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Patent Analysis of

Antagonat compositions and methods of use

Updated Time 12 June 2019

Patent Registration Data

Publication Number

US10000752

Application Number

US13/988063

Application Date

17 November 2011

Publication Date

19 June 2018

Current Assignee

CURNA, INC.

Original Assignee (Applicant)

COLLARD, JOSEPH,KHORKOVA SHERMAN, OLGA,DE LEON, BELINDA

International Classification

C12N15/11,C12N15/113,A61K31/00,A61K31/7088,A61K31/7115

Cooperative Classification

C12N15/113,A61K31/00,A61K31/7088,A61K31/7115,C12N15/111

Inventor

COLLARD, JOSEPH,KHORKOVA SHERMAN, OLGA,DE LEON, BELINDA

Patent Images

This patent contains figures and images illustrating the invention and its embodiment.

US10000752 Antagonat compositions 1 US10000752 Antagonat compositions 2 US10000752 Antagonat compositions 3
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Abstract

Provided herein are compositions, compounds, and methods of modulating gene expression. In certain embodiments described herein is a composition, wherein the composition comprises an antagoNAT. In some embodiments, the antagoNAT is an oligonucleotide comprising modified and unmodified sugar subunits, wherein the antagoNAT hybridizes with a natural antisense transcript. Certain embodiments of the present invention provide a method for modulating gene expression in a cell comprising contacting the cell with an antagoNAT. In some embodiments, the method includes forming a hybrid comprising the antagoNAT and a natural antisense transcript of the gene, wherein the hybrid sterically blocks the normal function of the natural antisense transcript.

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Claims

1. A method of upregulating the expression of a single gene in a cell comprising contacting the cell with an antagoNAT, wherein the antagoNAT is a single stranded oligonucleotide comprising 12-27 nucleoside subunits and which: is 100% complementary to and specifically hybridizes with a complementary 12-27 nucleotide region of a non-coding natural antisense transcript of the gene; and comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus; and further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, and at least one internal nucleoside is modified; andwherein said antagoNAT upregulates the expression of said gene and wherein the antagoNAT comprises a compound of Formula (I), or a salt thereof:

C-Au-[Bv-A′w]x-By-A″2-C   Formula (I) wherein each A, A′, and A″ independently has the structure of: each B independently has the structure of: each C is independently hydroxy, phosphate, substituted or unsubstituted alkoxyl, or any suitable 5′ or 3′ terminus cap; each u, v, w, x, y and z are independently integers greater than or equal to one; each D is a heterocyclic base; each E is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amine, halogen, substituted or unsubstituted aminoalkoxy, substituted or unsubstituted alkenyl, or thiol; each G is independently —OP(O)2O—, —OP(O)(OR)O—, —OP(O)(S)O—, —OP(O)(SR)O—, —OP(S)2O—, —OP(R)(O)O—, —OP(NR2)(O)O—, —OC(O)O—, —OCH2C(O)NHCH2—, —OCH2S—, —CH2SCH2—, —OP(O)(BH3)O—, —NP(O)2O—, —OP(R)(O)O—, or absent when (Ia) is connected to C; each R is independently hydrogen or substituted or unsubstituted alkyl; each J is hydrogen or J and E taken together form a ring structure that optionally includes an additional heteroatom selected from N or O; and each K is independently hydroxy or hydrogen and further wherein SEQ ID NOS: 13, 18, 19, 20, 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 38 and 39 are excluded and wherein the single gene is selected from the group consisting of SCN1A, SIRT1, ABCA1, VEGF, BDNF and GDNF.

2. The method according to claim 1 wherein the sugar modified and sugar unmodified nucleoside subunits each comprise a pyrimidine base or purine base and said modified sugar comprises a locked nucleic acid.

3. The method according to claim 1 wherein the internal sugar modified nucleoside subunits each comprises a pyrimidine base or purine base and wherein said internal modified subunits comprise at least one locked nucleic acid.

4. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, or O-alkylene bound to the C4′ carbon.

5. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, halogen, or O-alkylene bound to the C4′ carbon.

6. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′ position with methoxy.

7. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methoxyethyl.

8. The method according to claim 1, wherein the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methylene bound to the C4′ carbon (2′-OCH2-4′) or O-ethylene bound to the C4′ carbon (2′-OCH2CH2-4′).

9. The method according to claim 1, wherein the unmodified nucleoside subunit comprises a ribose sugar.

10. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphodiester, phosphotriester, phosphorothioate, phosphorodithiate, alkylphosphonate, phosphoramidate, boranophosphate, carbonate, carbamate, acetamidate, thioether, thioformacetal internucleotide linkages, or combinations thereof.

11. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphodiester and phosphorothioate internucleotide linkages.

12. The method according to claim 1, wherein the antagoNAT comprises a backbone of phosphorothioate internucleotide linkages.

13. The method according to claim 1, wherein no more than five internal unmodified nucleosides with 2′-deoxyribose sugar moieties are consecutive, wherein(a) the 3′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside; or(b) the 3′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside.

14. The method according to claim 1 wherein the antagoNAT comprises a compound of Formula (I), or a salt thereof:

C-Au-[Bv-A′w]x-By-A″z-C   Formula (I) wherein each A, A′, and A″ independently has the structure of: each B independently has the structure of: each C is independently hydroxy, phosphate, substituted or unsubstituted alkoxyl, or any suitable 5′ or 3′ terminus cap; each u, v, w, x, y and z are independently integers greater than or equal to one; each D is a heterocyclic base; each E is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amine, halogen, substituted or unsubstituted aminoalkoxy, substituted or unsubstituted alkenyl, or thiol; each G is independently —OP(O)2O—, —OP(O)(OR)O—, —OP(O)(S)O—, —OP(O)(SR)O—, —OP(S)2O—, —OP(R)(O)O—, —OP(NR2)(O)O—, —OC(O)O—, —OCH2C(O)NHCH2—, —OCH2S—, —CH2SCH2—, —OP(O)(BH3)O—, —NP(O)2O—, —OP(R)(O)O—, or absent when (Ia) is connected to C; each R is independently hydrogen or substituted or unsubstituted alkyl; each J is hydrogen or J and E taken together form a ring structure that optionally includes an additional heteroatom selected from N or O; and each K is independently hydroxy or hydrogen and wherein the upregulated targets are selected from the group consisting of SCN1A, BDNF, GDNF, or ABCA1 and further wherein SEQ ID NOS:13, 18, 19, 20, 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 38 and 39 are excluded.

15. The method according to claim 14 wherein each heterocyclic base is independently selected from a purine or pyrimidine base and wherein a internucleoside linkage is selected from a phosphorthioate.

16. The method according to claim 14 wherein each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, cytosine, 2-aminoadenine, 5-methylcytosine, 5-bromouracil, or hypoxanthine and wherein at least one internucleoside linkage is selected from a phosphorothiate.

17. The method of claim 14, wherein each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, or cytosine.

18. The method of claim 14, wherein the heterocyclic base of each A′ is independently selected from uracil, thymine, or cytosine.

19. The method of claim 14, wherein each A, A′, or A″ independently has the structure of:

20. The method of claim 14, wherein each E is independently methoxy, ethoxy, O-methylethyl, or fluoro.

21. The method of claim 14, wherein each E is methoxy.

22. The method of claim 14, wherein each E is O-methylethyl.

23. The method of claim 14, wherein each G is independently —OP(O)2O—, —OP(O)(OR)O—, or —OP(O)(S)O—.

24. The method of claim 14, wherein each G is —OP(O)(S)O—.

25. The method of claim 14, wherein each C is hydroxy.

26. The method of claim 14, wherein v and y are independently integers of 1, 2, or 3 when K is hydroxy and x is at least one.

27. The method of claim 14, wherein v and y are independently integers of 1, 2, 3, 4, or 5 when K is hydrogen, and(a) wherein at least one A has the structure of (Id) or (Ie) and at least one A″ has the structure of (Ic); or(b) wherein at least one A has the structure of (Ic) and at least one A″ has the structure of (Id) or (Ie).

