (e.g., introducing Kozak consensus sequences), and removing sequence elements capable of forming secondary structures (e.g., stem-loops) in the transcribed mRNA.

As discussed herein, various nomenclature exists herein to refer to the components of the invention. "CS-numbering" (e.g., "CS 04") refers to codon-altered polynucleotides and/or encoded polypeptides, including variants, that encode FVIII polypeptides. For example, CS04-FL refers to a full-length codon-altered CS04 polynucleotide sequence or amino acid sequence encoded by a CS04 polynucleotide sequence (sometimes referred to herein as "CS 04-FL-AA" for amino acid sequences and "CS 04-FL-NA" for nucleic acid sequences). Similarly, "CS 04-LC" refers to a codon-altered nucleic acid sequence ("CS 04-LC-NA") encoding the light chain of a FVIII polypeptide or the amino acid sequence of a FVIII light chain encoded by a CS04 polynucleotide sequence (also sometimes referred to herein as "CS 04-LC-AA"). Likewise, CS04-HC, CS04-HC-AA and CS04-HC-NA are identical for the FVIII heavy chain. As will be appreciated by those skilled in the art, for constructs that are only codon-altered, such as CS04 (e.g., which does not contain other amino acid substitutions as compared to Refacto), the amino acid sequence will be the same, as the amino acid sequence is not altered by codon optimization. Thus, sequence constructs of the present disclosure include, but are not limited to, CS04-FL-NA, CS04-FL-AA, CS04-LC-NA, CS04-LC-AA, CS04-HC-AA, and CS 04-HC-NA.

Codon-altered factor VIII variants

In some embodiments, the present disclosure provides a codon altered polynucleotide encoding a factor VIII variant. These codon-altered polynucleotides provide significantly improved factor VIII expression when administered in AAV-based gene therapy constructs. The codon-altered polynucleotides also exhibit improved AAV virion encapsulation compared to conventional codon-optimized constructs. As demonstrated in example 2 and table 4, applicants have realized these advantages from the discovery of a codon-altered polynucleotide (CS04-FL-NA) encoding a factor VIII polypeptide having human wild-type factor VIII heavy and light chains and a short, 14 amino acid, B-domain substituted linker ("SQ" linker) containing a furin cleavage site to facilitate in vivo maturation of active FVIIIa proteins.

In one embodiment, the codon altered polynucleotides provided herein have a nucleotide sequence with high sequence identity to a sequence within CS04(SEQ ID NO:1) encoding at least the factor VIII heavy chain and the factor VIII light chain. As known in the art, the B domain of factor VIII is not essential for in vivo activity. Thus, in some embodiments, the codon altered polynucleotides provided herein lack the factor VIII B domain entirely. In some embodiments, the native factor VIII B domain is replaced by a short amino acid linker containing a furin cleavage site, such as the "SQ" linker consisting of amino acids 760-773 of the CS04(SEQ ID NO 2) construct. The "SQ" linker is also known as BDLO04 (BDLO 04-AA for the amino acid sequence and BDLO04-NA for the nucleotide sequence).

In one embodiment, the factor VIII heavy and light chains encoded by the codon altered polynucleotide are human factor VIII heavy and light chains, respectively. In other embodiments, the codon altered polynucleotide encodes a factor VIII heavy and light chain that is a heavy and light chain sequence from another mammal (e.g., porcine factor VIII). In other embodiments, the factor VIII heavy and light chains are chimeric heavy and light chains (e.g., a combination of human and second mammalian sequences). In other embodiments, the factor VIII heavy and light chains are humanized versions of heavy and light chains from another mammal, e.g., from another mammal's heavy and light chain sequences in which human residues are substituted at selected positions to reduce the immunogenicity of the resulting peptide when administered to a human.

The GC content of human genes varies widely from below 25% to above 90%. However, in general, human genes with higher GC content are expressed at higher levels. For example, Kudla et al (PLoS biol.,4(6):80(2006)) demonstrated that increasing the GC content of a gene increases expression of the encoded polypeptide, primarily by increasing transcription and achieving higher steady-state levels of mRNA transcription. Generally, the desired GC content of the codon-optimized gene construct is equal to or higher than 60%. However, the GC content of the native AAV genome is approximately 56%.

Thus, in some embodiments, the CG content of the codon-altered polynucleotides provided herein more closely matches the GC content of native AAV virions (e.g., about 56% GC), which is lower than the preferred CG content (e.g., at or above 60% GC) of polynucleotides that are conventionally codon-optimized for expression in mammalian cells. As outlined in example 1, CS04-FL-NA (SEQ ID NO:1) with a GC content of about 56% has an improved virion encapsulation compared to a coding sequence with a similar codon change with a higher GC content.

Thus, in some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is less than 60%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is less than 59%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is less than 58%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is less than 57%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide does not exceed 56%.

In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 54% to 59%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 55% to 59%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56% to 59%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 54% to 58%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 55% to 58%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56% to 58%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 54% to 57%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 55% to 57%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56% to 57%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 54% to 56%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 55% to 56%.

In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56 ± 0.5%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56 ± 0.4%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56 ± 0.3%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is 56 ± 0.2%. In some embodiments, the total GC content of the codon altered polynucleotide encoding the factor VIII polypeptide is 56 ± 0.1%. In some embodiments, the codon altered polynucleotide encoding the factor VIII polypeptide has an overall GC content of 56%.

A. Substituted linkers for the factor VIII B domain

In some embodiments, the linkage between the FVIII heavy chain and the light chain is further altered (e.g., B domain in wild-type factor VIII). Due to size limitations of AAV encapsulation capacity, B domain deleted, truncated, and or linker substituted variants should improve the efficacy of FVIII gene therapy constructs. The most commonly used B domain substituted linker is the linker of SQ FVIII, which retains only 14 amino acids of the B domain as linker sequence. Another variant of porcine VIII ("OBI-1" described in U.S. patent No. 6,458,563) is well expressed in CHO cells and has a slightly longer linker of 24 amino acids. In some embodiments, the factor VIII construct encoded by the codon altered polynucleotides described herein comprises a SQ type B domain linker sequence. In other embodiments, the factor VIII construct encoded by the codon altered polynucleotides described herein comprises an OBI-type 1B domain linker sequence.

In some embodiments, the encoded factor VIII polypeptide described herein comprises a SQ-type B domain linker (SFSQNPPVLKRHQR; BDL-SQ-AA; SEQ ID NO:13) that comprises amino acids 760-762/1657-1667 (FVIII-FL-AA; SEQ ID NO:12) of the wild-type human factor VIII B domain (Sandberg et al, Thromb. Haemost.85:93 (2001)). In some embodiments, the SQ type B domain linker has one amino acid substitution relative to the corresponding wild type sequence. In some embodiments, the SQ-type B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptide described herein comprises a Greenene type B domain linker comprising amino acids 760/1582-1667 (FVIII-FL-AA; SEQ ID NO:12) of the wild-type human factor VIII B domain (Oh et al, Biotechnol. prog.,17:1999 (2001)). In some embodiments, the greenene-type B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the greenene-type B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptide described herein comprises an extended SQ-type B domain linker comprising amino acids 760-769/1657-1667 (FVIII-FL-AA; SEQ ID NO:12) of the wild-type human factor VIII B domain (Thim et al, Haemophilia,16:349 (2010)). In some embodiments, the extended SQ B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the extended SQ B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptides described herein comprise a porcine OBI-1 type B domain linker comprising amino acid SFAQNSRPPSASAPKPPVLRRHQR (SEQ ID NO:14) from a wild-type porcine factor VIII B domain (Toschi et al, curr. opin. mol. ther.12:517 (2010)). In some embodiments, the porcine OBI-1 type B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the porcine OBI-1 type B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptide described herein comprises a human OBI-1 type B domain linker comprising amino acids 760-772/1655-1667 (FVIII-FL-AA; SEQ ID NO:12) of a wild-type human factor VIII B domain. In some embodiments, the human OBI-1 type B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the human OBI-1 type B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

In some embodiments, the encoded factor VIII polypeptides described herein comprise a type 08B domain linker comprising amino acid SFSQNSRHQAYRYRRG (SEQ ID NO:15) from the wild-type porcine factor VIII B domain (tosci et al, curr. opin. mol. ther.12:517 (2010)). In some embodiments, the porcine OBI-1 type B domain linker has one amino acid substitution relative to the corresponding wild-type sequence. In some embodiments, the porcine OBI-1 type B domain linker has two amino acid substitutions relative to the corresponding wild-type sequence.

B. Codon-altered polynucleotides encoding factor VIII variants with cleavable linkers

CS04 codon altered polynucleotides

In one embodiment, the codon altered polynucleotides provided herein comprise a nucleotide sequence encoding a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence having high sequence identity to CS04-HC-NA (SEQ ID NO:3), which CS04-HC-NA (SEQ ID NO:3) is the portion of CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence having high sequence identity to CS04-LC-NA (SEQ ID NO:4), which CS04-LC-NA (SEQ ID NO:4) is part of the CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII light chain. The polypeptide linker includes a furin cleavage site that causes in vivo maturation (e.g., following expression or administration of a precursor polypeptide in vivo).

In some embodiments, the first and second nucleotide sequences have at least 95% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 96% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 97% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 98% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.5% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences have at least 99.9% sequence identity to CS04-HC-NA and CS04-LC-NA (SEQ ID NOs 3 and 4), respectively. In some embodiments, the first and second nucleotide sequences are identical to CS04-HC-NA and CS04-LC-NA (SEQ ID NOS 3 and 4), respectively.

In some embodiments, the codon altered polynucleotide has a nucleotide sequence with high sequence identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 96% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 97% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 98% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is identical to CS04-FL-NA (SEQ ID NO: 1).

In some embodiments, the factor VIII variant encoded by the codon altered polynucleotide has an amino acid sequence with high sequence identity to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 97% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 98% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99.5% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99.9% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is identical to CS04-FL-AA (SEQ ID NO: 2).

C. Factor VIII expression vectors

In some embodiments, the codon altered polynucleotides described herein are integrated into an expression vector. Non-limiting examples of expression vectors include viral vectors (e.g., vectors suitable for gene therapy), plasmid vectors, phage vectors, cosmids, phagemids, artificial chromosomes, and the like.

Non-limiting examples of viral vectors include: retroviruses, such as Moloney (Moloney) murine leukemia virus (MMLV), Harvey (Harvey) murine sarcoma virus, murine mammary tumor virus, and Rous (Rous) sarcoma virus; adenovirus, adeno-associated virus; SV 40-type virus; a polyoma virus; Epstein-Barr (Epstein-Barr) virus; papilloma virus; herpes virus; vaccinia virus and poliovirus.

In some embodiments, the codon altered polynucleotides described herein are integrated into a gene therapy vector. In some embodiments, the gene therapy vector is a retrovirus, and in particular a replication defective retrovirus. Protocols for generating replication-defective retroviruses are known in the art. For a review see Kriegler, m., Gene Transfer and Expression, a Laboratory Manual, w.h.freeman co., New York (1990) and Murry, e.j., Methods in Molecular Biology, volume 7, Humana Press, inc., Cliffton, n.j. (1991).

In one embodiment, the gene therapy vector is an adeno-associated virus (AAV) -based gene therapy vector. AAV systems have been previously described and are generally well known in the art (Kelleher and Vos, Biotechniques,17(6):1110-17 (1994); Cotten et al, Proc Natl Acad Sci USA,89(13):6094-98 (1992); Curiel, Nat Immun,13(2-3):141-64 (1994); Muzyzka, Curr Top Microbiol Immunol,158:97-129 (1992); and Asokan A et al, mol. Ther.,20(4):699-708(2012), each incorporated herein by reference in its entirety for all purposes). Details regarding the production and use of rAAV vectors are described, for example, in U.S. patent nos. 5,139,941 and 4,797,368, each of which is incorporated herein by reference for all purposes. In a particular embodiment, the AAV vector is an AAV-8 vector.

In some embodiments, the codon altered polynucleotides described herein are integrated into a retroviral expression vector. These systems have been described previously and are generally well known in the art (Mann et al, Cell 33: 153-. In particular embodiments, the retroviral vector is a lentiviral vector (see, e.g., Naldini et al, Science,272(5259):263- -267, 1996; Zufferey et al, Nat Biotechnol,15(9):871 875, 1997; Blomer et al, J Virol,71(9): 6641-.

A wide variety of vectors are available for expressing factor VIII polypeptides from codon altered polypeptides in cell culture, including eukaryotic and prokaryotic expression vectors. In certain embodiments, plasmid vectors are contemplated for expression of the factor VIII polypeptide in cell culture. In general, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in conjunction with these hosts. The vector may carry a replication site as well as a marker sequence capable of providing phenotypic selection in transformed cells. The plasmid will comprise a codon altered polynucleotide encoding a factor VIII polypeptide operably linked to one or more control sequences (e.g., a promoter).

Non-limiting examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, pBAD, etc., wherein promoters for prokaryotic expression vectors include lac, trc, trp, recA, araBAD, etc. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors using promoters such as AOX1, GAP, GAL1, AETG1, and the like, such as pAO, pPIC, pYES, pMET; (ii) for expression in insect cells, vectors using promoters such as PH, p1O, MT, Ac5, OpIE2, gp64, polh, etc., such as pMT, pAc5, pIB, pMIB, pBAC, etc.; and (iii) vectors such as pSVL, pCMV, prb/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β -actin, for expression in mammalian cells.

D. Administration of drugs

The present invention provides for administering the codon-optimized constructs of the invention to a human patient who has been diagnosed with hemophilia a ("hemophilia a patient" or "patient"). In general, administration is performed using AAV particles containing the codon-optimized constructs of the invention, as outlined herein. In addition, as described more fully below, administration of the constructs of the invention may be augmented by also administering prednisolone or prednisone.

2×10¹²Individual adeno-associated virus (AAV) particles per kilogram body weight

In one aspect, the invention provides a method for treating hemophilia a comprising intravenous infusion (e.g., by peripheral intravenous infusion) of 2x 10 into a patient with hemophilia a¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of a human patient, wherein the AAV particles comprise a codon-altered polynucleotide encoding a factor VIII polypeptide and having high sequence identity to SEQ ID NO:1(CS 04-FL-NA).

In one embodiment, at 2 × 10¹²A codon-altered polynucleotide having high sequence identity to SEQ ID NO:1(CS04-FL-NA) administered to a human patient at a dose of individual adeno-associated virus (AAV) particles per kilogram of body weight of the human patient encodes a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence having high sequence identity to CS04-HC-NA (SEQ ID NO:3), which CS04-HC-NA (SEQ ID NO:3) is the portion of CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence having high sequence identity to CS04-LC-NA (SEQ ID NO:4), which CS04-LC-NA (SEQ ID NO:4) is part of the CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII light chain. The polypeptide linker comprises furinA cleavage site that causes in vivo maturation (e.g., following expression or administration of a precursor polypeptide in vivo).

In some embodiments, the polypeptide linker of the factor VIII construct is encoded by a third nucleotide sequence having high sequence identity to BDLO04(SEQ ID NO:5), which BDLO04(SEQ ID NO:5) encodes a 14 amino acid linker corresponding to amino acids 760-773 of CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the third nucleotide sequence is at least 95% identical to BDLO04(SEQ ID NO: 5). In some embodiments, the third nucleotide sequence has at least 96% identity to BDLO04(SEQ ID NO: 5). In some embodiments, the third nucleotide sequence is at least 97% identical to BDLO04(SEQ ID NO: 5). In some embodiments, the third nucleotide sequence has at least 98% identity to BDLO04(SEQ ID NO: 5). In some embodiments of the present invention, the substrate is,the third nucleotide sequence was identical to BDLO04(SEQ ID NO: 5). In these embodiments, the amino acid sequence encoded by these nucleotide sequences is identical to amino acids 760-773 of CS04-FL-AA (SEQ ID NO: 2). In some embodiments, at 2 × 10¹²The codon-altered polynucleotide administered to a human patient at a dose of individual adeno-associated virus (AAV) particles per kilogram of body weight of the human patient has a nucleotide sequence with high sequence identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 96% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 97% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 98% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is identical to CS04-FL-NA (SEQ ID NO: 1). In these embodiments, the amino acid sequence encoded by these nucleotide sequences is identical to CS 04-FL-AA.

Accordingly, in one embodiment, the present invention provides a method for treating hemophilia a comprising intravenous infusion of 2x 10 into a patient having hemophilia a¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of a human patient, wherein the AAV particles comprise a polynucleotide having the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA).

In some embodiments, the AAV particles are administered in a single dose by intravenous infusion (e.g., into a vein in the arm of a patient). In some embodiments, a portion of the bolus dose is administered, the patient is monitored for a brief period of time (e.g., 30 minutes) of adverse reaction signs to the administration, and then (e.g., if no adverse reaction signs are present) the remaining portion of the bolus dose is administered to the patient.

In some embodiments, the human patient administered the AAV particle has severe hemophilia a. For example, in some embodiments, when not receiving factor VIII replacement therapy, the patient has a level of factor VIII activity in their blood stream that is 2% or more lower than the average factor VIII activity present in a blood sample of a subject determined not to have hemophilia a than the amount of factor VIII activity present in a reference blood sample, e.g., a blood sample having normal factor VIII activity (e.g., a blood sample from a subject determined not to have hemophilia a). In some embodiments, the subject has a level of factor VIII activity in its blood stream that is less than 2% of the amount of factor VIII activity present in the reference blood sample when not receiving factor VIII replacement therapy.

In some embodiments, a human patient administered an AAV particle does not have a FVIII inhibitor (e.g., factor VIII inhibitor antibody), does not have a hemostatic deficiency other than severe hemophilia a, does not have chronic liver dysfunction, and/or does not have severe kidney injury.

Thus, in some embodiments, the methods described herein comprise identifying a patient for administration of 2x 10¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the human patient, wherein the AAV particles comprise a polypeptide encoding factor VIII and have a sequence as set forth in SEQ ID NO:1(CS04-FL-NA)A codon-altered polynucleotide of high sequence identity. The method comprises determining the level of factor VIII activity in the blood stream of the patient when the patient is not receiving factor VIII replacement therapy, and identifying the patient for administration of AAV particles when the level of factor VIII activity in the blood stream of the patient is less than about 2% of the level of factor VIII in a reference sample or less than about 1% of the level of factor VIII in a reference sample. In some embodiments, the method comprises determining whether the patient has one or more FVIII inhibitors (e.g., factor VIII inhibitor antibodies), hemostasis deficiency other than severe hemophilia a, chronic liver dysfunction, and severe kidney injury, and culling the patient if the patient has any of the listed conditions.

6×10¹²Individual adeno-associated virus (AAV) particles per kilogram body weight

In one aspect, the invention provides a method for treating hemophilia a comprising intravenous infusion (e.g., by peripheral intravenous infusion) of 6 x 10 into a patient with hemophilia a¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of a human patient, wherein the AAV particles comprise a codon-altered polynucleotide encoding a factor VIII polypeptide and having high sequence identity to SEQ ID NO:1(CS 04-FL-NA).

In one embodiment, at 6 × 10¹²A codon-altered polynucleotide having high sequence identity to SEQ ID NO:1(CS04-FL-NA) administered to a human patient at a dose of individual adeno-associated virus (AAV) particles per kilogram of body weight of the human patient encodes a factor VIII variant polypeptide having an in vivo cleavable linker. The factor VIII polypeptide includes a factor VIII light chain, a factor VIII heavy chain, and a polypeptide linker connecting the C-terminus of the heavy chain to the N-terminus of the light chain. The heavy chain of the factor VIII polypeptide is encoded by a first nucleotide sequence having high sequence identity to CS04-HC-NA (SEQ ID NO:3), which CS04-HC-NA (SEQ ID NO:3) is the portion of CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII heavy chain. The light chain of the factor VIII polypeptide is encoded by a second nucleotide sequence having high sequence identity to CS04-LC-NA (SEQ ID NO:4), which CS04-LC-NA (SEQ ID NO:4) is part of the CS04-FL-NA (SEQ ID NO:1) encoding the factor VIII light chain. The polypeptide linker comprises furinA cleavage site that causes in vivo maturation (e.g., following expression or administration of a precursor polypeptide in vivo).

In some embodiments, at 6 × 10¹²The codon-altered polynucleotide administered to a human patient at a dose of individual adeno-associated virus (AAV) particles per kilogram of body weight of the human patient has a nucleotide sequence with high sequence identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 95% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 96% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 97% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 98% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is at least 99% identical to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.5% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence has at least 99.9% identity to CS04-FL-NA (SEQ ID NO: 1). In some embodiments, the nucleotide sequence is identical to CS04-FL-NA (SEQ ID NO: 1). In these embodiments, the amino acid sequence encoded by these nucleotide sequences is identical to CS 04-FL-AA.

In some embodiments, the codon altered polynucleotide encodes a factor VIII variant having an amino acid sequence with high sequence identity to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 97% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 98% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99.5% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is at least 99.9% identical to CS04-FL-AA (SEQ ID NO: 2). In some embodiments, the amino acid sequence is identical to CS04-FL-AA (SEQ ID NO: 2).

Accordingly, in one embodiment, the present invention provides a method for treating hemophilia a comprising intravenous infusion of 6 x 10 into a patient having hemophilia a¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of a human patient, wherein the AAV particles comprise a polynucleotide having the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA).

Thus, in some embodiments, the methods described herein comprise identifying a patient for administration of 6 x 10¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the human patient, wherein the AAV particles comprise a polypeptide encoding factor VIII anda codon altered polynucleotide having high sequence identity to SEQ ID NO. 1(CS 04-FL-NA). The method comprises determining the level of factor VIII activity in the blood stream of the patient when the patient is not receiving factor VIII replacement therapy, and identifying the patient for administration of AAV particles when the level of factor VIII activity in the blood stream of the patient is less than about 2% of the level of factor VIII in a reference sample or less than about 1% of the level of factor VIII in a reference sample. In some embodiments, the method comprises determining whether the patient has one or more FVIII inhibitors (e.g., factor VIII inhibitor antibodies), hemostasis deficiency other than severe hemophilia a, chronic liver dysfunction, and severe kidney injury, and culling the patient if the patient has any of the listed conditions.

Coadministration with prednisolone or prednisone

In some embodiments, the methods described above for treating hemophilia a by administering AAV particles at any dose further comprise administering prednisolone or a course of treatment of prednisone to a human patient, for example, to reduce the level of inflammatory response, e.g., by reducing cytokine and/or chemokine production in the subject. An example method of co-administering prednisolone or prednisone with gene therapy is described, for example, in international patent application publication No. WO 2008/069942, the contents of which are incorporated herein by reference in their entirety for all purposes.

In some embodiments, prednisolone or prednisone is administered to a human patient prior to administration of adeno-associated virus (AAV) particles having a polynucleotide with high sequence identity to SEQ ID NO:1(CS04-FL-NA) and encoding a factor VIII polypeptide. For example, in some embodiments, prednisolone or prednisone is administered about one week or about one or two days prior to administration of AAV particles to the patient. In some embodiments, the course of administration of prednisolone or prednisone begins about one week or about one or two days before administration of the AAV particles and continues after administration of the AAV particles.

In some embodiments, prednisolone or prednisone is co-administered to a human subject when adeno-associated virus (AAV) particles having a polynucleotide encoding a factor VIII polypeptide and having high sequence identity to SEQ ID NO:1(CS04-FL-NA) are administered. For example, in some embodiments, prednisolone or prednisone is administered on the same day (e.g., immediately before or after administration of AAV particles). In some embodiments, the prednisolone or the course of prednisone is administered on the same day as the AAV particles and continues after the AAV particles are administered.

In some embodiments, prednisolone or prednisone is administered to the patient after administration of adeno-associated virus (AAV) particles having a polynucleotide encoding a factor VIII polypeptide and having high sequence identity to SEQ ID NO:1(CS 04-FL-NA). For example, in some embodiments, prednisolone or prednisone is administered first about one or two days after the AAV particles are administered to the patient.

It should be noted that prednisolone or prednisone is a small molecule drug that is administered orally (although it may also be administered intravenously), and thus "co-administration" in this case does not require a single solution containing both drugs.

In some embodiments, prednisolone or a course of prednisone is administered to the patient over a period of at least two weeks, e.g., daily or every two days. In some embodiments, the prednisolone or the course of prednisone is administered over a period of at least three weeks. In some embodiments, the dose of prednisolone or prednisone is decreased during the course of treatment. For example, in one embodiment, a course of treatment begins with administration of about 60mg of prednisolone or prednisone per day, and decreases as the course of treatment progresses.

In one embodiment, the course of treatment comprises administering about 60mg of prednisolone or prednisone per day to a human patient during a first week of the course of treatment, about 40mg of prednisolone or prednisone per day to the patient during a second week of the course of treatment, and about 30mg of prednisolone or prednisone per day to the patient during a third week immediately following infusion of the AAV particles.

In some embodiments, the course of treatment comprises further tapering of the administration of prednisolone or prednisone after the third week, e.g., administering a tapering dose of prednisolone or prednisone. In one embodiment, the tapering of the dose of prednisolone or prednisone comprises administering a dose of about 20mg of prednisolone or prednisone per day, about 15mg of prednisolone or prednisone per day, about 10mg of prednisolone or prednisone per day, and about 5mg of prednisolone or prednisone per day (e.g., one or more doses at each concentration) sequentially.

In one embodiment, the tapering of the dose of prednisolone or prednisone includes administering about 20mg of prednisolone or prednisone to the patient for 5 consecutive days (e.g., immediately) after completion of an initial course of prednisolone or prednisone, administering about 15mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 20mg of prednisolone or prednisone to the patient for 5 days, administering about 10mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 15mg of prednisolone or prednisone to the patient for 3 days, and administering about 5mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 10mg of prednisolone or prednisone to the patient for 3 days.

In one embodiment, the tapering of the dose of prednisolone or prednisone comprises administering to the patient about 30mg of prednisolone or prednisone per day for 7 consecutive days following completion of an initial course of prednisolone or prednisone, administering to the patient about 20mg of prednisolone or prednisone per day for 7 consecutive days following administration of 30mg of prednisolone or prednisone to the patient for 7 days, administering to the patient about 15mg of prednisolone or prednisone per day for 7 days following administration of 20mg of prednisolone or prednisone to the human subject, administering to the patient 15mg of prednisolone or prednisone for 5 days followed by about 10mg of prednisolone or prednisone per day for 5 consecutive days and administering to the patient about 5mg of prednisolone or prednisone per day for 5 consecutive days after 10mg of prednisolone or prednisone for 5 days.

In some embodiments, the length of time that the patient is administered a tapered dose of prednisolone or prednisone is determined based on whether the patient still exhibits signs of hepatitis (e.g., as indicated by a decrease in factor VIII levels (e.g., factor VIII potency or factor VIII activity) or an increase in liver enzymes at the end of the initial course of prednisolone or prednisone.

For example, in one embodiment, following administration to a patient of a polynucleotide adeno-associated virus (AAV) particle comprising a protein encoding factor VIII, a first level (e.g., potency or activity) of factor VIII in the patient's bloodstream (e.g., in a blood sample collected from the patient) is determined concurrently with an initial course of treatment of the patient with a glucocorticoid steroid. After completion of the initial course of glucocorticoid steroid treatment, a second level of factor VIII (e.g., potency or activity) in the patient's bloodstream is determined. The second level of factor VIII is then compared to the first level of factor VIII. When the second level of factor VIII is not decreased (e.g., when the second level of factor VIII is not less than the first level of factor VIII or is not less than a threshold amount less than the first level of factor VIII), the patient is administered a first escalating dose of a glucocorticoid steroid for a period of no more than three weeks. When the second level of factor VIII is decreased (e.g., when the second level of factor VIII is below the first level of factor VIII or below a threshold amount less than the first level of factor VIII), the patient is administered a second, progressively decreasing dose of a glucocorticoid steroid for a period of more than three weeks.

Similarly, in some embodiments, a first level of liver enzymes (e.g., liver enzyme titer or activity) in the patient's blood stream is determined prior to (e.g., or shortly after) administering to the patient an adeno-associated virus (AAV) particle comprising a polynucleotide encoding a factor VIII protein. After completion of the initial course of glucocorticoid steroid treatment, a second level of liver enzyme levels (e.g., liver enzyme titer or activity) in the patient's bloodstream is determined. The second level of liver enzyme is then compared to the first level of liver enzyme. When the second level of liver enzyme is not increased (e.g., when the second level of liver enzyme does not exceed the first level of liver enzyme or does not exceed a threshold amount greater than the first level of liver enzyme), administering to the patient a first progressively decreasing dose of a glucocorticoid steroid for a period of no more than three weeks. When the second level of liver enzymes is increased (e.g., when the second level of liver enzymes exceeds the first level of liver enzymes or exceeds a threshold amount greater than the first level of liver enzymes), a second, progressively decreasing dose of a glucocorticoid steroid is administered to the patient for a period of more than three weeks.

In some embodiments, the first tapering dose of prednisolone or prednisone includes administering about 20mg of prednisolone or prednisone to the patient for 5 consecutive days (e.g., immediately) after completion of an initial course of prednisolone or prednisone, administering about 15mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 20mg of prednisolone or prednisone to the patient for 5 days, administering about 10mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 15mg of prednisolone or prednisone to the human subject for 3 days, and administering about 5mg of prednisolone or prednisone to the patient for 3 consecutive days (e.g., immediately) after administering 10mg of prednisolone or prednisone to the patient.

In some embodiments, the second tapering dose of prednisolone or prednisone includes administering to the patient about 30mg of prednisolone or prednisone per day for 7 consecutive days following completion of the initial course of prednisolone or prednisone, administering to the patient about 20mg of prednisolone or prednisone per day for 7 consecutive days following administration of 30mg of prednisolone or prednisone to the patient for 7 days, administering to the patient about 15mg of prednisolone or prednisone per day for 7 days following administration of 20mg of prednisolone or prednisone to the patient for 5 consecutive days, administering to the patient 15mg of prednisolone or prednisone for 5 days followed by about 10mg of prednisolone or prednisone per day for 5 consecutive days and administering to the patient about 5mg of prednisolone or prednisone per day for 5 consecutive days after 10mg of prednisolone or prednisone for 5 days.

In some embodiments, the prednisolone or course of treatment of prednisone is administered after the signs of an immune response are detected in the patient following administration of the AAV particles. In some embodiments, the prednisolone or the course of prednisone therapy is administered after detection of evidence of hepatitis in the patient. For example, in some embodiments, the hepatitis of the patient is monitored after administration of the AAV particles, and prednisolone or a course of prednisone is administered to the patient after hepatitis is detected.

In some embodiments, a rapid or large decrease in factor VIII expression or factor VIII activity in the patient's blood stream is indicative of hepatitis in the subject. In some embodiments, it is possible that an early peak in factor VIII activity may be observed followed by a small and/or gradual decrease, followed by the formation of factor VIII protein at slightly lower levels, without the need for prednisolone or a course of prednisone administration. For example, in some embodiments, the amount of factor VIII in the patient's blood stream (e.g., factor VIII titer or factor VIII activity level) is monitored after administration of AAV particles, and if a rapid or large decrease in the amount of factor VIII is detected (e.g., a threshold decrease in the level in the patient's blood stream above the factor VIII titer or factor VIII activity level as compared to the level in the patient's blood stream after administration of AAV particles), a course of prednisolone or prednisone is administered to the subject.

In some embodiments, an increase in liver enzyme levels in the patient is indicative of hepatitis in the subject. For example, in some embodiments, liver enzyme levels in the patient are monitored after administration of the AAV particles, and if an increase in liver enzyme levels is detected (e.g., a threshold increase in the amount of liver enzyme is exceeded, e.g., compared to a baseline level of liver enzyme in the patient prior to administration of the AAV particles or shortly after administration of the AAV particles), the patient is administered prednisolone or a course of prednisone.

Post-administration monitoring

In some embodiments, methods are provided for monitoring adverse reactions and/or therapeutic efficacy in a patient following administration of adeno-associated virus (AAV) particles having a polynucleotide encoding a factor VIII polypeptide, e.g., a polynucleotide having high sequence identity to SEQ ID NO:1(CS 04-FL-NA). In some embodiments, the patient is monitored for one or more of: (a) evidence of hepatitis (e.g., via a rapid or major decrease in factor VIII levels (e.g., potency or activity) and/or an increase in liver enzymes (e.g., potency or activity), (b) an increase in factor VIII inhibitor antibodies in the patient's bloodstream, (c) an increase in capsid protein in the patient's bloodstream, (d) an increase in anti-capsid protein antibodies in the patient's bloodstream, and (e) an increase in a polynucleotide encoding a factor VIII polypeptide or fragment thereof in the patient's bloodstream. In some embodiments, the subject is further treated after one or more adverse reactions are detected and/or the treatment is ineffective.

For example, in one embodiment, a method is provided for monitoring the efficacy of factor VIII gene therapy for hemophilia a using adeno-associated virus (AAV) particles comprising a polynucleotide encoding a factor VIII polypeptide. The methods include determining whether factor VIII inhibitor antibodies are present in the blood stream of the patient (e.g., in a blood sample collected from the patient) after administration of AAV particles to the patient. In some embodiments, when a factor VIII inhibitor antibody is detected in the bloodstream of a patient (e.g., when an increase in the level of the factor VIII inhibitor antibody is detected as compared to the level in the patient prior to administration of the AAV particles), the method comprises administering to the patient an alternative agent for treating hemophilia a.

In some embodiments, an alternative agent for treating hemophilia a is an alternative form of factor VIII (e.g., one that does not include or mask one or more of the targeted epitopes by the detected factor VIII inhibitor antibody). In some embodiments, the alternative form of factor VIII is a chemically modified factor VIII protein (e.g., a chemically modified human or porcine factor VIII protein). In some embodiments, the factor VIII alternative is a factor VIII protein derived from a non-human factor VIII protein, such as a porcine factor VIII protein. In some embodiments, the alternative agent for treating hemophilia a is factor VIII bypass therapy, e.g., a therapeutic agent comprising factor II, factor IX, and factor X. For example, in some embodiments, the factor VIII bypass therapy is a factor VIII inhibitor bypass activity (FEIBA) complex, recombinant activated factor vii (fviia), prothrombin complex concentrate, or activated prothrombin complex concentrate.

In one embodiment, a method is provided for monitoring the level of a polynucleotide encoding a factor VIII polypeptide, or fragment thereof, in the blood stream of a patient following administration of AAV particles. In one embodiment, the method comprises administering to a hemophilia a patient a dose of adeno-associated virus (AAV) particles per kilogram body weight of the patient at a first time point, wherein the AAV particles comprise a polynucleotide encoding a factor VIII protein. The method further comprises measuring the level of a polynucleotide encoding a factor VIII protein, or fragment thereof, in the bloodstream of the patient at a later time point, wherein the later time point is 7 days or longer. In one embodiment, the method comprises administering 2x 10 to a hemophilia a patient at a first time point¹²Related to each glandA dose of viral (AAV) particles per kilogram of patient body weight, wherein the AAV particles comprise a polynucleotide having the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of the nucleic acid of SEQ ID NO:1 or fragment thereof in the bloodstream of the patient at a later time point, wherein the later time point is 7 days or longer. In one embodiment, the method comprises administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a polynucleotide having the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of the nucleic acid of SEQ ID NO:1 or fragment thereof in the bloodstream of the patient at a later time point, wherein the later time point is 7 days or longer. In some embodiments of the methods, the later time point is at least 14 days later or at least 21 days later. In some embodiments, the later time point is 7 days, 14 days, or 21 days after administration of the AAV particles.

In one embodiment, a method is provided for monitoring the level of capsid protein in the blood stream of a patient following administration of AAV particles. In one embodiment, the method comprises administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of said patient, wherein AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring capsid protein levels in the patient's blood stream at a later time point, wherein the later time point is 7 days or more. In one embodiment, the method comprises administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring capsid protein levels in the patient's blood stream at a later time point, wherein the later time point is 7 days or more. In one embodiment, the method comprises administering to a patient having hemophilia a dose of adeno-associated virus (AAV) particles per kilogram body weight of the patient at a first time point, wherein the AAV particles comprise a capsid protein and a coding forA polynucleotide encoding a factor VIII protein. The method further comprises measuring capsid protein levels in the patient's blood stream at a later time point, wherein the later time point is 7 days or more. In some embodiments of the methods, the later time point is at least 14 days later or at least 21 days later. In some embodiments, the later time point is 7 days, 14 days, or 21 days after administration of the AAV particles.