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Claim Tree

  • 1
    1. A method of upregulating the expression of a single gene in a cell comprising
    • contacting the cell with an antagoNAT, wherein the antagoNAT is a single stranded oligonucleotide comprising 12-27 nucleoside subunits and which: is 100% complementary to and specifically hybridizes with a complementary 12-27 nucleotide region of a non-coding natural antisense transcript of the gene
    • and comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus
    • and further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, and at least one internal nucleoside is modified
    • andwherein said antagoNAT upregulates the expression of said gene and wherein the antagoNAT comprises a compound of Formula (I), or a salt thereof: C-Au-[Bv-A′w]x-By-A″2-C   Formula (I) wherein each A, A′, and A″ independently has the structure of: each B independently has the structure of: each C is independently hydroxy, phosphate, substituted or unsubstituted alkoxyl, or any suitable 5′ or 3′ terminus cap
    • each u, v, w, x, y and z are independently integers greater than or equal to one
    • each D is a heterocyclic base
    • each E is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amine, halogen, substituted or unsubstituted aminoalkoxy, substituted or unsubstituted alkenyl, or thiol
    • each G is independently —OP(O)2O—, —OP(O)(OR)O—, —OP(O)(S)O—, —OP(O)(SR)O—, —OP(S)2O—, —OP(R)(O)O—, —OP(NR2)(O)O—, —OC(O)O—, —OCH2C(O)NHCH2—, —OCH2S—, —CH2SCH2—, —OP(O)(BH3)O—, —NP(O)2O—, —OP(R)(O)O—, or absent when (Ia) is connected to C
    • each R is independently hydrogen or substituted or unsubstituted alkyl
    • each J is hydrogen or J and E taken together form a ring structure that optionally includes an additional heteroatom selected from N or O
    • and each K is independently hydroxy or hydrogen and further wherein SEQ ID NOS: 13, 18, 19, 20, 21, 22, 23, 24, 25, 27, 31, 32, 34, 35, 38 and 39 are excluded and wherein the single gene is selected from the group consisting of SCN1A, SIRT1, ABCA1, VEGF, BDNF and GDNF.
    • 2. The method according to claim 1 wherein
      • the sugar modified and sugar unmodified nucleoside subunits each comprise
    • 3. The method according to claim 1 wherein
      • the internal sugar modified nucleoside subunits each comprises
    • 4. The method according to claim 1, wherein
      • the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, or O-alkylene bound to the C4′ carbon.
    • 5. The method according to claim 1, wherein
      • the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, halogen, or O-alkylene bound to the C4′ carbon.
    • 6. The method according to claim 1, wherein
      • the sugar modified nucleoside subunits are each substituted at the 2′ position with methoxy.
    • 7. The method according to claim 1, wherein
      • the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methoxyethyl.
    • 8. The method according to claim 1, wherein
      • the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methylene bound to the C4′ carbon (2′-OCH2-4′) or O-ethylene bound to the C4′ carbon (2′-OCH2CH2-4′).
    • 9. The method according to claim 1, wherein
      • the unmodified nucleoside subunit comprises
    • 10. The method according to claim 1, wherein
      • the antagoNAT comprises
    • 11. The method according to claim 1, wherein
      • the antagoNAT comprises
    • 12. The method according to claim 1, wherein
      • the antagoNAT comprises
    • 13. The method according to claim 1, wherein
      • no more than five internal unmodified nucleosides with 2′-deoxyribose sugar moieties are consecutive, wherein
    • 14. The method according to claim 1 wherein
      • the antagoNAT comprises
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Description

FIELD OF THE INVENTION

Embodiments of the invention comprise compositions, compounds, and methods of modulating gene expression.

BACKGROUND OF THE INVENTION

DNA-RNA and RNA-RNA hybridization are important to many aspects of nucleic acid function including DNA replication, transcription, and translation. Hybridization is also central to a variety of technologies that either detect a particular nucleic acid or alter its expression. Antisense nucleotides, for example, disrupt gene expression by hybridizing to target RNA, thereby interfering with RNA splicing, transcription, translation, and replication. Antisense DNA has the added feature that DNA-RNA hybrids serve as a substrate for digestion by ribonuclease H, an activity that is present in most cell types. Antisense molecules can be delivered into cells, as is the case for oligodeoxynucleotides, or they can be expressed from endogenous genes as RNA molecules.

SUMMARY OF THE INVENTION

Provided herein are compositions, compounds, and methods of modulating the function of a polynucleotide in a cell. In certain embodiments described herein is a composition, wherein the composition comprises a pharmaceutically acceptable diluent or carrier and an antagoNAT. In some embodiments, the antagoNAT is a modified oligonucleotide that hybridizes with a natural antisense transcript. Certain embodiments of the present invention provide a method for modulating the function of a polynucleotide in a cell comprising contacting the cell with an antagoNAT. In some embodiments, the method includes forming a hybrid comprising the antagoNAT and the polynucleotide, wherein the hybrid sterically blocks the normal function of the polynucleotide.

Some embodiments of the present invention describe a composition comprising a pharmaceutically acceptable diluent or carrier and an antagoNAT, wherein the antagoNAT is 10 to 30 nucleoside subunits in length. In some embodiments, the antagoNAT hybridizes with a preselected natural antisense transcript. In other embodiments, the antagoNAT comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In further or additional embodiments, the antagoNAT comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between the internal sugar unmodified nucleoside subunits.

In some embodiments, the antagoNAT of a composition described herein comprises sugar modified and sugar unmodified nucleoside subunits, wherein the sugar modified and sugar unmodified nucleoside subunits each comprise a pyrimidine base or purine base. In other embodiments, the internal sugar modified nucleoside subunits each comprise a pyrimidine base or purine base. In further or additional embodiments, the internal sugar modified nucleoside subunits each comprise a pyrimidine base.

In other embodiments, the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, or O-alkylene bound to the C4′ carbon. In some embodiments, the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, halogen, or O-alkylene bound to the C4′ carbon. In specific embodiments, the sugar modified nucleoside subunits are each substituted at the 2′ position with methoxy. In certain specific embodiments, the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methoxyethyl. In other embodiments of the present invention, the sugar modified nucleoside subunits are each substituted at the 2′ position with O-methylene bound to the C4′ carbon (2′-OCH2-4′) or O-ethylene bound to the C4′ carbon (2′-OCH2CH2-4′).

In some preferred compositions of the invention, each unmodified nucleoside subunit independently comprises a ribose or 2′-deoxyribose sugar.

In some embodiments of the composition, the antagoNAT of a composition described herein comprises a backbone of phosphodiester, phosphotriester, phosphorothioate, phosphorodithiate, alkylphosphonate, phosphoramidate, boranophosphate, carbonate, carbamate, acetamidate, thioether, thioformacetal intemucleotide linkages, or combinations thereof. In other embodiments, the antagoNAT comprises a backbone of phosphodiester and phosphorothioate internucleotide linkages. In specific embodiments, the antagoNAT comprises a backbone of phosphorothioate intemucleotide linkages.

In certain embodiments, the antagoNAT of a composition described herein is at least 50% complementary to the preselected natural antisense transcript.

In certain embodiments, the antagoNAT of a composition described herein does not include more than five consecutive internal unmodified nucleosides comprising 2′-deoxyribose sugars, wherein (a) the 3′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside; or (b) the 3′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside.

Some embodiments of the present invention describe an antagoNAT of Formula (I), or a salt thereof:

C-Au-[Bv-A′w]x-By-A″z-C   Formula (I)

wherein:

    • each A, A′, and A″ independently has the structure of:

    • each B independently has the structure of:

    • each C is independently hydroxy, phosphate, substituted or unsubstituted alkoxyl, or any suitable 5′ or 3′ terminus cap;
    • each u, v, w, x, y and z are independently integers greater than or equal to one;
    • each D is a heterocyclic base;
    • each E is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amine, halogen, substituted or unsubstituted aminoalkoxy, substituted or unsubstituted alkenyl, or thiol;
    • each G is independently —OP(O)2O—, —OP(O)(OR)O—, —OP(O)(S)O—, —OP(O)(SR)O—, —OP(S)2O—, —OP(R)(O)O—, —OP(NR2)(O)O—, —OC(O)O—, —OCH2C(O)NHCH2—, —OCH2S—, —CH2SCH2—, —OP(O)(BH3)O—, —NP(O)2O—, —OP(R)(O)O—, or absent when (Ia) is connected to C;
    • each R is independently hydrogen or substituted or unsubstituted alkyl;
    • each J is hydrogen or J and E taken together form a ring structure that optionally includes an additional heteroatom selected from N or O; and
    • each K is independently hydroxy or hydrogen.