In one embodiment, a method is provided for monitoring the level of factor VIII inhibitor antibodies in the blood stream of a patient following administration of AAV particles. In one embodiment, the method comprises administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of anti-factor VIII antibody in the blood stream of the patient at a later time point, wherein the later time point is 7 days or more. In one embodiment, the method comprises administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of anti-factor VIII antibody in the blood stream of the patient at a later time point, wherein the later time point is 7 days or longer. In one embodiment, the method comprises administering a dose of adeno-associated virus (AAV) particles to a hemophilia a patient at a first time point, wherein the AAV particles comprise a polynucleotide encoding a factor VIII protein. The method further comprises measuring the level of anti-factor VIII antibody in the blood stream of the patient at a later time point, wherein the later time point is 7 days or longer. In some embodiments of the methods, the later time point is at least 14 days later or at least 21 days later. In some embodiments, the later time point is 7 days, 14 days, or 21 days after administration of the AAV particles.

In one embodiment, a method is provided for monitoring the level of anti-capsid protein antibodies in the blood stream of a subject following administration of AAV particles. In one embodiment, theThe method comprises administering 2x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of said patient, wherein AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of anti-capsid protein antibodies in the blood stream of the patient at a later time point, wherein the later time point is 7 days or more. In one embodiment, the method comprises administering 6 x 10 to a hemophilia a patient at a first time point¹²A dose of individual adeno-associated virus (AAV) particles per kilogram body weight of the patient, wherein the AAV particles comprise a capsid protein and a polynucleotide comprising the nucleic acid sequence of SEQ ID NO:1(CS 04-FL-NA). The method further comprises measuring the level of anti-capsid protein antibodies in the patient's blood stream at a later time point, wherein the later time point is 7 days or more. In one embodiment, the method comprises administering to a hemophilia a patient a dose of adeno-associated virus (AAV) particles per kilogram body weight of the patient at a first time point, wherein the AAV particles comprise a capsid protein and a polynucleotide encoding a factor VIII protein. The method further comprises measuring the level of anti-capsid protein antibodies in the patient's blood stream at a later time point, wherein the later time point is 7 days or more. In some embodiments of the methods, the later time point is at least 14 days later or at least 21 days later. In some embodiments, the later time point is 7 days, 14 days, or 21 days after administration of the AAV particles.

Example IV

Example 1 construction of codon-altered factor VIII variant expression sequences

Two obstacles must be overcome to create factor VIII coding sequences that are effective for gene therapy of hemophilia a. First, due to the genomic size limitations of conventional gene therapy delivery vectors (e.g., AAV virions), the encoded factor VIII polypeptide must be greatly shortened. Second, the coding sequence must be altered to: (i) stabilizing the encapsulation interaction within the delivery vector, (ii) stabilizing the mRNA intermediate, and (iii) improving the robustness of transcription/translation of the mRNA.

To achieve the first object, the applicant has providedA B domain deleted factor VIII variant construct referred to herein as "FVIII-BDD-SQ". In this construct, the B domain is replaced with a fourteen amino acid sequence called the "SQ" sequence. Recombinant FVIII-BDD-SQ under the trade nameAre sold and have been shown to be effective in controlling haemophilia a. However, the native coding sequence of FVIII-BDD-SQ, which includes the human wild-type nucleic acid sequences of the factor VIII heavy and light chains, cannot be efficiently expressed in gene therapy vectors.

To address the poor expression of native FVIII-BDD-SQ, a codon optimization algorithm described in Fath et al (ploS ONE,6: e17596(2011)) modified as described in Ward et al (Blood,117:798(2011)) and McIntosh et al (Blood,121, 3335-. However, applicants recognized that the CS04a sequence formed using the modified algorithm could be improved by further modifying the sequence. Thus, applicants reintroduce CpG dinucleotides, reintroduce CGC codons for arginine, alter leucine and serine codon distributions, reintroduce highly conserved codon pairs, and remove the recessive TATA box, CCAAT box and splice site elements while avoiding local over-expression of CpG islands and AT-and GC-rich stretches.

First, the modified algorithm replaces codons containing CpG dinucleotides (e.g., arginine codons) with non-CpG dinucleotide codon systems and eliminates/avoids CpG dinucleotides formed by adjacent codons. This strict avoidance of CpG dinucleotides is usually performed to prevent TLR-induced immunity after intramuscular injection of DNA vaccines. However, the possibility of limiting codon optimization is performed as such. For example, the modified algorithm excludes the use of the complete set of CGX arginine codons. This is particularly disruptive in gene coding for expression in human cells, since CGC is the most commonly used arginine codon in highly expressed human genes. In addition, avoiding the formation of CpG from adjacent codons further limits the optimization possibilities (e.g. limits the number of codon pairs that can be used in common).

Since TLR-induced immunity is not expected to be a problem associated with AAV-based liver-targeted gene therapy, codons that include CpG and adjacent codons that form CpG are reintroduced into the intermediate coding sequence CS04a, preferably into the sequence encoding the factor VIII light chain (e.g., at the 3' end of the FVIII-BDD-SQ coding sequence). This allows for more frequent use of the preferred human codons, especially for arginine. Care was taken, however, to avoid the formation of CpG islands, which are regions of the coding sequence with high frequency of CpG sites. This is in contrast to the teachings of Kriner et al (Nucleic Acids Res.,42(6):3551-64(2014)), which propose that CpG domains downstream of the transcription start site promote high levels of gene expression.

Second, the modified algorithm exclusively applies certain codons, such as CTG for leucine, GTG for valine, and CAG for glutamine. However, this violates the principle of balanced codon usage, as proposed, for example, by Haas et al (Current Biology,6(3):315-24 (1996)). In view of the overuse of preferred codons by the modified algorithm, alternative leucine codons were reintroduced as allowed by other rules applied for codon changes (e.g., CpG frequency and GC content).

Third, when certain criteria are met (e.g., the presence of CG dinucleotides), the modified algorithm replaces codon pairs without regard to how well they are conserved in nature. Considering the beneficial properties that may have been retained by evolution, most of the conserved codon pairs and most of the conserved preferred codon pairs replaced by the algorithm are analyzed and adjusted as allowed by other rules applied to codon changes (e.g., CpG frequency and GC content), e.g., as described in Tats et al (BMC Genomics 9:463 (2008)).

Fourth, the serine codon for the intermediate coding sequence was also re-engineered. Specifically, AGC, TCC and TCT serine codons were introduced at higher frequencies into modified coding sequences to better match overall for human codon usage (Haas et al, supra).

Fifth, TATA box, CCAAT box elements and intron/exon splice sites were screened and removed from the modified coding sequence. When modifying the coding sequence, care was taken to avoid local overexpression of AT-rich or GC-rich stretches.

Finally, in addition to optimizing codon usage within the coding sequence, the structural requirements of the potential AAV virions were considered when further optimizing the intermediate coding sequence CS04 a. AAV vectors (e.g., the nucleic acid portion of AAV virions) are encapsulated in their capsid as single-stranded DNA molecules (for review see Daya and Berns, clin. microbiol rev.,21(4):583-93 (2008)). The GC content of the vector may thus affect the encapsulation of the genome and thus the vector yield during production. As with many algorithms, the modified algorithm used herein results in an optimized gene sequence with a GC content of at least 60% (see Fath et al, PLoS One,6(3): e17596(2011) (error in: PLoS One, (6)3 (2011)).

The overall GC content of the resulting CS04 coding sequence shown in fig. 2 was 56%. The CpG dinucleotide content of the sequence is moderate. However, CpG dinucleotides are predominantly in the downstream part of the coding sequence (e.g. the part encoding the factor VIII light chain). The CS04 sequence has 79.77% nucleotide sequence identity to the corresponding coding sequence in wild-type factor VIII (Genbank accession number M14113).

For comparison purposes, several other codon optimized refecto constructs were prepared. The CS08 refecto construct was codon optimized as described in Radcliff p.m. et al, Gene Therapy,15:289-97(2008), the contents of which are hereby expressly incorporated herein in their entirety by reference for all purposes. The CS10 codon-optimized ReFactor construct was obtained from Eurofms Genomics (Ebersberg, Germany). The CS11 codon-optimized ReFactor construct was obtained from Integrated DNA Technologies, Inc. (Coralville, ETSA). The CH25 codon optimized refecto construct was obtained from the GeneArt service (Regensburg, Germany) of ThermoFischer Scientific. The CS40 refecto construct consists of a wild-type factor VIII coding sequence. The consensus sequence identity between each of the factor coding sequences is shown in table 2 below.

Table 2-percentage identity matrix for codon-altered factor VIII constructs.

CS04

CS08

CS10

CS11

CS40

CH25

CS04

100％

CS08

82.2.％

100％

CS10

79.4％

78.4％

100％

CS11

78.3％

78.1％

77.5％

100％

CS40

79.8％

76.7％

77.6％

75.4％

100％

CH25

85.1％

85.0％

79.9％

79.4％

75.8％

100％

Plasmids for each construct were constructed by cloning different synthetic DNA fragments into the same vector backbone plasmid (pCh-BBO 1). DNA synthesis with Refacto type BDD-FVIII fragments flanked by AscI and NotI enzyme restriction sites was performed by ThermoFischer Scientific (Regensburg, Germany). The vector backbone contains two flanking AAV 2-derived Inverted Terminal Repeats (ITRs) encompassing the promoter/enhancer sequences derived from the liver-specific murine transthyretin gene; AscI and NotI enzyme restriction sites and synthetic polyA sites for insertion of the corresponding Refacto type BDD-FVIII. The prepared vector backbones were ligated and, after insertion through the AscI and NotI sites, the resulting plasmids were amplified on a milligram scale. The Refacto type BDD-FVIII sequence of the construct was verified by direct sequencing (Microsynth, Balgach, Switzerland). Cloning yielded seven different plasmid constructs designated pCS40, pCS04, pCS08, pCS1O, pCS11 and pCh25 (fig. 14). The constructs have the same vector backbone and encode the same B domain deleted FVIII protein (Refacto type BDD-FVIII), but differ in their FVIII coding sequence.

AAV 8-based Vectors are prepared by three plasmid transfection methods as described in Grieger JC et al (viruses Vectors Using Suspension HEK293 Cells and Continuous harbor harborst of Vectors From the Culture Media for GMP FIX and FLT1 Clinical Vectors, Mol Ther., 10.6.10 (2015) doi:10.1038/mt.2015.187.[ electronic publication before printing ]), the contents of which are hereby expressly incorporated herein by reference in their entirety for all purposes. HEK293 suspension cells were used for plasmid transfection using the corresponding FVIII vector plasmid, helper plasmid pXX6-80 (carrying adenovirus helper gene) and encapsulating plasmid pGSK2/8 (contributing rep2 and cap8 genes). To isolate the AAV8 construct, cell pellets of one liter of culture were treated with an iodixanol gradient as described in Grieger et al (2015, supra). The procedure resulted in a vector formulation designated vCS04, vCS08, vCS10, vCS11, and vCH 25. Vectors were quantified by qPCR using a general qPCR program targeting the inverted terminal repeat of AAV2 (Aurnhammer, Human Gene Therapy Methods: Part B23: 18-28 (2012)). Control vector plasmids carrying the inverted terminal repeat of AAV2 were used to prepare standard curves. The resulting vCS04 construct is presented in FIGS. 7A-7C as SEQ ID NO 8.

The integrity of the vector genome was analyzed by AAV agarose gel electrophoresis. Electrophoresis was performed as described in Fagon et al, Human Gene Therapy Methods 23:1-7 (2012). Briefly, AAV vector formulations were incubated in the presence of 0.5% SDS for 10 minutes at 75 ℃ and then cooled to room temperature. Approximately 1.5E10 vector genomes (vg) were loaded per lane on a 1% 1XTAE agarose gel and run for 60 min at 7V/cm gel length. The gel was then stained in 2x GelRed (Biotium catalog No. 41003) solution and imaged by chemidoctmmp (biorad). The results shown in FIG. 15 confirm that vCS04 and vCS40 viral vectors have the same size genome, as indicated by distinct bands in the 5kb range (FIG. 15, lanes 2-4). Although the vector size was approximately 5.2kb, the genome was a uniform band confirming correct encapsulation of a slightly oversized genome (relative to the 4.7kb AAV wild type genome). All other vCS vector preparations showed the same genome size (data not shown).

To confirm the expected pattern of capsid proteins, SDS PAGE followed by silver staining was performed with vectors vCS04 and vCS40 (fig. 16). As shown in the figure, the downstream purification procedure yielded highly purified material exhibiting the expected protein pattern of VP1, VP2 and VP3 (fig. 16, lanes 2-4). The same pattern was seen with all other virus preparations (not shown). The SDS-PAGE procedure for AAV preparations was performed according to standard procedures. Each lane contains vg of 1E10 corresponding viral constructs and is at 4-12% Bis-Tris (S:. sup.)Novex, Life Technologies) were isolated on a gel according to the manufacturer's instructions. Silver staining was performed with the SilverQuest kit (Novex, Life Technologies) according to the manufacturer's instructions.

Surprisingly, AAV vector vCS04 has higher virion encapsulation as measured by higher yield in AAV virus production compared to vCS40 wild-type encoding constructs and other codon-optimized constructs. As shown in table 3, the vCS04 vector replicated substantially better than vCS40, providing a 5-7 fold increase in yield in AAV titers.

Table 3-yields obtained with AAV vector constructs vCS04 and vCD40 per liter of cell culture as purified from cell pellets.

Example 2 in vivo expression of codon-altered factor VIII variant expression sequences

To test the biological efficacy of the codon altered factor VIII variant sequences, the refecto-type FVIII construct described in example 1 was administered to mice lacking factor VIII. Briefly, the analysis was performed by tail vein injection of 4E12 vector genomes (vg) per kilogram mouse body weight in C57B1/6FVIII knockout (ko) mice (6-8 animals per group). Blood was withdrawn by retroorbital puncture 14 days after injection, and plasma was prepared and frozen using standard procedures. The expression level at day 14 was chosen as the effect of the inhibitory antibody was minimal at this time, which was seen in some animals of this mouse model at a later time. As suggested by the manufacturer (Technoclone, austria, vienna) FVIII activity in mouse plasma was determined using a Technochrome FVIII assay that was only performed with minor modifications. For the assay, plasma samples are suitably diluted and mixed with assay reagents containing thrombin, activated factor ix (fixa), phospholipids, factor X and calcium. Upon activation of FVIII by thrombin, a complex with FIXa, phospholipids and calcium is formed. This complex activates FX to activated FX (fxa), which in turn cleaves paranitroaniline (pNA) from the chromogenic substrate. The kinetics of pNA formation were measured at 405 nm. The rate is proportional to the FVIII concentration in the sample. FVIII concentrations were read from the reference curve and results were provided as IU FVIII/ml.

The results presented in table 4 below demonstrate that the sequences of codon changes (CS10, CS11 and CH25) designed using commercial algorithms only modestly increased (3-4 fold) BDD-factor VIII compared to the wild-type BDD-factor VIII construct (CS 40). Similarly, the codon-altered BDD-factor VIII construct (CS08) prepared as described in Radcliffe et al increased BDD-FVIII expression by only 3-4 fold. This result is the same as reported in Radcliff et al. Surprisingly, the CS04 construct provided much higher BDD-FVIII expression (e.g., a 74-fold increase) in an in vivo bioperformance assay.

Table 4-FVIII gene expression in plasma of knockout mice induced by different AAV vector constructs.

Example 3 non-clinical efficacy and toxicology assessment of human FVIII Gene therapy vectors in mice

Hemophilia a is an inherited bleeding disorder caused by a deficiency of factor viii (fviii) or deficient factor viii (fviii) and is treated with plasma-derived or recombinant factor concentrates. These concentrates require regular infusion to maintain appropriate FVIII levels to control and prevent bleeding episodes. Given the challenges of protein replacement therapy, gene therapy may provide an alternative treatment for patients with hemophilia a. By introducing a functional F8 gene copy into target hepatocytes to induce endogenous FVIII expression, frequent infusion of coagulation factors may no longer be required.

Adeno-associated virus (AAV) -based gene therapy may provide clinical benefit in patients with hemophilia a. Recombinant (r) AAV 8-based gene therapy vectors containing a CS04 factor VIII codon-optimized construct were designed to deliver a human codon-optimized B domain deleted fviii (bddfviii) transgene under the control of a liver-specific transthyretin promoter. This construct was used to examine the dose-response relationship of FVIII activity in F8 knockout (ko) mice and to evaluate toxicity following a single intravenous administration.

Briefly to test the efficacy of treatment, 3.0 × 10 male FVIII knockout mice were administered to each group of 12 male FVIII gene knockout mice¹¹、1.2×10¹²Or 3.0X 10¹²Single intravenous doses of individual vector capsid particles (cp)/kg or 10mL/kg buffer. Retroorbital blood samples were collected every other week for 8 weeks and analyzed for FVIII using a chromogenic assay. Plasma samples obtained from blood samples taken during final life were also used for the analysis of FVIII binding and neutralizing antibodies. At the end of the observation period, hemostasis control was assessed using the tail tip bleeding assay.

At the end of the study, 4 animals tested positive for the neutralizing antibody except for binding (using 3.0X 10)¹²Individual cp/kg vector treatment) was negative for anti-BDD-FVIII binding antibodies. These animals were excluded from statistical analysis of FVIII activity levels and blood loss measurements in tail tip bleeding. Application of 1.2X 10¹²Or 3.0X 10¹²Individual cp/kg vectors caused a dose-dependent increase in mean plasma FVIII activity, up to 0.6 and 1.9IU/mL, respectively, calculated over the study period, but in buffer or 3.0X 10¹¹FVIII activity was below the lower limit of quantitation (LLOQ) in individual cp/kg vector treated mice (figure 17).

Efficacy was assessed on day 63 in the tail tip bleeding assay. The amount of blood lost in mg/g body weight over 60 minutes is presented in figure 18. Using buffer or 3.0X 10¹¹Individual cp/kg gene therapy vector treated animals showed similar blood loss volumes (6.1 mg/g and 7.5mg/g, respectively), as if no detectable FVIII activity was present. Higher doses of gene therapy vectors significantly reduced blood loss in a dose-dependent manner (1.2X 10)¹²：0.6mg/g，3.0×10¹²: 0.4 mg/g; Jonckheer-Terpstra test: 1-side P value<0.001)。

To test the toxicology of the constructs, male C57BL/6J mice (n-20/group) were injected intravenously with 1 × 10¹³、3×10¹³Or 5X 10¹³Single dose of cp/kg vector or formulation buffer (table 5). Toxicity was assessed based on clinical signs, body weight, food intake, ophthalmology, and clinical and anatomical pathology. To 5Complete necropsy was performed only on animals from each group and macroscopic results, organ weights and microscopic examination results were recorded. Tissues were collected from 5 additional animals from each group for evaluation of biodistribution by quantitative polymerase chain reaction. Blood was collected prior to dosing and at autopsy. FVIII activity, BDD-FVIII antigen binding, anti-BDD-FVIII antibody binding, neutralizing anti-BDD-FVIII antibody binding and anti-AAV 8 antibody binding were analyzed.

Table 5-design of toxicity study.

Found at most 5X 10¹³Individual cp/kg single bolus intravenous administration gene therapy vectors are well tolerated. No mortality occurred during the study and no clinical signs or post-dose observations were considered to be associated with vehicle administration. No negative ophthalmic results were observed. No effect on body weight or food intake was observed. No changes in clinical chemistry, hematology or urinalysis parameters were observed. And non-toxicologically-related macroscopic or microscopic results are associated with administration of gene therapy vectors.

FVIII activity and BDD-FVIII antigen evaluation are prone to extensive variation, most likely due to the generation of neutralizing antibodies to human BDD-FVIII. However, individual animals in all vehicle groups had activity above the normal baseline level on day 3 and weeks 3 and 18 (data not shown). In the collected tissue samples, the vector DNA was mainly detected in the liver. The biodistribution of the liver and other tissues is dose-related and is generally highest at the earliest time point and decreases over time. The presence of vector DNA in brain and testis decreased significantly over time and was lower than the measured LLOQ in many animals at week 18 (fig. 19).

Taken together, the results show that the dosage is ≧ 1.2X 10¹²Individual cp/kg FVIII gene knockout miceCodon-optimized BDD-FVII gene therapy is effective when administered. No observed adverse effect level was considered to be 5.0 > < 10¹³Individual cp/kg, highest dose tested in toxicity studies.

In some embodiments, the Dose administered to the mouse may be converted to an Adult Human Dose according to the guidelines provided in the Industry Evaluation guidelines for Maximum Safe Starting Dose in an Initial Clinical trial of therapeutic agents for Adult Healthy Volunteers (guidelines for Industry-timing the Maximum Safe Starting Dose in the Adult Human Health care subjects for Therapeutics in the Administration Health Services), the U.S. department of Health and public Services (U.S. department of Health and Human Services), the Food and Drug Administration (Food and Drug Administration), the Drug Evaluation and Research Center (Center for Drug Evaluation and Research, CDER), month 2005, Pharmacology and Toxicology (pharmaceutical and Toxicology), the contents of which are incorporated herein by reference in their entirety for all purposes.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Sequence listing

<110> Bai deep company (BAXALTA INCORPORATED)

Baishen Limited liability company (BAXALTA GMBH)

<120> Gene therapy for hemophilia A Using viral vectors encoding recombinant FVIII variants with increased expression

<130> 008073-5202-WO

<140>

<141>

<150> 62/698,680

<151> 2018-07-16

<150> 62/867,171

<151> 2019-06-26

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 1

atgcagattg agctgagcac ctgcttcttc ctgtgcctgc tgaggttctg cttctctgcc 60

accaggagat actacctggg ggctgtggag ctttcttggg actacatgca gtctgacctg 120

ggggagctgc ctgtggatgc caggttccca cccagagtgc ccaaatcctt cccattcaac 180

acctctgtgg tctacaagaa gaccctcttt gtggagttca ctgaccacct gttcaacatt 240

gccaaaccca ggccaccctg gatgggactc ctgggaccca ccattcaggc tgaggtgtat 300

gacactgtgg tcatcaccct caagaacatg gcctcccacc ctgtgagcct gcatgctgtg 360

ggggtcagct actggaaggc ctctgagggg gctgagtatg atgaccagac ctcccagagg 420

gagaaggagg atgacaaagt gttccctggg ggcagccaca cctatgtgtg gcaggtcctc 480

aaggagaatg gccccatggc ctctgaccca ctctgcctga cctactccta cctttctcat 540

gtggacctgg tcaaggacct caactctgga ctgattgggg ccctgctggt gtgcagggag 600

ggctccctgg ccaaagagaa gacccagacc ctgcacaagt tcattctcct gtttgctgtc 660

tttgatgagg gcaagagctg gcactctgaa accaagaact ccctgatgca ggacagggat 720

gctgcctctg ccagggcctg gcccaagatg cacactgtga atggctatgt gaacaggagc 780

ctgcctggac tcattggctg ccacaggaaa tctgtctact ggcatgtgat tggcatgggg 840

acaacccctg aggtgcactc cattttcctg gagggccaca ccttcctggt caggaaccac 900

agacaggcca gcctggagat cagccccatc accttcctca ctgcccagac cctgctgatg 960

gacctcggac agttcctgct gttctgccac atcagctccc accagcatga tggcatggag 1020

gcctatgtca aggtggacag ctgccctgag gagccacagc tcaggatgaa gaacaatgag 1080

gaggctgagg actatgatga tgacctgact gactctgaga tggatgtggt ccgctttgat 1140

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gatgtcaggc ccctgtacag ccgcaggctg ccaaaggggg tgaaacacct caaggacttc 1560

cccattctgc ctggggagat cttcaagtac aagtggactg tcactgtgga ggatggacca 1620

accaaatctg accccaggtg cctcaccaga tactactcca gctttgtgaa catggagagg 1680

gacctggcct ctggcctgat tggcccactg ctcatctgct acaaggagtc tgtggaccag 1740

aggggaaacc agatcatgtc tgacaagagg aatgtgattc tgttctctgt ctttgatgag 1800

aacaggagct ggtacctgac tgagaacatt cagcgcttcc tgcccaaccc tgctggggtg 1860

cagctggagg accctgagtt ccaggccagc aacatcatgc actccatcaa tggctatgtg 1920

tttgacagcc tccagctttc tgtctgcctg catgaggtgg cctactggta cattctttct 1980

attggggccc agactgactt cctttctgtc ttcttctctg gctacacctt caaacacaag 2040

atggtgtatg aggacaccct gaccctcttc ccattctctg gggagactgt gttcatgagc 2100

atggagaacc ctggcctgtg gattctggga tgccacaact ctgacttccg caacaggggc 2160

atgactgccc tgctcaaagt ctcctcctgt gacaagaaca ctggggacta ctatgaggac 2220

agctatgagg acatctctgc ctacctgctc agcaagaaca atgccattga gcccaggagc 2280

ttcagccaga atccacctgt cctgaaacgc caccagaggg agatcaccag gaccaccctc 2340

cagtctgacc aggaggagat tgactatgat gacaccattt ctgtggagat gaagaaagag 2400

gactttgaca tctatgacga ggacgagaac cagagcccaa ggagcttcca gaagaagacc 2460

aggcactact tcattgctgc tgtggagcgc ctgtgggact atggcatgag ctccagcccc 2520

catgtcctca ggaacagggc ccagtctggc tctgtgccac agttcaagaa agtggtcttc 2580

caagagttca ctgatggcag cttcacccag cccctgtaca gaggggagct gaatgagcac 2640

ctgggactcc tgggcccata catcagggct gaggtggagg acaacatcat ggtgaccttc 2700

cgcaaccagg cctccaggcc ctacagcttc tacagctccc tcatcagcta tgaggaggac 2760

cagaggcagg gggctgagcc acgcaagaac tttgtgaaac ccaatgaaac caagacctac 2820

ttctggaaag tccagcacca catggccccc accaaggatg agtttgactg caaggcctgg 2880

gcctacttct ctgatgtgga cctggagaag gatgtgcact ctggcctgat tggcccactc 2940

ctggtctgcc acaccaacac cctgaaccct gcccatggaa ggcaagtgac tgtgcaggag 3000

tttgccctct tcttcaccat ctttgatgaa accaagagct ggtacttcac tgagaacatg 3060

gagcgcaact gcagggcccc atgcaacatt cagatggagg accccacctt caaagagaac 3120

taccgcttcc atgccatcaa tggctacatc atggacaccc tgcctgggct tgtcatggcc 3180

caggaccaga ggatcaggtg gtacctgctt tctatgggct ccaatgagaa cattcactcc 3240

atccacttct ctgggcatgt cttcactgtg cgcaagaagg aggagtacaa gatggccctg 3300

tacaacctct accctggggt ctttgagact gtggagatgc tgccctccaa agctggcatc 3360

tggagggtgg agtgcctcat tggggagcac ctgcatgctg gcatgagcac cctgttcctg 3420

gtctacagca acaagtgcca gacccccctg ggaatggcct ctggccacat cagggacttc 3480

cagatcactg cctctggcca gtatggccag tgggccccca agctggccag gctccactac 3540

tctggatcca tcaatgcctg gagcaccaag gagccattca gctggatcaa agtggacctg 3600

ctggccccca tgatcatcca tggcatcaag acccaggggg ccaggcagaa gttctccagc 3660

ctgtacatca gccagttcat catcatgtac agcctggatg gcaagaaatg gcagacctac 3720

agaggcaact ccactggaac actcatggtc ttctttggca atgtggacag ctctggcatc 3780

aagcacaaca tcttcaaccc cccaatcatc gccagataca tcaggctgca ccccacccac 3840

tacagcatcc gcagcaccct caggatggag ctgatgggct gtgacctgaa ctcctgcagc 3900

atgcccctgg gcatggagag caaggccatt tctgatgccc agatcactgc ctccagctac 3960

ttcaccaaca tgtttgccac ctggagccca agcaaggcca ggctgcacct ccagggaagg 4020

agcaatgcct ggaggcccca ggtcaacaac ccaaaggagt ggctgcaggt ggacttccag 4080

aagaccatga aggtcactgg ggtgaccacc cagggggtca agagcctgct caccagcatg 4140

tatgtgaagg agttcctgat cagctccagc caggatggcc accagtggac cctcttcttc 4200

cagaatggca aggtcaaggt gttccagggc aaccaggaca gcttcacccc tgtggtgaac 4260

agcctggacc cccccctcct gaccagatac ctgaggattc acccccagag ctgggtccac 4320

cagattgccc tgaggatgga ggtcctggga tgtgaggccc aggacctgta ctga 4374

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<213> Artificial Sequence (Artificial Sequence)

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<223> description of artificial sequences: synthesis of

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Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

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Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

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Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

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Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

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Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

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Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

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Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

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Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

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Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

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Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Pro Pro Val Leu