In certain embodiments of the antagoNAT, each heterocyclic base of the antagoNAT described herein is independently selected from a purine or pyrimidine base. In other embodiments, each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, cytosine, 2-aminoadenine, 5-methylcytosine, 5-bromouracil, or hypoxanthine. In certain specific embodiments, each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, or cytosine. In other specific embodiments, the heterocyclic base of each A′ is independently selected from uracil, thymine, or cytosine.

In some embodiments, an antagoNAT is described, wherein each A, A′, or A″ independently has the structure of:

In some preferred compounds of the invention, each E is independently methoxy, ethoxy, O-methylethyl, or fluoro. In specific embodiments, each E is methoxy. In certain specific embodiments, each E is O-methylethyl.

In some embodiments of an antagoNAT described herein, each G is independently —OP(O)2O—, —OP(O)(OR)O—, or —OP(O)(S)O—. In specific embodiments, each G is —OP(O)(S)O—.

In some embodiments of an antagoNAT described herein, each C is hydroxy or any suitable terminus cap structure.

In certain preferred antagoNATs of the invention, v and y are independently integers of 1, 2, or 3 when K is hydroxy and x is at least one. In other embodiments, v and y are independently integers of 1, 2, 3, 4, or 5 when K is hydrogen, and (a) wherein at least one A has the structure of (Id) or (Ie) and at least one A″ has the structure of (Ic); or (b) wherein at least one A has the structure of (Ic) and at least one A″ has the structure of (Id) or (Ie).

In certain embodiments, provided herein is a method for modulating expression of a gene in a cell. In some embodiments, the method includes contacting the cells with an antagoNAT described herein, wherein the antagoNAT is 10 to 30 nucleoside subunits in length. In some embodiments, the antagoNAT of a composition or used in a method described herein specifically hybridizes with a natural antisense transcript of the gene. In other embodiments, the antagoNAT includes at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In specific embodiments, the antagoNAT additionally includes internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between the internal sugar unmodified nucleoside subunits.

In some embodiments, any method of modulating gene expression described herein further comprises forming a hybrid comprising the antagoNAT and the natural antisense transcript; thereby modulating the expression of said gene. In some embodiments, any method of modulating gene expression described herein further comprises forming a hybrid comprising the antagoNAT and the natural antisense transcript, wherein the hybrid is not a substrate for ribonuclease cleavage. In certain embodiments, the method comprises sterically blocking the normal function of the natural antisense transcript. In some embodiments, the antagoNAT has at least 50% sequence identity to a complement of the natural antisense transcript. In other embodiments, expression of the gene is up-regulated in the cell with respect to a control. In certain embodiments, expression of the gene is down-regulated in the cell with respect to a control.

In some embodiments, the type of cell contacted with an antagoNAT according to a method described herein is a mammalian cell.

Further in accordance with certain embodiments of the present invention, there is provided a method of modulating function of a polynucleotide in a cell comprising contacting the cell with an antagoNAT. In some embodiments, the antagoNAT is 10 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT hybridizes with the polynucleotide. In specific embodiments, the antagoNAT comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In further or additional embodiments, the antagoNAT comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between the internal sugar unmodified nucleoside subunits.

In some embodiments, the targeted polynucleotide according to a method described herein is a natural antisense strand to a sense strand. In certain embodiments, the antagoNAT has at least 50% sequence identity to a complement of the polynucleotide.

In some embodiments, any method of modulating function of a polynucleotide described herein further comprises forming a hybrid comprising the antagoNAT and the polynucleotide; thereby modulating said function of said polynucleotide. In certain embodiments, the hybrid is not a substrate for ribonuclease cleavage. In some embodiments, the method comprises sterically blocking the normal function of the polynucleotide. In certain embodiments, expression of the sense strand is elevated in the cell with respect to a control. In other embodiments, expression of the sense strand is decreased in the cell with respect to a control.

In some embodiments, the type of cell contacted with an antagoNAT according to a method described herein is a mammalian cell.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF FIGURES

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying figures of which:

FIG. 1—cDNA Sequence of BF133827 (SEQ ID NO: 40), a potential non-coding antisense transcript for mouse ABCA1. Specific target sites are highlighted.

FIG. 2—RT-PCR analysis of macrophage ABCA1 mRNA expression at 48 hours following treatment with ABCA1-AS antisense oligonucleotides (50 nM, n=3). Values represent mean±SEM. * indicates statistical significance compared to CUR586 control (p<0.05; 1-way ANOVA; n=3).

FIG. 3a—Western immunoblot analysis of macrophage ABCA1 protein expression at 48 hours following treatment with ABCA1-AS antisense oligonucleotides (50 nM, n=3), using ABCA1 primary polyclonal antibody (Novus).

FIG. 3b—Densitometric analysis of the macrophage Western immunoblot. Values represent mean±SEM. * indicates statistical significance compared to CUR586 control (p<0.05; 1-way ANOVA; n=3).

FIG. 4—Immunostaining of macrophage ABCA1 protein at 48 hours following treatment with ABCA1-AS antisense oligonucleotides (50 nM). Nuceli were stained with Hoechst 33258 while ABCA1 was visualized using an Alexa Fluor 488-conjugated secondary antibody.

FIG. 5—RT-PCR analysis of astrocyte ABCA1 mRNA expression following treatment with ABCA1-AS antisense oligonucleotides (50 nM, n=3).

FIG. 6a—Western immunoblot analysis of astrocyte ABCA1 protein expression following treatment with ABCA1-AS antisense oligonucleotides (50 nM, n=3), using ABCA1 primary polyclonal antibody.

FIG. 6b—Densitometric analysis of the astrocyte Western immunoblot following treatment with ABCA1-AS antisense oligonucleotides (50 nM, n=3). Values represent mean±SEM. * indicates statistical significance compared to CUR586 control (p<0.05; 1-way ANOVA; n=3).

FIG. 7—ABCA1 protein expression in macrophages, at 48 hours following treatment with chemically modified ABCA1-AS antisense oligonucleotides, including an antagoNAT CUR1463.

FIG. 8—RT-PCR Analysis of ABCA1 mRNA levels in mouse NIH 3T3 cells following treatment with AntagoNAT CUR1463.

FIG. 9—Western Immunoblot Analysis of ABCA1 protein levels in mouse NIH 3T3 cells following treatment with AntagoNAT CUR1463.

FIG. 10—Expression of ABCA1 mRNA in the livers of wild-type mice treated with ABCA-AS antisense oligonucleotides and antagoNAT (5 mg/kg), twice a week for four weeks. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM. * indicates statistically significance differences between CUR1463 and both CUR1575 and saline controls (p<0.05; 1-way ANOVA; 4≤n≤9)

FIG. 11—Expression of ABCA1 protein in the livers of wild-type mice treated with ABCA1-AS antisense oligonucleotides (5 mg/kg), twice a week for four weeks, as assessed by densitometric analysis of the Western immunoblot assay. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM.

FIG. 12a—Total serum cholesterol in mice treated with ABCA1-AS antisense oligonucleotides. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM. * indicates statistically significance differences between CUR1463 and both CUR1575 and saline controls (p<0.05; 1-way ANOVA; 5≤n≤10).

FIG. 12b—LDL-cholesterol in mice treated with ABCA1-AS antagoNAT. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM. * indicates statistically significance differences between CUR1463 and both CUR1575 and saline controls (p<0.05; 1-way ANOVA; 5≤n≤10).

FIG. 13—Ratio of HDL:LDL in mice treated with ABCA1-AS antagoNAT (5 mg/kg), twice a week for four weeks. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM. * indicates statistically significance differences between CUR1463 and both CUR1575 and saline controls (p<0.05; 1-way ANOVA; 5≤n≤10).