755 760 765

Lys Arg His Gln Arg Glu Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln

770 775 780

Glu Glu Ile Asp Tyr Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu

785 790 795 800

Asp Phe Asp Ile Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe

805 810 815

Gln Lys Lys Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp

820 825 830

Asp Tyr Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln

835 840 845

Ser Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

850 855 860

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu His

865 870 875 880

Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp Asn Ile

885 890 895

Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser Phe Tyr Ser

900 905 910

Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly Ala Glu Pro Arg

915 920 925

Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr Tyr Phe Trp Lys Val

930 935 940

Gln His His Met Ala Pro Thr Lys Asp Glu Phe Asp Cys Lys Ala Trp

945 950 955 960

Ala Tyr Phe Ser Asp Val Asp Leu Glu Lys Asp Val His Ser Gly Leu

965 970 975

Ile Gly Pro Leu Leu Val Cys His Thr Asn Thr Leu Asn Pro Ala His

980 985 990

Gly Arg Gln Val Thr Val Gln Glu Phe Ala Leu Phe Phe Thr Ile Phe

995 1000 1005

Asp Glu Thr Lys Ser Trp Tyr Phe Thr Glu Asn Met Glu Arg Asn

1010 1015 1020

Cys Arg Ala Pro Cys Asn Ile Gln Met Glu Asp Pro Thr Phe Lys

1025 1030 1035

Glu Asn Tyr Arg Phe His Ala Ile Asn Gly Tyr Ile Met Asp Thr

1040 1045 1050

Leu Pro Gly Leu Val Met Ala Gln Asp Gln Arg Ile Arg Trp Tyr

1055 1060 1065

Leu Leu Ser Met Gly Ser Asn Glu Asn Ile His Ser Ile His Phe

1070 1075 1080

Ser Gly His Val Phe Thr Val Arg Lys Lys Glu Glu Tyr Lys Met

1085 1090 1095

Ala Leu Tyr Asn Leu Tyr Pro Gly Val Phe Glu Thr Val Glu Met

1100 1105 1110

Leu Pro Ser Lys Ala Gly Ile Trp Arg Val Glu Cys Leu Ile Gly

1115 1120 1125

Glu His Leu His Ala Gly Met Ser Thr Leu Phe Leu Val Tyr Ser

1130 1135 1140

Asn Lys Cys Gln Thr Pro Leu Gly Met Ala Ser Gly His Ile Arg

1145 1150 1155

Asp Phe Gln Ile Thr Ala Ser Gly Gln Tyr Gly Gln Trp Ala Pro

1160 1165 1170

Lys Leu Ala Arg Leu His Tyr Ser Gly Ser Ile Asn Ala Trp Ser

1175 1180 1185

Thr Lys Glu Pro Phe Ser Trp Ile Lys Val Asp Leu Leu Ala Pro

1190 1195 1200

Met Ile Ile His Gly Ile Lys Thr Gln Gly Ala Arg Gln Lys Phe

1205 1210 1215

Ser Ser Leu Tyr Ile Ser Gln Phe Ile Ile Met Tyr Ser Leu Asp

1220 1225 1230

Gly Lys Lys Trp Gln Thr Tyr Arg Gly Asn Ser Thr Gly Thr Leu

1235 1240 1245

Met Val Phe Phe Gly Asn Val Asp Ser Ser Gly Ile Lys His Asn

1250 1255 1260

Ile Phe Asn Pro Pro Ile Ile Ala Arg Tyr Ile Arg Leu His Pro

1265 1270 1275

Thr His Tyr Ser Ile Arg Ser Thr Leu Arg Met Glu Leu Met Gly

1280 1285 1290

Cys Asp Leu Asn Ser Cys Ser Met Pro Leu Gly Met Glu Ser Lys

1295 1300 1305

Ala Ile Ser Asp Ala Gln Ile Thr Ala Ser Ser Tyr Phe Thr Asn

1310 1315 1320

Met Phe Ala Thr Trp Ser Pro Ser Lys Ala Arg Leu His Leu Gln

1325 1330 1335

Gly Arg Ser Asn Ala Trp Arg Pro Gln Val Asn Asn Pro Lys Glu

1340 1345 1350

Trp Leu Gln Val Asp Phe Gln Lys Thr Met Lys Val Thr Gly Val

1355 1360 1365

Thr Thr Gln Gly Val Lys Ser Leu Leu Thr Ser Met Tyr Val Lys

1370 1375 1380

Glu Phe Leu Ile Ser Ser Ser Gln Asp Gly His Gln Trp Thr Leu

1385 1390 1395

Phe Phe Gln Asn Gly Lys Val Lys Val Phe Gln Gly Asn Gln Asp

1400 1405 1410

Ser Phe Thr Pro Val Val Asn Ser Leu Asp Pro Pro Leu Leu Thr

1415 1420 1425

Arg Tyr Leu Arg Ile His Pro Gln Ser Trp Val His Gln Ile Ala

1430 1435 1440

Leu Arg Met Glu Val Leu Gly Cys Glu Ala Gln Asp Leu Tyr

1445 1450 1455

<210> 3

<211> 2220

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 3

gccaccagga gatactacct gggggctgtg gagctttctt gggactacat gcagtctgac 60

ctgggggagc tgcctgtgga tgccaggttc ccacccagag tgcccaaatc cttcccattc 120

aacacctctg tggtctacaa gaagaccctc tttgtggagt tcactgacca cctgttcaac 180

attgccaaac ccaggccacc ctggatggga ctcctgggac ccaccattca ggctgaggtg 240

tatgacactg tggtcatcac cctcaagaac atggcctccc accctgtgag cctgcatgct 300

gtgggggtca gctactggaa ggcctctgag ggggctgagt atgatgacca gacctcccag 360

agggagaagg aggatgacaa agtgttccct gggggcagcc acacctatgt gtggcaggtc 420

ctcaaggaga atggccccat ggcctctgac ccactctgcc tgacctactc ctacctttct 480

catgtggacc tggtcaagga cctcaactct ggactgattg gggccctgct ggtgtgcagg 540

gagggctccc tggccaaaga gaagacccag accctgcaca agttcattct cctgtttgct 600

gtctttgatg agggcaagag ctggcactct gaaaccaaga actccctgat gcaggacagg 660

gatgctgcct ctgccagggc ctggcccaag atgcacactg tgaatggcta tgtgaacagg 720

agcctgcctg gactcattgg ctgccacagg aaatctgtct actggcatgt gattggcatg 780

gggacaaccc ctgaggtgca ctccattttc ctggagggcc acaccttcct ggtcaggaac 840

cacagacagg ccagcctgga gatcagcccc atcaccttcc tcactgccca gaccctgctg 900

atggacctcg gacagttcct gctgttctgc cacatcagct cccaccagca tgatggcatg 960

gaggcctatg tcaaggtgga cagctgccct gaggagccac agctcaggat gaagaacaat 1020

gaggaggctg aggactatga tgatgacctg actgactctg agatggatgt ggtccgcttt 1080

gatgatgaca acagcccatc cttcattcag atcaggtctg tggccaagaa acaccccaag 1140

acctgggtgc actacattgc tgctgaggag gaggactggg actatgcccc actggtcctg 1200

gcccctgatg acaggagcta caagagccag tacctcaaca atggcccaca gaggattgga 1260

cgcaagtaca agaaagtcag gttcatggcc tacactgatg aaaccttcaa gaccagggag 1320

gccattcagc atgagtctgg catcctgggc ccactcctgt atggggaggt gggggacacc 1380

ctgctcatca tcttcaagaa ccaggcctcc aggccctaca acatctaccc acatggcatc 1440

actgatgtca ggcccctgta cagccgcagg ctgccaaagg gggtgaaaca cctcaaggac 1500

ttccccattc tgcctgggga gatcttcaag tacaagtgga ctgtcactgt ggaggatgga 1560

ccaaccaaat ctgaccccag gtgcctcacc agatactact ccagctttgt gaacatggag 1620

agggacctgg cctctggcct gattggccca ctgctcatct gctacaagga gtctgtggac 1680

cagaggggaa accagatcat gtctgacaag aggaatgtga ttctgttctc tgtctttgat 1740

gagaacagga gctggtacct gactgagaac attcagcgct tcctgcccaa ccctgctggg 1800

gtgcagctgg aggaccctga gttccaggcc agcaacatca tgcactccat caatggctat 1860

gtgtttgaca gcctccagct ttctgtctgc ctgcatgagg tggcctactg gtacattctt 1920

tctattgggg cccagactga cttcctttct gtcttcttct ctggctacac cttcaaacac 1980

aagatggtgt atgaggacac cctgaccctc ttcccattct ctggggagac tgtgttcatg 2040

agcatggaga accctggcct gtggattctg ggatgccaca actctgactt ccgcaacagg 2100

ggcatgactg ccctgctcaa agtctcctcc tgtgacaaga acactgggga ctactatgag 2160

gacagctatg aggacatctc tgcctacctg ctcagcaaga acaatgccat tgagcccagg 2220

<210> 4

<211> 2052

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 4

gagatcacca ggaccaccct ccagtctgac caggaggaga ttgactatga tgacaccatt 60

tctgtggaga tgaagaaaga ggactttgac atctatgacg aggacgagaa ccagagccca 120

aggagcttcc agaagaagac caggcactac ttcattgctg ctgtggagcg cctgtgggac 180

tatggcatga gctccagccc ccatgtcctc aggaacaggg cccagtctgg ctctgtgcca 240

cagttcaaga aagtggtctt ccaagagttc actgatggca gcttcaccca gcccctgtac 300

agaggggagc tgaatgagca cctgggactc ctgggcccat acatcagggc tgaggtggag 360

gacaacatca tggtgacctt ccgcaaccag gcctccaggc cctacagctt ctacagctcc 420

ctcatcagct atgaggagga ccagaggcag ggggctgagc cacgcaagaa ctttgtgaaa 480

cccaatgaaa ccaagaccta cttctggaaa gtccagcacc acatggcccc caccaaggat 540

gagtttgact gcaaggcctg ggcctacttc tctgatgtgg acctggagaa ggatgtgcac 600

tctggcctga ttggcccact cctggtctgc cacaccaaca ccctgaaccc tgcccatgga 660

aggcaagtga ctgtgcagga gtttgccctc ttcttcacca tctttgatga aaccaagagc 720

tggtacttca ctgagaacat ggagcgcaac tgcagggccc catgcaacat tcagatggag 780

gaccccacct tcaaagagaa ctaccgcttc catgccatca atggctacat catggacacc 840

ctgcctgggc ttgtcatggc ccaggaccag aggatcaggt ggtacctgct ttctatgggc 900

tccaatgaga acattcactc catccacttc tctgggcatg tcttcactgt gcgcaagaag 960

gaggagtaca agatggccct gtacaacctc taccctgggg tctttgagac tgtggagatg 1020

ctgccctcca aagctggcat ctggagggtg gagtgcctca ttggggagca cctgcatgct 1080

ggcatgagca ccctgttcct ggtctacagc aacaagtgcc agacccccct gggaatggcc 1140

tctggccaca tcagggactt ccagatcact gcctctggcc agtatggcca gtgggccccc 1200

aagctggcca ggctccacta ctctggatcc atcaatgcct ggagcaccaa ggagccattc 1260

agctggatca aagtggacct gctggccccc atgatcatcc atggcatcaa gacccagggg 1320

gccaggcaga agttctccag cctgtacatc agccagttca tcatcatgta cagcctggat 1380

ggcaagaaat ggcagaccta cagaggcaac tccactggaa cactcatggt cttctttggc 1440

aatgtggaca gctctggcat caagcacaac atcttcaacc ccccaatcat cgccagatac 1500

atcaggctgc accccaccca ctacagcatc cgcagcaccc tcaggatgga gctgatgggc 1560

tgtgacctga actcctgcag catgcccctg ggcatggaga gcaaggccat ttctgatgcc 1620

cagatcactg cctccagcta cttcaccaac atgtttgcca cctggagccc aagcaaggcc 1680

aggctgcacc tccagggaag gagcaatgcc tggaggcccc aggtcaacaa cccaaaggag 1740

tggctgcagg tggacttcca gaagaccatg aaggtcactg gggtgaccac ccagggggtc 1800

aagagcctgc tcaccagcat gtatgtgaag gagttcctga tcagctccag ccaggatggc 1860

caccagtgga ccctcttctt ccagaatggc aaggtcaagg tgttccaggg caaccaggac 1920

agcttcaccc ctgtggtgaa cagcctggac ccccccctcc tgaccagata cctgaggatt 1980

cacccccaga gctgggtcca ccagattgcc ctgaggatgg aggtcctggg atgtgaggcc 2040

caggacctgt ac 2052

<210> 5

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Oligonucleotides

<400> 5

agcttcagcc agaatccacc tgtcctgaaa cgccaccaga gg 42

<210> 6

<211> 7827

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 6

tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60

cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120

ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180

accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240

attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300

tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360

tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cctcgagatt taaatgacgt 420

tggccactcc ctctctgcgc gctcgctcgc tcactgaggc cgggcgacca aaggtcgccc 480

gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg agcgcgcaga gagggagtgg 540

ccaactccat cactaggggt tcctgagttt aaacttcgtc gacgattcga gcttgggctg 600

caggtcgagg gcactgggag gatgttgagt aagatggaaa actactgatg acccttgcag 660

agacagagta ttaggacatg tttgaacagg ggccgggcga tcagcaggta gctctagagg 720

atccccgtct gtctgcacat ttcgtagagc gagtgttccg atactctaat ctccctaggc 780

aaggttcata tttgtgtagg ttacttattc tccttttgtt gactaagtca ataatcagaa 840

tcagcaggtt tggagtcagc ttggcaggga tcagcagcct gggttggaag gagggggtat 900

aaaagcccct tcaccaggag aagccgtcac acagactagg cgcgccaccg ccaccatgca 960

gattgagctg agcacctgct tcttcctgtg cctgctgagg ttctgcttct ctgccaccag 1020

gagatactac ctgggggctg tggagctttc ttgggactac atgcagtctg acctggggga 1080

gctgcctgtg gatgccaggt tcccacccag agtgcccaaa tccttcccat tcaacacctc 1140

tgtggtctac aagaagaccc tctttgtgga gttcactgac cacctgttca acattgccaa 1200

acccaggcca ccctggatgg gactcctggg acccaccatt caggctgagg tgtatgacac 1260

tgtggtcatc accctcaaga acatggcctc ccaccctgtg agcctgcatg ctgtgggggt 1320

cagctactgg aaggcctctg agggggctga gtatgatgac cagacctccc agagggagaa 1380

ggaggatgac aaagtgttcc ctgggggcag ccacacctat gtgtggcagg tcctcaagga 1440

gaatggcccc atggcctctg acccactctg cctgacctac tcctaccttt ctcatgtgga 1500

cctggtcaag gacctcaact ctggactgat tggggccctg ctggtgtgca gggagggctc 1560

cctggccaaa gagaagaccc agaccctgca caagttcatt ctcctgtttg ctgtctttga 1620

tgagggcaag agctggcact ctgaaaccaa gaactccctg atgcaggaca gggatgctgc 1680

ctctgccagg gcctggccca agatgcacac tgtgaatggc tatgtgaaca ggagcctgcc 1740

tggactcatt ggctgccaca ggaaatctgt ctactggcat gtgattggca tggggacaac 1800

ccctgaggtg cactccattt tcctggaggg ccacaccttc ctggtcagga accacagaca 1860

ggccagcctg gagatcagcc ccatcacctt cctcactgcc cagaccctgc tgatggacct 1920

cggacagttc ctgctgttct gccacatcag ctcccaccag catgatggca tggaggccta 1980

tgtcaaggtg gacagctgcc ctgaggagcc acagctcagg atgaagaaca atgaggaggc 2040

tgaggactat gatgatgacc tgactgactc tgagatggat gtggtccgct ttgatgatga 2100

caacagccca tccttcattc agatcaggtc tgtggccaag aaacacccca agacctgggt 2160

gcactacatt gctgctgagg aggaggactg ggactatgcc ccactggtcc tggcccctga 2220

tgacaggagc tacaagagcc agtacctcaa caatggccca cagaggattg gacgcaagta 2280

caagaaagtc aggttcatgg cctacactga tgaaaccttc aagaccaggg aggccattca 2340

gcatgagtct ggcatcctgg gcccactcct gtatggggag gtgggggaca ccctgctcat 2400

catcttcaag aaccaggcct ccaggcccta caacatctac ccacatggca tcactgatgt 2460

caggcccctg tacagccgca ggctgccaaa gggggtgaaa cacctcaagg acttccccat 2520

tctgcctggg gagatcttca agtacaagtg gactgtcact gtggaggatg gaccaaccaa 2580

atctgacccc aggtgcctca ccagatacta ctccagcttt gtgaacatgg agagggacct 2640

ggcctctggc ctgattggcc cactgctcat ctgctacaag gagtctgtgg accagagggg 2700

aaaccagatc atgtctgaca agaggaatgt gattctgttc tctgtctttg atgagaacag 2760

gagctggtac ctgactgaga acattcagcg cttcctgccc aaccctgctg gggtgcagct 2820

ggaggaccct gagttccagg ccagcaacat catgcactcc atcaatggct atgtgtttga 2880

cagcctccag ctttctgtct gcctgcatga ggtggcctac tggtacattc tttctattgg 2940

ggcccagact gacttccttt ctgtcttctt ctctggctac accttcaaac acaagatggt 3000

gtatgaggac accctgaccc tcttcccatt ctctggggag actgtgttca tgagcatgga 3060

gaaccctggc ctgtggattc tgggatgcca caactctgac ttccgcaaca ggggcatgac 3120

tgccctgctc aaagtctcct cctgtgacaa gaacactggg gactactatg aggacagcta 3180

tgaggacatc tctgcctacc tgctcagcaa gaacaatgcc attgagccca ggagcttcag 3240

ccagaatcca cctgtcctga aacgccacca gagggagatc accaggacca ccctccagtc 3300

tgaccaggag gagattgact atgatgacac catttctgtg gagatgaaga aagaggactt 3360

tgacatctat gacgaggacg agaaccagag cccaaggagc ttccagaaga agaccaggca 3420

ctacttcatt gctgctgtgg agcgcctgtg ggactatggc atgagctcca gcccccatgt 3480

cctcaggaac agggcccagt ctggctctgt gccacagttc aagaaagtgg tcttccaaga 3540

gttcactgat ggcagcttca cccagcccct gtacagaggg gagctgaatg agcacctggg 3600

actcctgggc ccatacatca gggctgaggt ggaggacaac atcatggtga ccttccgcaa 3660

ccaggcctcc aggccctaca gcttctacag ctccctcatc agctatgagg aggaccagag 3720

gcagggggct gagccacgca agaactttgt gaaacccaat gaaaccaaga cctacttctg 3780

gaaagtccag caccacatgg cccccaccaa ggatgagttt gactgcaagg cctgggccta 3840

cttctctgat gtggacctgg agaaggatgt gcactctggc ctgattggcc cactcctggt 3900

ctgccacacc aacaccctga accctgccca tggaaggcaa gtgactgtgc aggagtttgc 3960

cctcttcttc accatctttg atgaaaccaa gagctggtac ttcactgaga acatggagcg 4020

caactgcagg gccccatgca acattcagat ggaggacccc accttcaaag agaactaccg 4080

cttccatgcc atcaatggct acatcatgga caccctgcct gggcttgtca tggcccagga 4140

ccagaggatc aggtggtacc tgctttctat gggctccaat gagaacattc actccatcca 4200

cttctctggg catgtcttca ctgtgcgcaa gaaggaggag tacaagatgg ccctgtacaa 4260

cctctaccct ggggtctttg agactgtgga gatgctgccc tccaaagctg gcatctggag 4320

ggtggagtgc ctcattgggg agcacctgca tgctggcatg agcaccctgt tcctggtcta 4380

cagcaacaag tgccagaccc ccctgggaat ggcctctggc cacatcaggg acttccagat 4440

cactgcctct ggccagtatg gccagtgggc ccccaagctg gccaggctcc actactctgg 4500

atccatcaat gcctggagca ccaaggagcc attcagctgg atcaaagtgg acctgctggc 4560

ccccatgatc atccatggca tcaagaccca gggggccagg cagaagttct ccagcctgta 4620

catcagccag ttcatcatca tgtacagcct ggatggcaag aaatggcaga cctacagagg 4680

caactccact ggaacactca tggtcttctt tggcaatgtg gacagctctg gcatcaagca 4740

caacatcttc aaccccccaa tcatcgccag atacatcagg ctgcacccca cccactacag 4800

catccgcagc accctcagga tggagctgat gggctgtgac ctgaactcct gcagcatgcc 4860

cctgggcatg gagagcaagg ccatttctga tgcccagatc actgcctcca gctacttcac 4920

caacatgttt gccacctgga gcccaagcaa ggccaggctg cacctccagg gaaggagcaa 4980

tgcctggagg ccccaggtca acaacccaaa ggagtggctg caggtggact tccagaagac 5040

catgaaggtc actggggtga ccacccaggg ggtcaagagc ctgctcacca gcatgtatgt 5100

gaaggagttc ctgatcagct ccagccagga tggccaccag tggaccctct tcttccagaa 5160

tggcaaggtc aaggtgttcc agggcaacca ggacagcttc acccctgtgg tgaacagcct 5220

ggaccccccc ctcctgacca gatacctgag gattcacccc cagagctggg tccaccagat 5280

tgccctgagg atggaggtcc tgggatgtga ggcccaggac ctgtactgat gacgagcggc 5340

cgctcttagt agcagtatcg ataataaaag atctttattt tcattagatc tgtgtgttgg 5400

ttttttgtgt gttaattaag ctcgcgaagg aacccctagt gatggagttg gccactccct 5460

ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct 5520

ttgcccgggc ggcctcagtg agcgagcgag cgcgcagaga gggagtggcc aagacgattt 5580

aaatgacaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc 5640

tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat 5700

gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc 5760

tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg 5820

ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 5880

cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag 5940

gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 6000

tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 6060

agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 6120

tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 6180

cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 6240

ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 6300

ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 6360

ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 6420

ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 6480

cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 6540

gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 6600

atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 6660

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 6720

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 6780

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 6840

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 6900

taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 6960

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 7020

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 7080

ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc 7140

aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg 7200

gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag 7260

cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 7320

actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt 7380

caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac 7440

gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac 7500

ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag 7560

caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 7620

tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 7680

gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc 7740

cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa 7800

ataggcgtat cacgaggccc tttcgtc 7827

<210> 7

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 7

atgcagatcg aactgagcac ttgcttcttc ctgtgtctcc tgcgcttttg cttctccgcc 60

acaaggagat actatctcgg tgccgtggag ctcagctggg actacatgca gagcgacttg 120

ggtgaactgc ctgtggacgc caggtttcca ccccgcgtgc ccaagagttt cccgttcaac 180

accagtgtcg tgtacaagaa aaccctcttc gtggaattca ccgaccacct gttcaacatc 240

gccaaaccgc gccctccctg gatggggctg ctcggcccga cgatccaggc tgaggtctat 300

gacacggtgg tgattaccct caagaacatg gctagccacc cggtgagcct gcacgccgtg 360

ggcgtgtcct attggaaagc gtccgagggt gcggagtacg atgaccagac ttcacagcgg 420

gagaaggaag acgacaaagt gttccccggg ggttcccaca cctatgtctg gcaggtcctg 480

aaggagaatg gtcctatggc ctccgaccca ttgtgcctca cctactctta cctaagccat 540

gtggatctcg tcaaggacct gaactcgggg ctgatcggcg ccctgctcgt gtgccgggag 600

ggctcactgg ccaaggagaa gacccaaact ctgcacaagt tcatcctgct gttcgcggta 660

ttcgacgagg ggaagtcctg gcactccgag accaagaaca gcctgatgca ggaccgcgac 720

gcagcctcgg cccgtgcgtg gccaaagatg cacaccgtga acggctacgt taacaggagc 780

ctacccggcc tgatcggctg ccaccgcaaa tcggtctact ggcatgtgat cggaatgggc 840

acaacgcccg aggtccacag tatcttcctc gagggccaca ctttcctggt ccggaatcac 900

cgccaggcca gcctggagat cagccccata acctttctga cggcgcagac cttactcatg 960

gatctcggcc agttcctcct gttctgccac atttcgtccc accagcacga tgggatggaa 1020

gcatatgtga aagtggactc ctgccccgag gaaccccagc ttaggatgaa gaacaatgag 1080

gaggccgagg actacgacga tgaccttacc gattcagaaa tggacgtagt acgctttgac 1140

gacgacaact ctccatcctt catacagatt cgctccgtcg ccaagaagca ccctaagact 1200

tgggtgcact acatcgcggc cgaggaggag gactgggatt atgctcccct ggtgctggcc 1260

cccgacgacc gcagctacaa gagccagtac ctgaataacg ggccccagcg catcggccgg 1320

aagtacaaga aagtgcggtt catggcttac acggacgaga ccttcaagac ccgggaggct 1380

atccagcatg agagcggcat cttggggccc ctcctgtacg gcgaagttgg agacacactg 1440

ctgatcatct tcaagaacca ggcgagcagg ccctacaaca tctaccccca cggcattacc 1500

gatgtccggc cgttgtacag ccgacggctg cccaagggcg tgaagcacct gaaggacttt 1560

ccgatcctgc cgggcgagat cttcaagtac aagtggactg tgaccgtgga ggatgggccg 1620

accaagagcg atccgcgctg cctgacccgt tactactcca gctttgtcaa tatggagcgc 1680

gacctcgcta gcggcttgat tggccctctg ctgatctgct acaaggagtc cgtggaccag 1740

agggggaatc agatcatgag tgacaagagg aacgtgatcc tgttctccgt gttcgacgaa 1800

aaccgcagct ggtatctcac cgagaatatc cagcgcttcc tgcccaaccc ggccggtgtg 1860

cagctggagg accccgagtt tcaggccagc aacatcatgc attctatcaa cggatatgtg 1920

tttgattccc tgcagctctc agtgtgtctg cacgaggtcg cctactggta tatcctcagc 1980

attggggcac agaccgactt cctgagcgtg ttcttctccg ggtatacctt caagcacaag 2040

atggtgtacg aggataccct gaccctgttc ccctttagcg gcgaaaccgt gtttatgtct 2100

atggagaacc ccgggctctg gatccttggc tgccataact ccgacttccg caaccgcgga 2160

atgaccgcgc tcctgaaagt gtcgagttgt gacaagaaca ccggcgacta ttacgaggac 2220

agttacgagg acatctctgc gtacctcctt agcaagaata acgccatcga gccaagatcc 2280

ttcagccaga accccccagt gctgaagagg catcagcggg agatcacccg cacgaccctg 2340

cagtcggatc aggaggagat tgattacgac gacacgatca gtgtggagat gaagaaggag 2400

gacttcgaca tctacgacga agatgaaaac cagtcccctc ggtccttcca aaagaagacc 2460

cggcactact tcatcgccgc tgtggaacgc ctgtgggact atggaatgtc ttctagccct 2520

cacgttttga ggaaccgcgc ccagtcgggc agcgtgcccc agttcaagaa agtggtgttc 2580

caggagttca ccgacggctc cttcacccag ccactttacc ggggcgagct caatgaacat 2640

ctgggcctgc tgggacccta catcagggct gaggtggagg acaacatcat ggtgacattc 2700

cggaatcagg ccagcagacc atacagtttc tacagttcac tcatctccta cgaggaggac 2760

cagcgccagg gggctgaacc ccgtaagaac ttcgtgaagc caaacgaaac aaagacctac 2820

ttctggaagg tccagcacca catggcacct accaaggacg agttcgattg caaggcctgg 2880

gcctacttct ccgacgtgga cctggagaaa gatgtgcaca gcggcctgat tggccctctg 2940

ctggtgtgtc acacgaacac actcaaccct gcacacgggc ggcaggtcac tgtgcaggaa 3000

ttcgccctgt tctttaccat ctttgatgag acgaagtcct ggtatttcac cgaaaacatg 3060

gagaggaact gccgcgcacc ctgcaacatc cagatggaag atccgacatt caaggagaac 3120

taccggttcc atgccatcaa tggctacatc atggacaccc tgcctggcct cgtgatggcc 3180

caagaccagc gtatccgctg gtatctgctg tcgatgggct ccaacgagaa catccatagt 3240

atccacttca gcgggcatgt cttcacggtg aggaaaaagg aggagtacaa gatggcactg 3300

tacaacctct atcccggcgt gttcgagacc gtggagatgc tgccctccaa ggccggcatc 3360

tggagagtgg aatgcctgat cggcgagcac ctccacgctg ggatgtccac gctgttcctc 3420

gtttacagca ataagtgcca gacccctctg ggcatggcga gcggccacat ccgcgacttc 3480

cagattacag ccagcggcca gtacggtcag tgggctccaa agctggcccg tctgcactac 3540

tccggatcca tcaacgcctg gtccaccaag gaaccgttct cctggatcaa agtagacctg 3600

ctagccccca tgatcattca cggcatcaag acacaaggcg cccgacagaa gttctcgagc 3660

ctctatatct cccagttcat catcatgtat agcctggacg gaaagaagtg gcagacttac 3720

cgcggaaact cgacagggac cctgatggta ttcttcggta acgtggacag ctccggaatc 3780

aagcacaaca tcttcaaccc acccattatc gcccgctaca tccgcctgca ccccactcac 3840

tatagcatta ggtccaccct gcgaatggag ctcatgggct gtgacctgaa cagctgtagc 3900

atgcccctcg gcatggagtc taaggcgatc tccgacgcac agataacggc atcatcctac 3960

tttaccaaca tgttcgctac ctggtccccc tccaaggccc gactccacct gcaagggaga 4020

tccaacgcct ggcggccaca ggtcaacaat cccaaggagt ggctgcaagt ggactttcag 4080

aaaactatga aagtcaccgg agtgaccaca cagggagtga agtctctgct gaccagcatg 4140

tacgtgaagg agttcctcat ctccagttcg caggatggcc accagtggac gttgttcttc 4200

caaaacggta aagtcaaagt cttccaaggg aaccaggaca gctttacacc cgtcgtgaac 4260

tccctggacc ccccgcttct cactagatac ctccgcatcc accctcagag ctgggtgcac 4320

cagattgccc tgcgcatgga ggttctgggg tgtgaagccc aggacctgta ctaa 4374

<210> 8

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 8

atgcagattg agctctccac ctgcttcttt ctctgccttc ttcgcttctg cttttctgcc 60

acacgcaggt actatttggg agcagtggaa ctgagctggg attacatgca gagtgacctt 120

ggtgaacttc ctgtggacgc tcgttttcca cctagagttc ccaagtcctt ccccttcaac 180

acctcagtgg tctacaagaa aacgctgttt gtggagttca ctgaccacct cttcaacatt 240

gccaaaccaa gacccccttg gatgggattg ctgggaccca caatacaagc agaagtctac 300

gacacggtgg tgattaccct gaagaacatg gcgtcacacc ctgtttcact tcacgctgtt 360

ggggtcagtt attggaaagc ctcagagggt gcggaatacg atgatcaaac cagccagagg 420

gagaaggaag atgacaaggt ctttcctggg ggtagccata cctatgtttg gcaggtgctg 480

aaagagaatg ggcctatggc ctctgatccc ttgtgcctca catactctta cctgagtcac 540

gtcgacctgg tgaaagacct gaatagcggt ctgattggtg cactgcttgt ttgtagagag 600

gggagtttgg ccaaggagaa aactcagact ctccacaagt ttatcctcct gtttgctgtg 660

ttcgacgagg gcaagtcttg gcactctgaa acaaagaact ccctgatgca ggacagagat 720

gctgcatctg caagggcttg gccaaaaatg cacacagtga acggctatgt gaatcgatca 780

ctgccaggac tgataggctg tcatcgcaag tcagtgtatt ggcacgttat cgggatggga 840

acaactccag aagtgcacag catcttcctt gagggccaca ctttcctggt tcggaatcat 900

agacaggcca gccttgagat cagcccaatc acctttctga ctgcccaaac cttgctgatg 960

gatctgggac agttcctcct gttttgtcac atctcctccc accaacatga cgggatggag 1020

gcttatgtga aggtcgatag ctgtccggag gaaccacaac tgaggatgaa gaacaacgaa 1080

gaggcagagg actatgacga cgatctgact gacagtgaaa tggacgtggt tcggttcgac 1140

gatgacaatt ctccttcatt tatccagatc cgttccgtgg ccaagaagca ccccaagact 1200

tgggttcatt acatcgctgc tgaggaggag gattgggact acgcgccctt ggtgttggcc 1260

ccagacgatc gctcatacaa gagccagtac cttaacaatg gtccacaaag gatcggccgg 1320

aagtacaaga aggttagatt tatggcttat accgacgaga cttttaaaac tagggaagca 1380

attcagcatg aaagtggcat tcttggaccc ctgctgtatg gcgaggttgg cgacaccctg 1440

ctgattatct ttaagaacca ggcaagccgg ccctacaaca tctacccgca cggcataacc 1500

gatgtacgac ccctgtacag tcgcagactt cctaaagggg tgaaacacct gaaggacttc 1560

ccaattctgc ccggggagat cttcaagtat aaatggaccg tgacggttga ggatggtccc 1620

acaaagtccg atccgagatg ccttacccga tattattcca gcttcgtgaa catggaaagg 1680

gacctggcca gcgggctgat tggcccactg ctgatttgtt acaaggagtc tgtcgatcaa 1740

agaggaaacc aaataatgag cgacaaacgt aacgtcatcc tgttcagcgt ctttgatgag 1800

aatagaagct ggtacctcac agaaaatatt cagcggtttc tgcctaaccc cgcaggcgtc 1860

cagctggaag atcccgagtt ccaagcctca aacatcatgc atagcatcaa cggatacgta 1920

ttcgatagcc tgcagctgtc cgtctgtctc catgaagtgg catattggta catcctgagt 1980

atcggggcgc agaccgactt cctgagcgtg ttcttttctg gatacacgtt caaacacaaa 2040

atggtctatg aagataccct gactctgttt ccattctcag gagagacagt ctttatgagt 2100

atggaaaatc ctggactgtg gatcctgggc tgtcacaatt ctgattttcg gaacagaggc 2160

atgacagccc tgcttaaagt gagctcatgc gacaagaaca ccggtgatta ctacgaagat 2220

agctatgagg acatcagtgc gtatttgctc tccaagaaca acgctatcga gccacggtct 2280

ttcagtcaga atcctcccgt tctgaagcgg catcagcgcg aaataacacg cacaaccctt 2340

cagtcagacc aagaggaaat cgactacgat gatactatct ctgtggagat gaagaaggag 2400

gatttcgaca tttacgacga ggacgagaat cagtccccaa ggagctttca gaagaaaaca 2460

agacactatt tcattgccgc cgtggagcga ctgtgggact acggcatgtc tagctctccg 2520

catgtactta gaaatagggc acaaagcgga tccgtgcctc agtttaagaa agttgtcttt 2580

caggagttta cagatggctc cttcacccag cccttgtatc gcggggaact caatgaacac 2640

ctgggcctcc tgggtcctta tattagggcc gaagtcgagg acaatatcat ggtgaccttt 2700

aggaaccagg catctagacc ttactctttc tactcctccc tgatatccta tgaggaggac 2760

cagcggcaag gcgctgagcc tcggaagaac tttgtgaagc caaatgaaac caaaacatac 2820

ttttggaaag ttcagcacca catggctccc acgaaggacg aatttgactg taaagcctgg 2880

gcctacttct cagatgtaga tctcgagaaa gacgtgcact cagggctcat tggtcccctc 2940

ctggtctgtc atactaatac cctcaatcca gcacacggac gtcaggtaac cgtccaggaa 3000

tttgccctgt tctttaccat tttcgatgag actaaatcct ggtactttac cgaaaacatg 3060

gagaggaatt gcagagcccc atgcaacatc cagatggagg accctacctt caaagagaac 3120