FIG. 14a—Serum HDL cholesterol in mice treated with ABCA1-AS antagoNAT (5 mg/kg), twice a week for four weeks. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM.

FIG. 14b—Triglyceride levels in mice treated with ABCA1-AS antagoNAT (5 mg/kg), twice a week for four weeks. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM.

FIG. 15—Serum alanine transaminase (ALT) activity in mice treated with ABCA1-AS antagoNAT (5 mg/kg), twice a week for four weeks. Values represent pooled data obtained over two repeated studies, and are expressed as mean±SEM.

FIG. 16—ABCA1 mRNA expression in human 518A2 melanoma cells 48 hours after treatment with 20 nM siRNA (n=5). Values indicate mean+Std Dev. * indicates statistical significance.

FIG. 17—RT-PCR analysis of ABCA1 mRNA levels in HepG2 cells. ABCA1 mRNA expression is increased with antagoNAT CUR-1719.

FIG. 18—ABCA1 mRNA expression in human HepG2 hepatocellular carcinoma cells 48 hours after treatment with 2′ O-methyl modified antagoNATs. Values indicate mean+Std Dev. * indicates statistical significance.

FIG. 19a—Western immunoblot analysis of ABCA1 protein expression in human epithelial colorectal adenocarcinoma (CaCo2) cells 48 hours following treatment of cells with ABCA1-AS antisense oligonucleotides (50 nM, n=3). ABCA1 primary polyclonal antibody (Novus) was used in the assay.

FIG. 19b—Densitometric analysis of CaCo2 Western immunoblot assay. Values represent mean±SEM. Data indicates statistical significance with an antagoNAT compared to vehicle treated cells and control oligonucleotide (p<0.05; 1-way ANOVA; n=3).

FIG. 20—RT-PCR analysis of SCN1A mRNA levels in HepG2 cells following treatment with an antagoNAT.

FIG. 21—RT-PCR analysis of SIRT1 mRNA levels in mouse NIH 3T3 cells following treatment with an antagoNAT.

FIG. 22—FIG. 22 shows the mouse Sirt1 mRNA expression is up-regulated in NIH3T3 cells with a phosphothioate oligonucleotide (CUR-1099), 2′Omethyl gapmer/pyrimidine modified oligonucleotide (CUR-1578) and a LNA modified gapmer oligonucleotide (CUR-1748). The control LNA modified gapmer configuration oligonucleotide (CUR-1750) as well as the LNA modified gapmer oligonucleotides (CUR-1658 and CUR-1749) did not significantly up-regulate mouse Sirt1 mRNA expression.

Sequence Listing Description—SEQ ID NO: 1: Homo sapiens ATP-binding cassette, sub-family A (ABC1), member 1(ABCA1), mRNA (NCBI Accession No.: NM_005502); SEQ ID NO: 2: Homo sapiens sodium channel, voltage-gated, type I, alpha subunit (SCN1A), transcript variant 1, mRNA (NCBI Accession No.: NM_001165963); SEQ ID NO: 3: Homo sapiens sodium channel, voltage-gated, type I, alpha subunit (SCN1A), transcript variant 2, mRNA (NCBI Accession No.: NM_006920); SEQ ID NO: 4: Homo sapiens sodium channel, voltage-gated, type I, alpha subunit (SCN1A), transcript variant 3 mRNA (NCBI Accession No.: NM_001165964); SEQ ID NO: 5: Homo sapiens sodium channel, voltage-gated, type I, alpha subunit (SCN1A), transcript variant 4, mRNA (NCBI Accession No.: NM_001202435); SEQ ID NO: 6: Mus musculus sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae) (Sirt1), transcript variant 2, mRNA (NCBI Accession Number: NM_001159589); SEQ ID NO: 7 Mus musculus sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae) (Sirt1), transcript variant 1, mRNA (NCBI Accession Number: NM_019812); SEQ ID NO: 8 Mus musculus sirtuin 1 (silent mating type information regulation 2, homolog) 1 (S. cerevisiae) (Sirt1), transcript variant 3, mRNA (NCBI Accession Number: NM_001159590); SEQ ID NO: 9: Mouse Natural ABCA1 antisense sequence (AK311445); SEQ ID NO: 10 and 11: Natural SCN1A antisense sequence, original and extended respectively (BG724147); SEQ ID NO: 12: Mouse Natural SIRT1 antisense sequence (ak044604); SEQ ID NOs: 13 to 21: Sequences of 2′-Unmodified and 2′-Modified ABCA1-AS Antisense Oligonucleotides; SEQ ID NOs: 22 to 26: Sequences of AntagoNAT and Control Oligonucleotides Targeted to ABCA1-AS Antisense Oligonucleotides; SEQ ID NOs: 27 to 29: Sequences of Chemically Modified Oligonucleotides Targeted to SCN1A-AS Antisense Oligonucleotide; SEQ ID NOs: 30 to 39: Sequences of Chemically Modified Oligonucleotides Targeted to SIRT1 Antisense Oligonucleotide. * indicates phosphorothioate bond, +indicates 2′-bicyclic sugar modified nucleoside or LNA, and m indicates 2′-O-Methyl sugar modified nucleoside.

DETAILED DESCRIPTION OF THE INVENTION

While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes or nucleic acid sequences are human.

Certain Definitions

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

As used herein, the term “nucleoside” or “nucleoside subunit” means a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides having mimetic bases and/or sugar groups.

As used herein, the term “unmodified nucleoside” or “natural nucleoside” means a nucleoside comprising a natural nucleobase and a natural sugar. Natural nucleosides include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) nucleosides.

As used herein, the term “unmodified sugar” or “sugar unmodified” refers to a sugar of a nucleoside that is unmodified from its naturally occurring form in RNA (2′-OH) or DNA (2′-H).

As used herein, the term “modified sugar”, “2′-modified sugar” or “sugar unmodified” refers to a pentofuranosyl sugar of a nucleoside comprising a substituent at the 2′ position other than H or OH. 2′-modified sugars include, but are not limited to, pentofuranosyl sugar substituted at the 2′ position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, or O-alkylene bound to the C4′ carbon.

As used herein, the term “nucleobase” refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “unmodified nucleobase” refers to a nucleobase that is unmodified from its naturally occurring form in RNA or DNA.

As used herein, the term “heterocyclic base” refers to a nucleobase comprising a heterocycle.

As used herein, the term “nucleotide” or “nucleotide subunit” refers to a nucleoside having a phosphate group covalently linked to the sugar. Nucleotides may be modified with any of a variety of substituents.

As used herein, “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides.

As used herein, “natural internucleotide linkage” refers to a 3′ to 5′ phosphodiester linkage.

As used herein, the term “modified internucleoside linkage” refers to any linkage between nucleosides or nucleotides other than a naturally occurring internucleoside linkage.

The term “oligomeric compound” is meant to be inclusive of the terms oligonucleotides and oligonucleosides, either used singly or in combination, as well as other oligomeric compounds including chimeric compounds formed.

In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. The term “oligonucleotide”, also includes linear or circular oligomers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha-anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like. Oligonucleotides are capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoögsteen or reverse Hoögsteen types of base pairing, or the like.

In the context of this invention, “chimeric” compounds are oligonucleotides, which contain two or more chemical regions, for example, DNA region(s), RNA region(s), modified nucleotide regions, etc. Each chemical region is made up of at least one subunit, i.e., a nucleotide in the case of an oligonucleotide. These oligonucleotides typically comprise at least one region wherein the oligonucleotide is modified in order to exhibit one or more desired properties. The desired properties of the oligonucleotide include, but are not limited, for example, increased resistance to nuclease degradation, increased cellular uptake, reduced toxicity effects, and/or increased binding affinity for the target nucleic acid. Different regions of the oligonucleotide may therefore have different properties. The chimeric oligonucleotides of the present invention can be formed as mixed structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide analogs as described above.

As used herein, the term “antagoNAT” refers to a polymeric structure comprising two or more nucleoside structures and capable of hybridizing to a region of a nucleic acid molecule. In some embodiments, antagoNATs are chemically engineered oligonucleotides that are complementary to specific natural antisense molecules, wherein the oligonucleotides comprise sugar unmodified nucleoside subunits and sugar modified nucleoside subunits. In other embodiments, the antagoNAT includes at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In some embodiments, the antagoNAT additionally includes internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between the internal sugar unmodified nucleoside subunits. In some embodiments, the antagoNAT hybridizes with a natural antisense transcript of a sense strand. In certain embodiments, antagoNATs are antisense oligonucleotides. In some embodiments, the specific hybridization of an antagoNAT and the target nucleic acid molecule interferes with the normal function of the nucleic acid molecule. In specific embodiments, the product of hybridization of a certain antagoNAT with the target molecule is not a substrate for ribonuclease cleavage. Oligomeric double-stranded compounds can be two strands hybridized to form double-stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound.

As used herein, the term “mixed-backbone antagoNAT” refers to an antagoNAT wherein a least one internucleoside linkage of the antagoNAT is different from at least one other internucleotide linkage of the antagoNAT.

As used herein, the term “terminus segment”, refers to a consecutive sequence of modified sugar nucleoside subunits at the 3′ terminus and/or the 5′ terminus of a chemically modified oligonucleotide.

As used herein, the term “antisense compound” or “antisense molecule” or “antisense transcript” refers to an oligomeric compound that is at least partially complementary to a target nucleic acid molecule to which it hybridizes. In certain embodiments, an antisense compound modulates (increases or decreases) expression of a target nucleic acid. Antisense compounds include, but are not limited to, compounds that are oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. Such molecules include, for example, antisense RNA or DNA molecules, interference RNA (RNAi), micro RNA, decoy RNA molecules, short interfering RNA (siRNA), enzymatic RNA, therapeutic editing RNA and agonist and antagonist RNA, antisense oligomeric compounds, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds that hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, partially single-stranded, or circular oligomeric compounds. While all antisense compounds are oligomeric compounds, not all oligomeric compounds are antisense compounds.

As used herein, the term “antisense oligonucleotide” refers to an antisense compound that is an oligonucleotide.

As used herein, the term “natural antisense transcript” refers to an oligomeric compound encoded within a cell that is at least partially complementary to other RNA transcripts and/or other endogenous sense transcripts. In certain embodiments, the natural antisense transcript does not code for a protein. In certain embodiments, the natural antisense transcript contains a stop codon early in the transcript that prevents significant protein coding.

As used herein, the term “antisense activity” refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. Such detection and or measuring may be direct or indirect. For example, in certain embodiments, antisense activity is assessed by detecting and or measuring the amount of target protein. In certain embodiments, antisense activity is assessed by detecting and/or measuring the amount of target nucleic acids.

As used herein, the term “detecting antisense activity” or “measuring antisense activity” means that a test for detecting or measuring antisense activity is performed on a sample and compared to that of a control sample. Such detection and/or measuring may include values of zero.

As used herein, the term “control sample” refers to a sample that has not been contacted with a test compound. In certain embodiments, a control sample is obtained prior to administration of an oligomeric compound to an animal. In certain embodiments, a control sample is obtained from an animal to which oligomeric compound is not administered. In certain embodiments, a reference standard is used as a surrogate for a control sample.

As used herein, the term “mock treated sample” refers to a sample that has not been contacted with a test compound. In certain embodiments, a mock treated sample is a control sample comprising liquid vehicle. In certain embodiments, a mock treated sample is a control sample comprising aqueous vehicle. In specific embodiments, a mock treated sample is a control sample comprising saline, water, or buffered solutions.

As used herein, the term “motif” refers to a pattern of unmodified and modified nucleotides or linkages in an oligomeric compound.

As used herein the term “target gene” refers to a gene encoding a target.

As used herein the term targeting or “targeted to” refers to the association of an antisense compound to a particular target nucleic acid molecule or a particular region of nucleotides within a target nucleic acid molecule.

As used herein, the term “oligonucleotide specific for” or “oligonucleotide which targets” refers to an oligonucleotide having a sequence (i) capable of forming a stable complex with a portion of the targeted nucleic acid molecule, or (ii) capable of forming a stable duplex with a portion of an RNA transcript and/or natural antisense transcript of the targeted gene. Stability of the complexes and duplexes can be determined by theoretical calculations and/or in vitro assays.

As used herein, the term “target nucleic acid” encompasses DNA, RNA (comprising premRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA, coding, noncoding sequences, sense or antisense polynucleotides. The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid. This modulation of function of a target nucleic acid by compounds, which specifically hybridize to it, is generally referred to as “antisense”. The functions of DNA to be interfered include, for example, replication and transcription. The functions of RNA to be interfered, include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA. The overall effect of such interference with target nucleic acid function is modulation of the expression of an encoded product or oligonucleotides.

As used herein, the term “percent complementary” refers to the number of nucleobases of an oligomeric compound that have nucleobases complementarity with a corresponding nucleobase of another oligomeric compound or nucleic acid divided by the total length (number of nucleobases) of the oligomeric compound.

As used herein, the term “modulation” refers to a perturbation of function or activity when compared to the level of function or activity when compared to the level of the function or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. Modulation also includes up-regulation (stimulation or induction) or down-regulation (inhibition or reduction) of gene expression.

As used herein, the term “expression” refers to all the functions and steps by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.

Further in the context of this invention, “hybridization” refers to hydrogen bonding, which may be Watson-Crick, Hoögsteen or reversed Hoögsteen hydrogen bonding, between complementary nucleobases.

“Complementary” as used herein, refers to the capacity for precise pairing between two nucleobases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds.

The terms “complementary” and “specifically hybridizable” as used herein, refer to precise pairing or sequence complementarity between a first and a second nucleic acid-like oligomers containing nucleoside subunits. For example, if a nucleobase at certain position of the first nucleic acid is capable of hydrogen bonding with a nucleobase at the same position of the second nucleic acid, then the first nucleic acid and the second nucleic acid are considered to be complementary to each other at that position. The first and second nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target nucleic acid molecule. It is understood that an oligomeric compound of the invention need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligomeric compound is specifically hybridizable when binding of the oligomeric compound to the target antisense molecule interferes with the normal function of the target antisense molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of vitro assays, under conditions in which the assays are performed.

As used herein, the term “side effects” refers to physiological responses attributable to a treatment other than desired effects. In certain embodiments, side effects include, without limitation, liver function test abnormalities, injections site reactions, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.

As used herein, the term “SIRT1” refers to Silencing mating type information regulator 2 homolog and is a member of the SIRTuin deacetylase protein family. The amino acid sequence of SIRT1 may be found at Genbank Accession number NP.sub.-08509. SIRT1 is the human homolog of the yeast Sir2 protein and exhibits NAD-dependent deacetylase activity.

As used herein, the term “ABCA1” refers ATP-binding cassette transporter A1 (ABCA1) and is an integral membrane protein that exports cholesterol from cells and initiates the formation of mature HDL by facilitating apolipoprotein A-I (apoA-I) lipidation.

As used herein, the term “SCN1” refers sodium channel, voltage-gated, type I, alpha subunit.

The term “alkyl” as used herein refers to saturated or unsaturated, straight- or branched-chain hydrocarbon radicals derived from a hydrocarbon moiety containing between one and twenty carbon atoms by removal of a single hydrogen atom. Alkyl groups as used herein may optionally include one or more further substituent groups.

The term “halo” and “halogen” as used herein refer to an atom selected from fluorine, chlorine, bromine and iodine.

The term “cycloalkyl” as used herein refers to a monovalent group derived from a monocyclic or bicyclic saturated carbocyclic ring compound containing between three and twenty carbon atoms by removal of a single hydrogen atom. Cycloalkyl groups as used herein may optionally include one or more further substituent groups.

The alkyl group or cycloalkyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, alkoxycarbonyl, alkylaminocarbonyl, di-(alkyl)-aminocarbonyl, hydroxyl, alkoxy, formyloxy, alkylcarbonyloxy, alkylthio, cycloalkyl or phenyl.