tatcgcttcc atgccattaa cggttacatt atggatactc tcccaggact tgtgatggca 3180

caggatcagc ggataagatg gtatctgttg agcatgggct ccaacgagaa tattcacagc 3240

atccatttct ccggtcacgt gtttacagtg agaaagaaag aagagtacaa gatggctctg 3300

tataatctct atccaggcgt attcgaaacg gtggagatgt tgcctagcaa ggccggcatt 3360

tggcgagtag aatgccttat cggggaacat ctgcatgccg gaatgagcac gctcttcctg 3420

gtgtatagta acaagtgcca gactccgctg ggcatggcat ctggccatat acgggacttt 3480

cagattacgg ctagcgggca gtatgggcag tgggcaccca aacttgcgcg actgcactat 3540

tcaggctcta tcaatgcatg gtccaccaag gaacccttct cttggattaa ggtggacctt 3600

ttggcgccca tgataatcca tgggatcaaa acccagggcg ctcgtcagaa attctcatca 3660

ctctacatct ctcagttcat aataatgtat tcactggatg ggaagaaatg gcagacttac 3720

agaggaaaca gcaccgggac gctgatggtg ttctttggca acgtggacag cagcggcatc 3780

aaacacaaca tcttcaatcc tcccattatt gcccgttata ttagactgca tcccactcac 3840

tactctatac gcagcacact taggatggag ctcatgggat gcgacctgaa cagttgtagt 3900

atgcccttgg ggatggagtc caaagctata agcgacgcac aaattacagc tagctcttac 3960

tttacgaata tgttcgccac gtggagccca agcaaagccc ggctgcattt gcagggtcgg 4020

agtaatgctt ggcgcccaca ggtgaataac cctaaggaat ggttgcaagt agatttccag 4080

aaaactatga aggtaaccgg cgtcactaca cagggagtca agtccctctt gacctctatg 4140

tacgtcaagg agttcctgat tagcagcagt caggatgggc accaatggac actgttcttc 4200

cagaatggga aagttaaagt atttcagggt aaccaggact cctttacacc tgtggtgaat 4260

agcctcgacc cacccctgct gacacgatac ctccgcatcc accctcagtc ttgggtgcat 4320

caaattgccc tgcgaatgga ggtgttggga tgcgaagctc aggacctcta ctga 4374

<210> 9

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 9

atgcagatcg aactctctac ttgcttcttc ctgtgccttc tgaggttctg cttctctgcc 60

actcgccgat attacctcgg ggccgtggag ttgagttggg actacatgca atcagatctg 120

ggcgaactcc ctgtggatgc ccgattccca ccgcgcgtgc ccaagtcttt cccatttaat 180

acttctgtgg tgtacaagaa gacattgttt gtggagttta ccgatcacct gttcaacatc 240

gccaaaccgc ggcccccatg gatgggtctg cttgggccca ccattcaagc ggaggtctat 300

gatacagtgg tgataacgct taagaacatg gcgagccacc cagtgtctct gcatgccgtt 360

ggtgtatcat attggaaggc cagcgaagga gcggagtacg atgaccagac ctctcagaga 420

gagaaggaag acgataaggt ttttcctggc ggaagtcata catatgtatg gcaggtcctg 480

aaagagaatg ggccgatggc ttctgacccc ctttgtctta cctatagtta tctgagccac 540

gtggacctgg tcaaggacct caacagtggt ctgattgggg ctctgcttgt ttgtagagag 600

ggtagcttgg ctaaggagaa aacccaaaca ctccataagt tcattttgct gttcgcggtg 660

ttcgacgagg gaaagagttg gcacagcgaa acaaagaatt cactgatgca agacagggac 720

gccgcttccg caagggcttg gcctaagatg catacggtga atgggtatgt gaaccggagc 780

ctcccggggc tgatcgggtg ccatcgcaag tctgtttact ggcacgtcat tggaatgggg 840

acaacgccag aggtacatag tatatttctt gaaggccaca cgttcctcgt acggaaccac 900

cgacaggctt ccctggagat aagccccatt acctttctga ccgctcagac tctgctgatg 960

gaccttggcc agtttctcct gttctgccat attagcagcc accagcacga cggtatggaa 1020

gcatacgtga aagtcgatag ctgtcctgag gagcctcagc tcagaatgaa gaacaacgag 1080

gaggccgaag actatgacga tgaccttaca gattccgaga tggacgtggt gcgctttgac 1140

gacgataaca gtcctagttt cattcaaatc agatccgtag ccaaaaagca tccaaagaca 1200

tgggtgcatt acattgcagc cgaagaggag gattgggatt atgcgcccct tgttctggct 1260

ccagatgaca ggagctataa gtcccagtac ttgaacaacg ggccacagcg aatcggtaga 1320

aaatataaga aggtaagatt catggcctac actgacgaaa catttaaaac cagggaagct 1380

atccaacacg aatctggaat tctcggccct ctgctctacg gtgaggtggg ggacaccttg 1440

ctgatcattt tcaaaaatca ggcatccagg ccttacaaca tataccccca tggcatcacc 1500

gatgtccgcc cgctgtattc cagaagactc cccaagggag tgaaacatct gaaagatttt 1560

cccatcctgc cgggcgagat ctttaaatac aaatggactg tgactgtaga ggacgggcct 1620

acaaaatcag acccacggtg cctgacaagg tattacagta gcttcgtcaa catggaacgc 1680

gacctcgcca gcggactcat tggcccactg ttgatctgtt acaaagagtc agtggatcag 1740

aggggaaatc agatcatgag cgataagaga aacgttatcc tgtttagtgt cttcgacgag 1800

aaccggtctt ggtaccttac tgagaacatc cagaggttcc tgccgaatcc ggctggcgtt 1860

cagctcgagg acccagagtt ccaggccagt aatataatgc actcaatcaa cggttatgtg 1920

ttcgatagcc tgcagctgag cgtctgcctc cacgaggtag cctattggta catattgtcc 1980

atcggggctc agaccgattt tctgtccgtg ttctttagcg ggtatacctt taaacataaa 2040

atggtctatg aagacaccct gaccctgttc ccattctccg gtgagactgt gttcatgtcc 2100

atggagaacc cagggctgtg gatcctgggg tgtcacaata gtgactttag gaatcgggga 2160

atgacggcac tgctgaaggt gagttcttgc gataaaaata caggagatta ctatgaggat 2220

agttacgagg atatcagtgc ctatctgctt tcaaaaaaca acgcaattga gccccggtct 2280

ttctcacaaa accccccggt gctgaagcgc caccagcgcg aaattacccg gacaaccttg 2340

cagtccgacc aggaggaaat cgattatgac gatactatca gtgtagaaat gaaaaaggag 2400

gattttgata tttacgacga agacgagaac cagtctccgc gaagttttca gaagaaaacg 2460

cgacactact ttatagctgc cgtggaacga ctctgggatt atggcatgtc ctccagccct 2520

catgtcctta ggaatcgagc gcagagtggc tctgtgcctc agttcaaaaa ggttgtgttc 2580

caggaattca ccgacggctc atttacccag ccgctgtaca gaggcgaact caacgaacac 2640

cttgggctgc ttgggccata tattcgagca gaggtggaag ataatatcat ggtaaccttt 2700

agaaaccagg cgtcaagacc ctattccttc tacagttctc tgatcagcta cgaggaggac 2760

caaagacagg gagctgaacc caggaagaac tttgtgaaac ctaatgagac caagacctac 2820

ttctggaagg tccagcacca tatggcccca actaaagatg aattcgattg caaggcctgg 2880

gcttatttca gcgacgtgga tctcgaaaag gatgtgcaca gcgggttgat cggaccgctt 2940

ttggtgtgcc acacaaatac cctcaatcct gcccacgggc ggcaggtcac agttcaagag 3000

tttgcactct tctttacaat atttgacgag acaaagtcat ggtattttac agagaatatg 3060

gagagaaatt gtcgcgcacc ttgcaacatt cagatggagg accccacatt taaggagaat 3120

tacagatttc atgctatcaa tgggtacatt atggatactc tgcctggtct ggtcatggcc 3180

caggatcagc gcataaggtg gtacttgctg agcatgggat ctaatgagaa tatacacagc 3240

attcacttca gtggccacgt ttttactgtt agaaagaagg aggagtacaa aatggcgctc 3300

tacaaccttt acccgggtgt gtttgagaca gtggagatgc tgccaagcaa ggcaggcatc 3360

tggagggttg agtgtcttat tggggagcat ctgcatgctg gaatgtccac cctctttctt 3420

gtgtacagca ataagtgcca gacaccgctt ggcatggcca gcggccacat tagggacttt 3480

cagataactg ccagtggaca gtacggccag tgggctccca agcttgcaag actccactac 3540

tccggaagca taaacgcatg gagcaccaag gaacccttct cttggattaa ggtggacctg 3600

ctggcgccaa tgatcattca cggcataaaa acccaagggg cacgacagaa attttcatct 3660

ttgtatatta gtcagtttat catcatgtac agcttggatg gaaagaagtg gcagacgtac 3720

aggggcaatt ctacaggaac acttatggtg ttttttggga atgtcgattc cagcgggatc 3780

aaacataaca tcttcaatcc tcctattatc gcccgatata tccgcctgca ccctacgcat 3840

tactccatca ggtccacatt gagaatggaa ctgatggggt gcgacctgaa tagttgtagt 3900

atgccactgg gcatggagtc taaagccatc agcgatgcac agatcactgc cagctcttac 3960

ttcaccaaca tgtttgcaac ttggtccccc tctaaagctc gcctgcatct gcagggacgc 4020

tcaaatgcat ggcgaccaca ggtgaacaat ccaaaagagt ggctccaggt cgactttcag 4080

aagacaatga aggtaacagg agtgacaacc cagggtgtaa aaagcctcct tacgagtatg 4140

tacgttaagg agtttctgat ttctagctcc caggacggac accagtggac tctgttcttc 4200

cagaacggca aagtgaaggt atttcaggga aaccaggatt cttttacccc ggtagtgaat 4260

agcctggatc caccgttgct gacccgctat ctgagaattc atccacaatc ctgggtgcat 4320

cagattgccc tccggatgga agtgctcggc tgtgaagctc aggatctgta ttag 4374

<210> 10

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 10

atgcaaatag agctctccac ctgcttcttt ctgtgccttt tgcgattctg ctttagtgcc 60

accagaagat actacctggg tgcagtggaa ctgtcatggg actatatgca aagtgatctc 120

ggtgagctgc ctgtggacgc aagatttcct cctagagtgc caaaatcttt tccattcaac 180

acctcagtcg tgtacaaaaa gactctgttt gtagaattca cggatcacct tttcaacatc 240

gctaagccaa ggccaccctg gatgggtctg ctaggtccta ccatccaggc tgaggtttat 300

gatacagtgg tcattacact taagaacatg gcttcccatc ctgtcagtct tcatgctgtt 360

ggtgtatcct actggaaagc ttctgaggga gctgaatatg atgatcagac cagtcaaagg 420

gagaaagaag atgataaagt cttccctggt ggaagccata catatgtctg gcaggtcctg 480

aaagagaatg gtccaatggc ctctgaccca ctgtgcctta cctactcata tctttctcat 540

gtggacctgg taaaagactt gaattcaggc ctcattggag ccctactagt atgtagagaa 600

gggagtctgg ccaaggaaaa gacacagacc ttgcacaaat ttatactact ttttgctgta 660

tttgatgaag ggaaaagttg gcactcagaa acaaagaact ccttgatgca ggatagggat 720

gctgcatctg ctcgggcctg gcctaaaatg cacacagtca atggttatgt aaacaggtct 780

ctgccaggtc tgattggatg ccacaggaaa tcagtctatt ggcatgtgat tggaatgggc 840

accactcctg aagtgcactc aatattcctc gaaggtcaca catttcttgt gaggaaccat 900

cgccaggcgt ccttggaaat ctcgccaata actttcctta ctgctcaaac actcttgatg 960

gaccttggac agtttctact gttttgtcat atctcttccc accaacatga tggcatggaa 1020

gcttatgtca aagtagacag ctgtccagag gaaccccaac tacgaatgaa aaataatgaa 1080

gaagcggaag actatgatga tgatcttact gattctgaaa tggatgtggt caggtttgat 1140

gatgacaact ctccttcctt tatccaaatt cgctcagttg ccaagaagca tcctaaaact 1200

tgggtacatt acattgctgc tgaagaggag gactgggact atgctccctt agtcctcgcc 1260

cccgatgaca gaagttataa aagtcaatat ttgaacaatg gccctcagcg gattggtagg 1320

aagtacaaaa aagtccgatt tatggcatac acagatgaaa cctttaagac tcgtgaagct 1380

attcagcatg aatcaggaat cttgggacct ttactttatg gggaagttgg agacacactg 1440

ttgattatat ttaagaatca agcaagcaga ccatataaca tctaccctca cggaatcact 1500

gatgtccgtc ctttgtattc aaggagatta ccaaaaggtg taaaacattt gaaggatttt 1560

ccaattctgc caggagaaat attcaaatat aaatggacag tgactgtaga agatgggcca 1620

actaaatcag atcctcggtg cctgacccgc tattactcta gtttcgttaa tatggagaga 1680

gatctagctt caggactcat tggccctctc ctcatctgct acaaagaatc tgtagatcaa 1740

agaggaaacc agataatgtc agacaagagg aatgtcatcc tgttttctgt atttgatgag 1800

aaccgaagct ggtacctcac agagaatata caacgctttc tccccaatcc agctggagtg 1860

cagcttgagg atccagagtt ccaagcctcc aacatcatgc acagcatcaa tggctatgtt 1920

tttgatagtt tgcagttgtc agtttgtttg catgaggtgg catactggta cattctaagc 1980

attggagcac agactgactt cctttctgtc ttcttctctg gatatacctt caaacacaaa 2040

atggtctatg aagacacact caccctattc ccattctcag gagaaactgt cttcatgtcg 2100

atggaaaacc caggtctatg gattctgggg tgccacaact cagactttcg gaacagaggc 2160

atgaccgcct tactgaaggt ttctagttgt gacaagaaca ctggtgatta ttacgaggac 2220

agttatgaag atatttcagc atacttgctg agtaaaaaca atgccattga accaagaagc 2280

ttctcccaga atccaccagt cttgaaacgc catcaacggg aaataactcg tactactctt 2340

cagtcagatc aagaggaaat tgactatgat gataccatat cagttgaaat gaagaaggaa 2400

gattttgaca tttatgatga ggatgaaaat cagagccccc gcagctttca aaagaaaaca 2460

cgacactatt ttattgctgc agtggagagg ctctgggatt atgggatgag tagctcccca 2520

catgttctaa gaaacagggc tcagagtggc agtgtccctc agttcaagaa agttgttttc 2580

caggaattta ctgatggctc ctttactcag cccttatacc gtggagaact aaatgaacat 2640

ttgggactcc tggggccata tataagagca gaagttgaag ataatatcat ggtaactttc 2700

agaaatcagg cctctcgtcc ctattccttc tattctagcc ttatttctta tgaggaagat 2760

cagaggcaag gagcagaacc tagaaaaaac tttgtcaagc ctaatgaaac caaaacttac 2820

ttttggaaag tgcaacatca tatggcaccc actaaagatg agtttgactg caaagcctgg 2880

gcttatttct ctgatgttga cctggaaaaa gatgtgcact caggcctgat tggacccctt 2940

ctggtctgcc acactaacac actgaaccct gctcatggga gacaagtgac agtacaggaa 3000

tttgctctgt ttttcaccat ctttgatgag accaaaagct ggtacttcac tgaaaatatg 3060

gaaagaaact gcagggctcc ctgcaatatc cagatggaag atcccacttt taaagagaat 3120

tatcgcttcc atgcaatcaa tggctacata atggatacac tacctggctt agtaatggct 3180

caggatcaaa ggattcgatg gtatctgctc agcatgggca gcaatgaaaa catccattct 3240

attcatttca gtggacatgt gttcactgta cgaaaaaaag aggagtataa aatggcactg 3300

tacaatctct atccaggtgt ttttgagaca gtggaaatgt taccatccaa agctggaatt 3360

tggcgggtgg aatgccttat tggcgagcat ctacatgctg ggatgagcac actttttctg 3420

gtgtacagca ataagtgtca gactcccctg ggaatggctt ctggacacat tagagatttt 3480

cagattacag cttcaggaca atatggacag tgggccccaa agctggccag acttcattat 3540

tccggatcaa tcaatgcctg gagcaccaag gagccctttt cttggatcaa ggtggatctg 3600

ttggcaccaa tgattattca cggcatcaag acccagggtg cccgtcagaa gttctccagc 3660

ctctacatct ctcagtttat catcatgtat agtcttgatg ggaagaagtg gcagacttat 3720

cgaggaaatt ccactggaac cttaatggtc ttctttggca atgtggattc atctgggata 3780

aaacacaata tttttaaccc tccaattatt gctcgataca tccgtttgca cccaactcat 3840

tatagcattc gcagcactct tcgcatggag ttgatgggct gtgatttaaa tagttgcagc 3900

atgccattgg gaatggagag taaagcaata tcagatgcac agattactgc ttcatcctac 3960

tttaccaata tgtttgccac ctggtctcct tcaaaagctc gacttcacct ccaagggagg 4020

agtaatgcct ggagacctca ggtgaataat ccaaaagagt ggctgcaagt ggacttccag 4080

aagacaatga aagtcacagg agtaactact cagggagtaa aatctctgct taccagcatg 4140

tatgtgaagg agttcctcat ctccagcagt caagatggcc atcagtggac tctctttttt 4200

cagaatggca aagtaaaggt ttttcaggga aatcaagact ccttcacacc tgtggtgaac 4260

tctctagacc caccgttact gactcgctac cttcgaattc acccccagag ttgggtgcac 4320

cagattgccc tgaggatgga ggttctgggc tgcgaggcac aggacctcta ctga 4374

<210> 11

<211> 4374

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Polynucleotide

<400> 11

atgcagatcg agctgtccac atgctttttt ctgtgcctgc tgcggttctg cttcagcgcc 60

acccggcggt actacctggg cgccgtggag ctgtcctggg actacatgca gagcgacctg 120

ggcgagctgc ccgtggacgc ccggttcccc cccagagtgc ccaagagctt ccccttcaac 180

accagcgtgg tgtacaagaa aaccctgttc gtggagttca ccgaccacct gttcaacatc 240

gccaagccca ggcccccctg gatgggcctg ctgggcccca ccatccaggc cgaggtgtac 300

gacaccgtgg tgatcaccct gaagaacatg gccagccacc ccgtgagcct gcacgccgtg 360

ggcgtgagct actggaaggc ctccgagggc gccgagtacg acgaccagac cagccagcgg 420

gagaaagagg acgacaaagt ctttcctggc ggcagccaca cctacgtgtg gcaggtcctg 480

aaagaaaacg gccccatggc ctccgacccc ctgtgcctga cctacagcta cctgagccac 540

gtggacctgg tgaaggacct gaacagcggg ctgattgggg ccctgctggt ctgccgggag 600

ggcagcctgg ccaaagagaa aacccagacc ctgcacaagt tcatcctgct gttcgccgtg 660

ttcgacgagg gcaagagctg gcacagcgag accaagaaca gcctgatgca ggaccgggac 720

gccgcctctg ccagagcctg gcccaagatg cacaccgtga acggctacgt gaacagaagc 780

ctgcccggcc tgattggctg ccaccggaag agcgtgtact ggcacgtgat cggcatgggc 840

accacacccg aggtgcacag catctttctg gaagggcaca cctttctggt gcggaaccac 900

cggcaggcca gcctggaaat cagccctatc accttcctga ccgcccagac actgctgatg 960

gacctgggcc agttcctgct gttttgccac atcagctctc accagcacga cggcatggaa 1020

gcctacgtga aggtggactc ctgccccgag gaaccccagc tgcggatgaa gaacaacgag 1080

gaagccgagg actacgacga cgacctgacc gacagcgaga tggacgtggt gcggttcgac 1140

gacgacaaca gccccagctt catccagatc agaagcgtgg ccaagaagca ccccaagacc 1200

tgggtgcact acatcgccgc cgaggaagag gactgggact acgcccccct ggtgctggcc 1260

cccgacgaca gaagctacaa gagccagtac ctgaacaatg gcccccagcg gatcggccgg 1320

aagtacaaga aagtgcggtt catggcctac accgacgaga ccttcaagac ccgggaggcc 1380

atccagcacg agagcggcat cctgggcccc ctgctgtacg gcgaagtggg cgacacactg 1440

ctgatcatct tcaagaacca ggccagccgg ccctacaaca tctaccccca cggcatcacc 1500

gacgtgcggc ccctgtacag caggcggctg cccaagggcg tgaagcacct gaaggacttc 1560

cccatcctgc ccggcgagat cttcaagtac aagtggaccg tgaccgtgga ggacggcccc 1620

accaagagcg accccagatg cctgacccgg tactacagca gcttcgtgaa catggaacgg 1680

gacctggcct ccgggctgat cggacctctg ctgatctgct acaaagaaag cgtggaccag 1740

cggggcaacc agatcatgag cgacaagcgg aacgtgatcc tgttcagcgt gttcgatgag 1800

aaccggtcct ggtatctgac cgagaacatc cagcggtttc tgcccaaccc tgccggggtg 1860

cagctggaag atcccgagtt ccaggccagc aacatcatgc actccatcaa tggctacgtg 1920

ttcgacagcc tgcagctgtc cgtgtgtctg cacgaggtgg cctactggta catcctgagc 1980

atcggcgccc agaccgactt cctgagcgtg ttcttcagcg gctacacctt caagcacaag 2040

atggtgtacg aggacaccct gaccctgttc cctttcagcg gcgagaccgt gttcatgagc 2100

atggaaaacc ccggcctgtg gatcctgggc tgccacaaca gcgacttccg gaaccggggc 2160

atgaccgccc tgctgaaggt gtccagctgc gacaagaaca ccggcgacta ctacgaggac 2220

agctacgagg atatcagcgc ctacctgctg tccaagaaca acgccatcga gcccagaagc 2280

ttcagccaga acccccctgt gctgaagcgg caccagagag agatcacccg gaccaccctg 2340

cagtccgacc aggaagagat cgattacgac gacaccatca gcgtggagat gaaaaaagaa 2400

gatttcgaca tctacgacga ggacgagaac cagagccccc ggtccttcca gaagaaaacc 2460

cggcactact ttatcgccgc cgtggagcgg ctgtgggact acggcatgag cagcagcccc 2520

cacgtgctgc ggaaccgggc ccagagcggc agcgtgcccc agttcaagaa agtggtgttc 2580

caggaattca ccgacggcag cttcacccag cccctgtacc ggggcgagct gaacgagcac 2640

ctggggctgc tggggcccta catcagggcc gaagtggagg acaacatcat ggtgaccttc 2700

cggaatcagg ccagcagacc ctactccttc tacagcagcc tgatcagcta cgaagaggac 2760

cagcggcagg gcgctgaacc ccggaagaac ttcgtgaagc ccaatgagac caagacctac 2820

ttctggaaag tgcagcacca catggccccc accaaggacg agttcgactg caaggcctgg 2880

gcctacttca gcgacgtgga tctggaaaag gacgtgcact ctggactgat tggccctctg 2940

ctggtgtgcc acaccaacac cctgaacccc gcccacggcc ggcaggtgac cgtgcaggaa 3000

ttcgccctgt tcttcaccat cttcgacgag accaagtcct ggtacttcac cgagaatatg 3060

gaacggaact gcagagcccc ctgcaacatc cagatggaag atcctacctt caaagagaac 3120

taccggttcc acgccatcaa cggctacatc atggacaccc tgcctggcct ggtgatggcc 3180

caggaccaga ggatccggtg gtatctgctg tccatgggca gcaacgagaa tatccacagc 3240

atccacttca gcggccacgt gttcaccgtg aggaagaaag aagagtacaa gatggccctg 3300

tacaacctgt accccggcgt gttcgagacc gtggagatgc tgcccagcaa ggccggcatc 3360

tggcgggtgg agtgtctgat cggcgagcac ctgcatgccg ggatgagcac cctgtttctg 3420

gtgtacagca acaagtgcca gacccccctg ggcatggcca gcggccacat ccgggacttc 3480

cagatcaccg cctccggcca gtacggccag tgggccccca agctggcccg gctgcactac 3540

agcggcagca tcaacgcctg gtccaccaaa gagcccttca gctggatcaa ggtggacctg 3600

ctggccccta tgatcatcca cggcattaag acccagggcg ccaggcagaa gttcagcagc 3660

ctgtacatca gccagttcat catcatgtac agcctggacg gcaagaagtg gcagacctac 3720

cggggcaaca gcaccggcac cctgatggtg ttcttcggca acgtggacag cagcggcatc 3780

aagcacaaca tcttcaaccc ccccatcatc gcccggtaca tccggctgca ccccacccac 3840

tacagcatca gatccaccct gcggatggaa ctgatgggct gcgacctgaa ctcctgcagc 3900

atgcctctgg gcatggaaag caaggccatc agcgacgccc agatcacagc cagcagctac 3960

ttcaccaaca tgttcgccac ctggtccccc tccaaggcca ggctgcacct gcagggccgg 4020

tccaacgcct ggcggcctca ggtgaacaac cccaaagaat ggctgcaggt ggactttcag 4080

aaaaccatga aggtgaccgg cgtgaccacc cagggcgtga aaagcctgct gaccagcatg 4140

tacgtgaaag agtttctgat cagcagcagc caggacggcc accagtggac cctgttcttt 4200

cagaacggca aggtgaaagt gttccagggc aaccaggact ccttcacccc cgtggtgaac 4260

tccctggacc cccccctgct gacccgctac ctgcggatcc acccccagtc ttgggtgcac 4320

cagatcgccc tgaggatgga agtgctggga tgtgaggccc aggatctgta ctga 4374

<210> 12

<211> 2351

<212> PRT

<213> Intelligent (Homo sapiens)