The term “aminoalkyl” as used herein, refers to an amino substituted alkyl radical. This term is meant to include alkyl groups having an amino substituent at any position and wherein the alkyl group attaches the aminoalkyl group to the parent molecule. The alkyl and/or amino portions of the aminoalkyl group can be further substituted with substituent groups.

As used herein, the term “alkoxy” refers to a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Alkoxy groups as used herein may optionally include further substituent groups.

As used herein, the term “alkenyl” refers to a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Alkenyl groups as used herein may optionally include one or more further substituent groups.

As used herein, the term “aryl” refers to a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include, but are not limited to, phenyl, naphthly, tetrahydronaphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Aryl groups as used herein may optionally include further substituent groups.

As used herein, the term “acyl” refers to a radical formed by removal of a hydroxyl group from an organic acid and has the general formula —C(O)—X where X is typically aliphatic, acyclic or aromatic. Acyl groups may optionally include further substituent groups.

As used herein, the terms “substituent” and “substituent group” refer to groups that are typically added to other groups or parent compounds to enhance desired properties or give desired effects. Substituent groups can be protected or unprotected and can be added to one available sites in a parent compound. Substituent groups may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl group to a parent compound.

2′-OH and 2′-H are utilized as an abbreviation for unmodified sugars, i.e. pentoribofuranosyl and pentodeoxyribofuranosyl sugars. For modified nucleosides, the abbreviations used are: 2′-O-alkyl for general alkyl groups at the 2′ position of a pentofuranosyl structure. (e.g., with a specific alkyl being noted as 2′-OMe for methyl).

As used herein, the term “bicyclic nucleoside” refers to a nucleoside wherein the furanose portion of the nucleosides includes a bridge connection two atoms on the furanose ring, thereby forming a bicyclic ring system.

As used herein, the term “LNA” or “locked nucleic acid” refers to the a nucleoside wherein the 2′ hydroxyl group of a ribosyl sugar ring is linked to the 4′ carbon of the sugar ring, thereby forming a bicyclic nucleoside.

As used herein, the term “substituted at 2′ position with O-methylene bound to the C4′ carbon” refers to a bicyclic nucleoside wherein the bridge connecting the two atoms of the furanose ring bridges the 4′ carbon atom and the 2′ carbon atom of the furanose ring, thereby forming a bicyclic ring system.

The term “pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated herein by reference for this purpose. The salts are prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base function with a suitable organic acid. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other documented methodologies such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

As used herein, the term “cap structure” or “terminal cap structure” refers to chemical modifications, which have been incorporated at either terminus of an antisense compound.

As used herein, the term “analogs” refers to nucleotides includes synthetic nucleotides having modified base moieties and/or modified sugar moieties (see e.g., described generally by Scheit, Nucleotide Analogs, John Wiley, New York, 1980; Freier & Altmann, (1997) Nucl. Acid. Res., 25(22), 4429-4443, Toulme, J. J., (2001) Nature Biotechnology 19:17-18; Manoharan M., (1999) Biochemica et Biophysica Acta 1489:117-139; Freier S. M., (1997) Nucleic Acid Research, 25:4429-4443, Uhlman, E., (2000) Drug Discovery & Development, 3: 203-213, Herdewin P., (2000) Antisense & Nucleic Acid Drug Dev., 10:297-310); 2′-O, 3′-C-linked [3.2.0]bicycloarabinonucleosides (see e.g. N. K Christiensen., et al, (1998) J. Am. Chem. Soc., 120: 5458-5463; Prakash T P, Bhat B. (2007) Curr Top Med Chem. 7(7):641-9; Eun Jeong Cho, Joo-Woon Lee, Andrew D. Ellington Applications of Aptamers as Sensors Annual Review of Analytical Chemistry, July 2009, Vol. 2, Pages 241-264. Such analogs include synthetic nucleotides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.

As used herein, the term “mammal” covers warm blooded mammals that are typically under medical care (e.g., humans and domesticated animals). Examples include feline, canine, equine, bovine, and human, as well as just human.

Overview

In certain embodiments, chemical modifications improve the potency and/or efficacy of antisense compounds, decreasing toxicological effects, decreasing the potential for side effects. In certain embodiments, antagoNATs comprising certain chemical modifications are less toxic than other oligomeric compounds comprising different modifications. Chemical modifications can alter oligonucleotide activity by, for example: increasing affinity of an antisense oligonucleotide for its target nucleic acid molecule, increasing nuclease resistance, altering the pharmacokinetics of the oligonucleotide and/or reducing toxicological effects.

Identifying a Natural Antisense Transcript

Some embodiments of the present invention describe an antagoNAT that targets a natural antisense transcript. In some embodiments, a scan of known gene databases, such as Genebank, is carried out to identify any potential naturally occurring antisense transcript. Certain scanning processes yield a non-coding RNA transcript within the intergenic region of a certain gene. In some embodiments, sequence identification and subsequent screening are used to identify single-stranded antisense oligonucleotides that inhibit expression of the certain gene.

Certain AntagoNATs

In certain embodiments, the antagoNAT of a composition described herein is an oligonucleotide that hybridizes with a natural antisense transcript. Certain oligonucleotides comprise modified nucleosides, unmodified nucleosides, and modified internucleoside linkages.

The compounds described herein according to some embodiments of this invention include one or more asymmetric center(s) and this gives rise to enantiomers, diastereomers, and other stereoisomeric configurations. The present invention includes all the enantiomers and diastereomers as well as mixtures thereof in any proportions. The invention also extends to isolated enantiomers or pairs of enantiomers. Methods of separating enantiomers and diastereomers are well known to persons skilled in the art.

Some embodiments of the present invention describe a composition comprising a pharmaceutically acceptable diluent or carrier and an antagoNAT, wherein the antagoNAT is 10 to 50 nucleoside subunits in length. In some embodiments, the antagoNAT is 10 to 45 nucleoside subunits in length, or 10 to 40 nucleoside subunits in length, or 10 to 35 nucleoside subunits in length, or 15 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT is 18 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT is 20 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT is 25 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT is 10 to 20 nucleoside subunits in length.

In other embodiments, the antagoNAT comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In further or additional embodiments, the antagoNAT comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between internal sugar unmodified nucleoside subunits. In some embodiments, the antagoNAT comprises an unmodified sugar nucleoside subunit at the 3′ terminus or an unmodified sugar nucleoside subunit at the 5′ terminus. In other embodiments, the antagoNAT comprises an unmodified sugar nucleoside subunit at the 3′ terminus and an unmodified sugar nucleoside subunit at the 5′ terminus. In other embodiments, the antagoNAT comprises a modified sugar nucleoside subunit at the 3′ terminus and an unmodified sugar nucleoside subunit at the 5′ terminus. In other embodiments, the antagoNAT comprises an unmodified sugar nucleoside subunit at the 3′ terminus and a modified sugar nucleoside subunit at the 5′ terminus.

In certain embodiments of the composition, there are no more than five internal unmodified nucleosides comprising 2′-deoxyribose sugars are consecutive, wherein (a) the 3′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside; or (b) the 3′ terminus segment comprises a non-bicyclic 2′-modified sugar nucleoside and the 5′ terminus segment comprises a bicyclic 2′-modified sugar nucleoside.

In some embodiments, the composition comprises sugar modified and sugar unmodified nucleoside subunits, wherein the sugar modified and sugar unmodified nucleoside subunits each comprise a pyrimidine base or purine base. In other embodiments, the internal sugar modified nucleoside subunits each comprise a pyrimidine base or purine base. In further or additional embodiments, the internal sugar modified nucleoside subunits each comprise a pyrimidine base. Natural or unmodified bases or heterocyclic bases include the purine bases adenine (A), and guanine (G), and the pyrimidine nucleobases thymine (T), cytosine (C), and uracil (U). Many modified nucleobases or nucleobase mimetics known to those skilled in the art are amenable with the compounds described herein. In addition, modified nucleobases or heterocyclic bases are optionally included such as 7-deazapurine, 5-methylcytosine, 2-aminoadenine, 5-bromouracil, or hypoxanthine. In specific embodiments, the internal sugar modified nucleoside subunits each independently comprise a pyrimidine base selected from uracil, thymine, or cytosine.

Any composition described herein comprises sugar modified nucleoside subunits that are substituted at the 2′ position with alkoxy, alkyl, halogen, amino, thiol, alkylamine, alkylthiol, alkylester, O-alkylene bound to the C4′ carbon, or combinations thereof. Many modified sugar nucleosides known to those skilled in the art are amenable with the compounds described herein. In some embodiments, the sugar modified nucleoside subunits are each substituted at the 2′ position with alkoxy, halogen, or O-alkylene bound to the C4′ carbon. Suitable substituents at the 2′ position include but are not limited to methoxy, fluoro, O-methoxyethyl, O-methylene bound to the C4′ carbon (2′-OCH2-4′), or O-ethylene bound to the C4′ carbon (2′-OCH2CH2-4′). In other embodiments, modified sugar subunits comprises one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C to CO alkyl or C2 to CO alkenyl and alkynyl. Particularly preferred are —O(CH2)nOCH3, —O[(CH2)nO]mCH3, —O(CH2)nNH2, —O(CH2)nCH3, —O(CH2)nONH2, and —O([(CH2)nON(CH2)nCH3)2 where n and m can be from 1 to about 10. Other oligonucleotides comprise one of the following at the 2′ position: C to CO, (lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures comprise, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514, 785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646, 265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference.

Any composition described herein comprises unmodified nucleoside subunit, wherein the sugar of the nucleoside is a ribose or 2′-deoxyribose sugar. In certain embodiments, the unmodified nucleoside subunits comprise ribose sugars. In other embodiments, the unmodified nucleoside subunits comprise 2′-deoxyribose sugars. In specific embodiments, the unmodified nucleoside subunits comprise ribose and 2′-deoxyribose sugars.

Any composition described herein comprises a backbone of phosphodiester, phosphotriester, phosphorothioate, phosphorodithiate, alkylphosphonate, phosphoramidate, boranophosphate, carbonate, carbamate, acetamidate, thioether, thioformacetal internucleotide linkages, or combinations thereof. In other embodiments, the antagoNAT comprises a backbone of phosphodiester and phosphorothioate internucleotide linkages. In specific embodiments, the antagoNAT comprises a backbone of phosphorothioate internucleotide linkages.

Some embodiments of the present invention describe an antagoNAT of Formula (I), or a salt thereof:

C-Au-[Bv-A′w]x-By-A″z-C   Formula (I)

wherein:

    • each A, A′, and A″ independently has the structure of:

    • each B independently has the structure of:

    • each C is independently hydroxy, phosphate, substituted or unsubstituted alkoxy, or any suitable 5′ or 3′ terminus cap;
    • each u, v, w, x, y and z are independently integers greater than or equal to one;
    • each D is a heterocyclic base;
    • each E is independently selected from the group consisting of substituted or unsubstituted alkyl, substituted or unsubstituted alkoxy, substituted or unsubstituted amine, halogen, substituted or unsubstituted aminoalkoxy, substituted or unsubstituted alkenyl, or thiol;
    • each G is independently —OP(O)2O—, —OP(O)(OR)O—, —OP(O)(S)O—, —OP(O)(SR)O—, —OP(S)2O—, —OP(R)(O)O—, —OP(NR2)(O)O—, —OC(O)O—, —OCH2C(O)NHCH2—, —OCH2S—, —CH2SCH2—, —OP(O)(BH3)O—, —NP(O)2O—, —OP(R)(O)O—, or absent when (Ia) is connected to C;
      • each R is independently hydrogen or substituted or unsubstituted alkyl;
    • each J is hydrogen or J and E taken together form a ring structure that optionally includes an additional heteroatom selected from N or O; and
    • each K is independently hydroxy or hydrogen.

In some embodiments, any antagoNAT described herein comprises a heterocyclic base that is independently selected from a purine or pyrimidine base. In other embodiments, each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, cytosine, 7-deazapurine, 2-aminoadenine, 5-methylcytosine, 5-bromouracil, or hypoxanthine. In certain specific embodiments, each heterocyclic base is independently selected from adenine, guanine, uracil, thymine, or cytosine. In other specific embodiments, the heterocyclic base of each A′ is independently selected from uracil, thymine, or cytosine.

In some embodiments, an antagoNAT is described, wherein each A, A′, or A″ independently has the structure of:

In some preferred compounds of the invention, each E is independently methoxy, ethoxy, O-methylethyl, or fluoro. In specific embodiments, each E is methoxy. In certain specific embodiments, each E is O-methylethyl.

In some preferred antagoNATs of the invention, each G is independently —OP(O)2O—, —OP(O)(OR)O—, or —OP(O)(S)O—. In specific embodiments, each G is —OP(O)(S)O—. In other embodiments, G is a combination of —OP(O)2O— and —OP(O)(S)O—.

In some preferred antagoNATs of the invention, each C is hydroxy or a suitable terminus cap structure.

In certain preferred antagoNATs of the invention, v and y are independently integers of 1, 2, or 3 when K is hydroxy and x is at least one. In other embodiments, v and y are independently integers of 1, 2, 3, 4, or 5 when K is hydrogen, and (a) wherein at least one A has the structure of (Id) or (Ie) and at least one A″ has the structure of (Ic); or (b) wherein at least one A has the structure of (Ic) and at least one A″ has the structure of (Id) or (Ie).

Complementarity

It is understood in the art that incorporation of nucleotide affinity modification may allow for a greater number of mismatches compared to an unmodified compound. Similarly, antagoNAT sequences may be more tolerant to mismatches than other oligonucleotide sequences. In some embodiments, the antagoNAT hybridizes with a natural antisense transcript of a gene.

Any antagoNAT or compound described herein is at least about 50% complementary to the preselected natural antisense transcript. In certain embodiments, the antagoNATs of the present invention comprise at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence complementarity to a target region within the target nucleic acid sequence to which they are targeted. For example, an antagoNAT in which 18 of 20 nucleotides of the compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. As such, an antagoNAT which is 18 nucleotides in length having four noncomplementary nucleotides which are flanked by two regions of complete complementarity with the target nucleic acid molecule would have 77.8% overall complementarity with the target nucleic acid molecule and would thus fall within the scope of the present invention. Percent complementarity of an antagoNAT with a region of a target nucleic acid molecule can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., (1990) J. Mol. Biol., 215, 403-410; Zhang and Madden, (1997) Genome Res., 7, 649-656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., (1981) 2, 482-489).

Selection of appropriate target nucleic acid molecules is facilitated by using computer programs that automatically align nucleic acid sequences and indicate regions of identity or homology. Such programs are used to compare nucleic acid sequences obtained, for example, by searching databases such as GenBank or by sequencing PCR products. Comparison of nucleic acid sequences from a range of species allows the selection of nucleic acid sequences that display an appropriate degree of identity between species. In the case of genes that have not been sequenced, Southern blots are performed to allow a determination of the degree of identity between genes in target species and other species. By performing Southern blots at varying degrees of stringency, as is well known in the art, it is possible to obtain an approximate measure of identity. These procedures allow the selection of target nucleic acid molecules that exhibit a high degree of complementarity to target nucleic acid sequences in a subject to be controlled and a lower degree of complementarity to corresponding nucleic acid sequences in other species. One skilled in the art will realize that there is considerable latitude in selecting appropriate regions of genes for use in the present invention.

Methods

In certain embodiments of the present invention, provided herein is a method for modulating expression of a gene in a cell. In some embodiments, the method includes contacting the cells with an antagoNAT, wherein the antagoNAT is 10 to 30 nucleoside subunits in length. In some embodiments, the antagoNAT specifically hybridizes with a natural antisense transcript of the gene. In other embodiments, the antagoNAT includes at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In some embodiments, the antagoNAT further comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal nucleoside is modified. In some embodiments, the antagoNAT additionally includes internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal ribonucleosides are consecutive and at least one internal modified nucleoside is present between internal sugar unmodified nucleoside subunits.