<400> 12

Met Gln Ile Glu Leu Ser Thr Cys Phe Phe Leu Cys Leu Leu Arg Phe

1 5 10 15

Cys Phe Ser Ala Thr Arg Arg Tyr Tyr Leu Gly Ala Val Glu Leu Ser

20 25 30

Trp Asp Tyr Met Gln Ser Asp Leu Gly Glu Leu Pro Val Asp Ala Arg

35 40 45

Phe Pro Pro Arg Val Pro Lys Ser Phe Pro Phe Asn Thr Ser Val Val

50 55 60

Tyr Lys Lys Thr Leu Phe Val Glu Phe Thr Asp His Leu Phe Asn Ile

65 70 75 80

Ala Lys Pro Arg Pro Pro Trp Met Gly Leu Leu Gly Pro Thr Ile Gln

85 90 95

Ala Glu Val Tyr Asp Thr Val Val Ile Thr Leu Lys Asn Met Ala Ser

100 105 110

His Pro Val Ser Leu His Ala Val Gly Val Ser Tyr Trp Lys Ala Ser

115 120 125

Glu Gly Ala Glu Tyr Asp Asp Gln Thr Ser Gln Arg Glu Lys Glu Asp

130 135 140

Asp Lys Val Phe Pro Gly Gly Ser His Thr Tyr Val Trp Gln Val Leu

145 150 155 160

Lys Glu Asn Gly Pro Met Ala Ser Asp Pro Leu Cys Leu Thr Tyr Ser

165 170 175

Tyr Leu Ser His Val Asp Leu Val Lys Asp Leu Asn Ser Gly Leu Ile

180 185 190

Gly Ala Leu Leu Val Cys Arg Glu Gly Ser Leu Ala Lys Glu Lys Thr

195 200 205

Gln Thr Leu His Lys Phe Ile Leu Leu Phe Ala Val Phe Asp Glu Gly

210 215 220

Lys Ser Trp His Ser Glu Thr Lys Asn Ser Leu Met Gln Asp Arg Asp

225 230 235 240

Ala Ala Ser Ala Arg Ala Trp Pro Lys Met His Thr Val Asn Gly Tyr

245 250 255

Val Asn Arg Ser Leu Pro Gly Leu Ile Gly Cys His Arg Lys Ser Val

260 265 270

Tyr Trp His Val Ile Gly Met Gly Thr Thr Pro Glu Val His Ser Ile

275 280 285

Phe Leu Glu Gly His Thr Phe Leu Val Arg Asn His Arg Gln Ala Ser

290 295 300

Leu Glu Ile Ser Pro Ile Thr Phe Leu Thr Ala Gln Thr Leu Leu Met

305 310 315 320

Asp Leu Gly Gln Phe Leu Leu Phe Cys His Ile Ser Ser His Gln His

325 330 335

Asp Gly Met Glu Ala Tyr Val Lys Val Asp Ser Cys Pro Glu Glu Pro

340 345 350

Gln Leu Arg Met Lys Asn Asn Glu Glu Ala Glu Asp Tyr Asp Asp Asp

355 360 365

Leu Thr Asp Ser Glu Met Asp Val Val Arg Phe Asp Asp Asp Asn Ser

370 375 380

Pro Ser Phe Ile Gln Ile Arg Ser Val Ala Lys Lys His Pro Lys Thr

385 390 395 400

Trp Val His Tyr Ile Ala Ala Glu Glu Glu Asp Trp Asp Tyr Ala Pro

405 410 415

Leu Val Leu Ala Pro Asp Asp Arg Ser Tyr Lys Ser Gln Tyr Leu Asn

420 425 430

Asn Gly Pro Gln Arg Ile Gly Arg Lys Tyr Lys Lys Val Arg Phe Met

435 440 445

Ala Tyr Thr Asp Glu Thr Phe Lys Thr Arg Glu Ala Ile Gln His Glu

450 455 460

Ser Gly Ile Leu Gly Pro Leu Leu Tyr Gly Glu Val Gly Asp Thr Leu

465 470 475 480

Leu Ile Ile Phe Lys Asn Gln Ala Ser Arg Pro Tyr Asn Ile Tyr Pro

485 490 495

His Gly Ile Thr Asp Val Arg Pro Leu Tyr Ser Arg Arg Leu Pro Lys

500 505 510

Gly Val Lys His Leu Lys Asp Phe Pro Ile Leu Pro Gly Glu Ile Phe

515 520 525

Lys Tyr Lys Trp Thr Val Thr Val Glu Asp Gly Pro Thr Lys Ser Asp

530 535 540

Pro Arg Cys Leu Thr Arg Tyr Tyr Ser Ser Phe Val Asn Met Glu Arg

545 550 555 560

Asp Leu Ala Ser Gly Leu Ile Gly Pro Leu Leu Ile Cys Tyr Lys Glu

565 570 575

Ser Val Asp Gln Arg Gly Asn Gln Ile Met Ser Asp Lys Arg Asn Val

580 585 590

Ile Leu Phe Ser Val Phe Asp Glu Asn Arg Ser Trp Tyr Leu Thr Glu

595 600 605

Asn Ile Gln Arg Phe Leu Pro Asn Pro Ala Gly Val Gln Leu Glu Asp

610 615 620

Pro Glu Phe Gln Ala Ser Asn Ile Met His Ser Ile Asn Gly Tyr Val

625 630 635 640

Phe Asp Ser Leu Gln Leu Ser Val Cys Leu His Glu Val Ala Tyr Trp

645 650 655

Tyr Ile Leu Ser Ile Gly Ala Gln Thr Asp Phe Leu Ser Val Phe Phe

660 665 670

Ser Gly Tyr Thr Phe Lys His Lys Met Val Tyr Glu Asp Thr Leu Thr

675 680 685

Leu Phe Pro Phe Ser Gly Glu Thr Val Phe Met Ser Met Glu Asn Pro

690 695 700

Gly Leu Trp Ile Leu Gly Cys His Asn Ser Asp Phe Arg Asn Arg Gly

705 710 715 720

Met Thr Ala Leu Leu Lys Val Ser Ser Cys Asp Lys Asn Thr Gly Asp

725 730 735

Tyr Tyr Glu Asp Ser Tyr Glu Asp Ile Ser Ala Tyr Leu Leu Ser Lys

740 745 750

Asn Asn Ala Ile Glu Pro Arg Ser Phe Ser Gln Asn Ser Arg His Pro

755 760 765

Ser Thr Arg Gln Lys Gln Phe Asn Ala Thr Thr Ile Pro Glu Asn Asp

770 775 780

Ile Glu Lys Thr Asp Pro Trp Phe Ala His Arg Thr Pro Met Pro Lys

785 790 795 800

Ile Gln Asn Val Ser Ser Ser Asp Leu Leu Met Leu Leu Arg Gln Ser

805 810 815

Pro Thr Pro His Gly Leu Ser Leu Ser Asp Leu Gln Glu Ala Lys Tyr

820 825 830

Glu Thr Phe Ser Asp Asp Pro Ser Pro Gly Ala Ile Asp Ser Asn Asn

835 840 845

Ser Leu Ser Glu Met Thr His Phe Arg Pro Gln Leu His His Ser Gly

850 855 860

Asp Met Val Phe Thr Pro Glu Ser Gly Leu Gln Leu Arg Leu Asn Glu

865 870 875 880

Lys Leu Gly Thr Thr Ala Ala Thr Glu Leu Lys Lys Leu Asp Phe Lys

885 890 895

Val Ser Ser Thr Ser Asn Asn Leu Ile Ser Thr Ile Pro Ser Asp Asn

900 905 910

Leu Ala Ala Gly Thr Asp Asn Thr Ser Ser Leu Gly Pro Pro Ser Met

915 920 925

Pro Val His Tyr Asp Ser Gln Leu Asp Thr Thr Leu Phe Gly Lys Lys

930 935 940

Ser Ser Pro Leu Thr Glu Ser Gly Gly Pro Leu Ser Leu Ser Glu Glu

945 950 955 960

Asn Asn Asp Ser Lys Leu Leu Glu Ser Gly Leu Met Asn Ser Gln Glu

965 970 975

Ser Ser Trp Gly Lys Asn Val Ser Ser Thr Glu Ser Gly Arg Leu Phe

980 985 990

Lys Gly Lys Arg Ala His Gly Pro Ala Leu Leu Thr Lys Asp Asn Ala

995 1000 1005

Leu Phe Lys Val Ser Ile Ser Leu Leu Lys Thr Asn Lys Thr Ser

1010 1015 1020

Asn Asn Ser Ala Thr Asn Arg Lys Thr His Ile Asp Gly Pro Ser

1025 1030 1035

Leu Leu Ile Glu Asn Ser Pro Ser Val Trp Gln Asn Ile Leu Glu

1040 1045 1050

Ser Asp Thr Glu Phe Lys Lys Val Thr Pro Leu Ile His Asp Arg

1055 1060 1065

Met Leu Met Asp Lys Asn Ala Thr Ala Leu Arg Leu Asn His Met

1070 1075 1080

Ser Asn Lys Thr Thr Ser Ser Lys Asn Met Glu Met Val Gln Gln

1085 1090 1095

Lys Lys Glu Gly Pro Ile Pro Pro Asp Ala Gln Asn Pro Asp Met

1100 1105 1110

Ser Phe Phe Lys Met Leu Phe Leu Pro Glu Ser Ala Arg Trp Ile

1115 1120 1125

Gln Arg Thr His Gly Lys Asn Ser Leu Asn Ser Gly Gln Gly Pro

1130 1135 1140

Ser Pro Lys Gln Leu Val Ser Leu Gly Pro Glu Lys Ser Val Glu

1145 1150 1155

Gly Gln Asn Phe Leu Ser Glu Lys Asn Lys Val Val Val Gly Lys

1160 1165 1170

Gly Glu Phe Thr Lys Asp Val Gly Leu Lys Glu Met Val Phe Pro

1175 1180 1185

Ser Ser Arg Asn Leu Phe Leu Thr Asn Leu Asp Asn Leu His Glu

1190 1195 1200

Asn Asn Thr His Asn Gln Glu Lys Lys Ile Gln Glu Glu Ile Glu

1205 1210 1215

Lys Lys Glu Thr Leu Ile Gln Glu Asn Val Val Leu Pro Gln Ile

1220 1225 1230

His Thr Val Thr Gly Thr Lys Asn Phe Met Lys Asn Leu Phe Leu

1235 1240 1245

Leu Ser Thr Arg Gln Asn Val Glu Gly Ser Tyr Asp Gly Ala Tyr

1250 1255 1260

Ala Pro Val Leu Gln Asp Phe Arg Ser Leu Asn Asp Ser Thr Asn

1265 1270 1275

Arg Thr Lys Lys His Thr Ala His Phe Ser Lys Lys Gly Glu Glu

1280 1285 1290

Glu Asn Leu Glu Gly Leu Gly Asn Gln Thr Lys Gln Ile Val Glu

1295 1300 1305

Lys Tyr Ala Cys Thr Thr Arg Ile Ser Pro Asn Thr Ser Gln Gln

1310 1315 1320

Asn Phe Val Thr Gln Arg Ser Lys Arg Ala Leu Lys Gln Phe Arg

1325 1330 1335

Leu Pro Leu Glu Glu Thr Glu Leu Glu Lys Arg Ile Ile Val Asp

1340 1345 1350

Asp Thr Ser Thr Gln Trp Ser Lys Asn Met Lys His Leu Thr Pro

1355 1360 1365

Ser Thr Leu Thr Gln Ile Asp Tyr Asn Glu Lys Glu Lys Gly Ala

1370 1375 1380

Ile Thr Gln Ser Pro Leu Ser Asp Cys Leu Thr Arg Ser His Ser

1385 1390 1395

Ile Pro Gln Ala Asn Arg Ser Pro Leu Pro Ile Ala Lys Val Ser

1400 1405 1410

Ser Phe Pro Ser Ile Arg Pro Ile Tyr Leu Thr Arg Val Leu Phe

1415 1420 1425

Gln Asp Asn Ser Ser His Leu Pro Ala Ala Ser Tyr Arg Lys Lys

1430 1435 1440

Asp Ser Gly Val Gln Glu Ser Ser His Phe Leu Gln Gly Ala Lys

1445 1450 1455

Lys Asn Asn Leu Ser Leu Ala Ile Leu Thr Leu Glu Met Thr Gly

1460 1465 1470

Asp Gln Arg Glu Val Gly Ser Leu Gly Thr Ser Ala Thr Asn Ser

1475 1480 1485

Val Thr Tyr Lys Lys Val Glu Asn Thr Val Leu Pro Lys Pro Asp

1490 1495 1500

Leu Pro Lys Thr Ser Gly Lys Val Glu Leu Leu Pro Lys Val His

1505 1510 1515

Ile Tyr Gln Lys Asp Leu Phe Pro Thr Glu Thr Ser Asn Gly Ser

1520 1525 1530

Pro Gly His Leu Asp Leu Val Glu Gly Ser Leu Leu Gln Gly Thr

1535 1540 1545

Glu Gly Ala Ile Lys Trp Asn Glu Ala Asn Arg Pro Gly Lys Val

1550 1555 1560

Pro Phe Leu Arg Val Ala Thr Glu Ser Ser Ala Lys Thr Pro Ser

1565 1570 1575

Lys Leu Leu Asp Pro Leu Ala Trp Asp Asn His Tyr Gly Thr Gln

1580 1585 1590

Ile Pro Lys Glu Glu Trp Lys Ser Gln Glu Lys Ser Pro Glu Lys

1595 1600 1605

Thr Ala Phe Lys Lys Lys Asp Thr Ile Leu Ser Leu Asn Ala Cys

1610 1615 1620

Glu Ser Asn His Ala Ile Ala Ala Ile Asn Glu Gly Gln Asn Lys

1625 1630 1635

Pro Glu Ile Glu Val Thr Trp Ala Lys Gln Gly Arg Thr Glu Arg

1640 1645 1650

Leu Cys Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg Glu

1655 1660 1665

Ile Thr Arg Thr Thr Leu Gln Ser Asp Gln Glu Glu Ile Asp Tyr

1670 1675 1680

Asp Asp Thr Ile Ser Val Glu Met Lys Lys Glu Asp Phe Asp Ile

1685 1690 1695

Tyr Asp Glu Asp Glu Asn Gln Ser Pro Arg Ser Phe Gln Lys Lys

1700 1705 1710

Thr Arg His Tyr Phe Ile Ala Ala Val Glu Arg Leu Trp Asp Tyr

1715 1720 1725

Gly Met Ser Ser Ser Pro His Val Leu Arg Asn Arg Ala Gln Ser

1730 1735 1740

Gly Ser Val Pro Gln Phe Lys Lys Val Val Phe Gln Glu Phe Thr

1745 1750 1755

Asp Gly Ser Phe Thr Gln Pro Leu Tyr Arg Gly Glu Leu Asn Glu

1760 1765 1770

His Leu Gly Leu Leu Gly Pro Tyr Ile Arg Ala Glu Val Glu Asp

1775 1780 1785

Asn Ile Met Val Thr Phe Arg Asn Gln Ala Ser Arg Pro Tyr Ser

1790 1795 1800

Phe Tyr Ser Ser Leu Ile Ser Tyr Glu Glu Asp Gln Arg Gln Gly

1805 1810 1815

Ala Glu Pro Arg Lys Asn Phe Val Lys Pro Asn Glu Thr Lys Thr

1820 1825 1830

Tyr Phe Trp Lys Val Gln His His Met Ala Pro Thr Lys Asp Glu

1835 1840 1845

Phe Asp Cys Lys Ala Trp Ala Tyr Phe Ser Asp Val Asp Leu Glu

1850 1855 1860

Lys Asp Val His Ser Gly Leu Ile Gly Pro Leu Leu Val Cys His

1865 1870 1875

Thr Asn Thr Leu Asn Pro Ala His Gly Arg Gln Val Thr Val Gln

1880 1885 1890

Glu Phe Ala Leu Phe Phe Thr Ile Phe Asp Glu Thr Lys Ser Trp

1895 1900 1905

Tyr Phe Thr Glu Asn Met Glu Arg Asn Cys Arg Ala Pro Cys Asn

1910 1915 1920

Ile Gln Met Glu Asp Pro Thr Phe Lys Glu Asn Tyr Arg Phe His

1925 1930 1935

Ala Ile Asn Gly Tyr Ile Met Asp Thr Leu Pro Gly Leu Val Met

1940 1945 1950

Ala Gln Asp Gln Arg Ile Arg Trp Tyr Leu Leu Ser Met Gly Ser

1955 1960 1965

Asn Glu Asn Ile His Ser Ile His Phe Ser Gly His Val Phe Thr

1970 1975 1980

Val Arg Lys Lys Glu Glu Tyr Lys Met Ala Leu Tyr Asn Leu Tyr

1985 1990 1995

Pro Gly Val Phe Glu Thr Val Glu Met Leu Pro Ser Lys Ala Gly

2000 2005 2010

Ile Trp Arg Val Glu Cys Leu Ile Gly Glu His Leu His Ala Gly

2015 2020 2025

Met Ser Thr Leu Phe Leu Val Tyr Ser Asn Lys Cys Gln Thr Pro

2030 2035 2040

Leu Gly Met Ala Ser Gly His Ile Arg Asp Phe Gln Ile Thr Ala

2045 2050 2055

Ser Gly Gln Tyr Gly Gln Trp Ala Pro Lys Leu Ala Arg Leu His

2060 2065 2070

Tyr Ser Gly Ser Ile Asn Ala Trp Ser Thr Lys Glu Pro Phe Ser

2075 2080 2085

Trp Ile Lys Val Asp Leu Leu Ala Pro Met Ile Ile His Gly Ile

2090 2095 2100

Lys Thr Gln Gly Ala Arg Gln Lys Phe Ser Ser Leu Tyr Ile Ser

2105 2110 2115

Gln Phe Ile Ile Met Tyr Ser Leu Asp Gly Lys Lys Trp Gln Thr

2120 2125 2130

Tyr Arg Gly Asn Ser Thr Gly Thr Leu Met Val Phe Phe Gly Asn

2135 2140 2145

Val Asp Ser Ser Gly Ile Lys His Asn Ile Phe Asn Pro Pro Ile

2150 2155 2160

Ile Ala Arg Tyr Ile Arg Leu His Pro Thr His Tyr Ser Ile Arg

2165 2170 2175

Ser Thr Leu Arg Met Glu Leu Met Gly Cys Asp Leu Asn Ser Cys

2180 2185 2190

Ser Met Pro Leu Gly Met Glu Ser Lys Ala Ile Ser Asp Ala Gln

2195 2200 2205

Ile Thr Ala Ser Ser Tyr Phe Thr Asn Met Phe Ala Thr Trp Ser

2210 2215 2220

Pro Ser Lys Ala Arg Leu His Leu Gln Gly Arg Ser Asn Ala Trp

2225 2230 2235

Arg Pro Gln Val Asn Asn Pro Lys Glu Trp Leu Gln Val Asp Phe

2240 2245 2250

Gln Lys Thr Met Lys Val Thr Gly Val Thr Thr Gln Gly Val Lys

2255 2260 2265

Ser Leu Leu Thr Ser Met Tyr Val Lys Glu Phe Leu Ile Ser Ser

2270 2275 2280

Ser Gln Asp Gly His Gln Trp Thr Leu Phe Phe Gln Asn Gly Lys

2285 2290 2295

Val Lys Val Phe Gln Gly Asn Gln Asp Ser Phe Thr Pro Val Val

2300 2305 2310

Asn Ser Leu Asp Pro Pro Leu Leu Thr Arg Tyr Leu Arg Ile His

2315 2320 2325

Pro Gln Ser Trp Val His Gln Ile Ala Leu Arg Met Glu Val Leu

2330 2335 2340

Gly Cys Glu Ala Gln Asp Leu Tyr

2345 2350

<210> 13

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> description of artificial sequences: synthesis of

Peptides

<400> 13

Ser Phe Ser Gln Asn Pro Pro Val Leu Lys Arg His Gln Arg

1 5 10

<210> 14

<211> 24

<212> PRT

<213> genus porcine (Sus sp.)

<400> 14

Ser Phe Ala Gln Asn Ser Arg Pro Pro Ser Ala Ser Ala Pro Lys Pro

1 5 10 15

Pro Val Leu Arg Arg His Gln Arg

<210> 15

<211> 16

<212> PRT

<213> genus porcine (Sus sp.)

<400> 15

Ser Phe Ser Gln Asn Ser Arg His Gln Ala Tyr Arg Tyr Arg Arg Gly

1 5 10 15

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