In some embodiments, any method of modulating gene expression described herein further comprises forming a hybrid comprising the antagoNAT and the natural antisense transcript, wherein the hybrid is not a substrate for ribonuclease cleavage. In certain embodiments, the method comprises sterically blocking the normal function of the natural antisense transcript, thereby modulating the function of the gene. In certain embodiments, the antagoNATs of the present invention comprise at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence complementarity to the natural antisense transcript. In other embodiments, expression of the gene is up-regulated in the cell with respect to a control cell. In certain embodiments, expression of the gene is down-regulated in the cell with respect to a control cell.

In some embodiments, the type of cell contacted with an antagoNAT according to a method described herein is a mammalian cell.

Further in accordance with certain embodiments of the present invention, there is provided a method of modulating function of a polynucleotide in a cell comprising contacting the cell with an antagoNAT. In some embodiments, the antagoNAT is 10 to 30 nucleoside subunits in length. In other embodiments, the antagoNAT hybridizes with the polynucleotide. In specific embodiments, the antagoNAT comprises at least one sugar modified nucleoside subunit at the 3′ terminus and at least one sugar modified nucleoside subunit at the 5′ terminus. In further or additional embodiments, the antagoNAT comprises internal sugar modified nucleoside subunits and internal sugar unmodified nucleoside subunits between the 5′ nucleoside subunit and the 3′ nucleoside subunit, wherein no more than three internal nucleosides comprising ribose sugars are consecutive and at least one internal modified nucleoside is present between the internal sugar unmodified nucleoside subunits.

In some embodiments, the polynucleotide targeted according to a method described herein is a natural antisense strand to a sense strand. In certain embodiments, the antagoNATs of the present invention comprise at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence complementarity to the polynucleotide.

In some embodiments, any method of modulating function of a polynucleotide described herein further comprises forming a hybrid comprising the antagoNAT and the polynucleotide, thereby modulating said function of said polynucleotide. In certain embodiments, the resultant hybrid is not a substrate for ribonuclease cleavage.

In some embodiments, the method comprises sterically blocking the normal function of the polynucleotide. In certain embodiments, expression of the sense strand is elevated in the cell with respect to a control. In other embodiments, expression of the sense strand is decreased in the cell with respect to a control.

In some embodiments, the type of cell contacted with an antagoNAT according to a method described herein is a mammalian cell.

The regulation of gene expression by targeting a natural antisense transcript has been described, e.g., in U. S. Pat. App. Pub. No. 2009/0258925, “Natural Antisense and Non-coding RNA Transcripts as Drug Targets”, incorporated herein by reference in its entirety. This publication reports targeting natural antisense transcripts to up-regulate and down-regulate sense transcripts, which can be coding or noncoding. Natural antisense targeting is also described in, e.g.: U. S. Pat. App. Pub. No. 2010/0105760, “Treatment of Apolipoprotein-A1 Related Diseases by Inhibition of Natural Antisense Transcript to Apolipoprotein-A1”; WO 2010/065671, “Treatment of Vascular Endothelial Growth Factor (VEGF) Related Diseases by Inhibition of Natural Antisense Transcript to VEGF”; WO 2010/065662, “Treatment of Sirtuin 1 (SIRT1) Related Disease by Inhibition of Natural Antisense Transcript to Sirtuin 1”; WO 2010/102058, “Treatment of Sirtuin 1 (SIRT1) Related Disease by Inhibition of Natural Antisense Transcript to Sirtuin 1”; WO 2010/065792, “Treatment of Erythropoietin (EPO) Related Diseases by Inhibition of Natural Antisense Transcript to EPO”; WO 2010/065787, “Treatment of Tumor Suppressor Gene Related Diseases by Inhibition of Natural Antisense Transcript to the Gene”; WO 2010/093904, “Treatment of Brain Derived Neurotrophic Factor (BDNF) Related Diseases by Inhibition of Natural Antisense Transcript to BDNF”, and; WO 2010/093906 GDNF, “Treatment of Glial Cell Derived Neurotrophic Factor (GDNF) Related Diseases by Inhibition of Natural Antisense Transcript to GDNF”, all incorporated herein by reference.

While various embodiments of the present invention have been described above, it should be understood that they have been presented by way of example only, and not limitation. Numerous changes to the disclosed embodiments can be made in accordance with the disclosure herein without departing from the spirit or scope of the invention. Thus, the breadth and scope of the present invention should not be limited by any of the above described embodiments.

All documents mentioned herein are incorporated herein by reference. All publications and patent documents cited in this application are incorporated by reference for all purposes to the same extent as if each individual publication or patent document were so individually denoted. By their citation of various references in this document, Applicants do not admit any particular reference is “prior art” to their invention. Embodiments of inventive compositions and methods are illustrated in the following examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selected embodiments of the invention. It will be appreciated that variations in proportions and alternatives in elements of the components shown will be apparent to those skilled in the art and are within the scope of embodiments of the present invention.

General

The sequences listed in the examples have been annotated to indicate the location and type of nucleoside and internucleoside linkage modifications. All the nucleosides that are not annotated in the examples are β-D-deoxyribonucleosides. Each modified nucleoside is preceded by a letter or symbol. In particular, “m” indicates a 2′-O-methyl group and “+” indicates a LNA or bicyclic nucleoside. The symbol “*” indicates a phosphorothioate internucleoside linkage.

Materials

RAW264.7 macrophage cells were purchased from ATCC and cultured in an Eagle Minimum Essential Medium supplemented with 10% FBS and 5% penicillin/streptomycin. Primary astrocytes were purchased from Sciencell Research Laboratories and cultured in Sciencell Astrocyte medium supplemented with 5% FBS and 2% astrocyte growth medium. HepG2 cells were grown in EMEM (ATCC cat #2003)+10% FBS. NIH 3T3 cells from ATCC were grown in DMEM (Mediatech cat#10-0 1 3-CV)+10% FCS (Mediatech cat #35-022-CV). 518A2 cells were grown in DMEM+5% FBS.

Small scale batches of antagoNATs for screening were manufactured by IDT Inc. (Coralville, Iowa). The oligonucleotides were applied to cells seeded in 6 well plates dropwise in OptiMEM+Lipofectamine mixture at the final concentration of 20 nM unless noted otherwise. After about 18 h incubation the media was replaced and the incubation continued for another 18-24 h when the cells were harvested for RNA isolation.

Example 1: Amidites for Oligonucleotide/Oligonucleoside Synthesis

2′-O-Methyl nucleoside amidites and 2′-OH nucleoside amidites are available from Glen Research, Sterling, Va. Other 2′-O-alkyl substituted nucleoside amidites are prepared as is described in U.S. Pat. Nos. 5,506,351, 5,466,786, or 5,514,786, herein incorporated by reference.

Example 2: Synthesis of 2′-O-Methyl Nucleoside Amidites

i. 2′-O-Methyl-5-methyluridine

2,2′-anhydro-5-methyluridine (10.0 g, 0.0416 mol) is dissolved in methanol (80 mL) in a stainless steel bomb (100 mL capacity). Trimethyl borate is generated by adding solutions (1 M in THF) of borane to methanol and allowing the resulting hydrogen gas to evolve. Trimethyl borate (5.6 mL, 0.049 mol) is added. The bomb is sealed and placed in an oil bath at 150° C. which generates pressure. After 40 h, the bomb is cooled in ice, opened and the contents concentrated under reduced pressure.

ii. 5′-O-Dimethoxytriphenylmethyl-2′-O-methyl-5-methyluridine

2′-O-methyl-5-methyluridine (12 g) is co-evaporated in pyridine (2×50 mL) and dissolved in dry pyridine (50 mL). Dimethoxytriphenylmethyl chloride (18.1 g, 0.054 mol) is added. The flask is covered and allowed to stand for 45 min at room temperature. The reaction mixture is treated with methanol (10 mL) and the resultant solution is concentrated under reduced pressure. The residue is partitioned between ethyl acetate (2×400 mL) and saturated sodium bicarbonate solution (500 mL). The organic layers are combined, dried (sodium sulfate), filtered and concentrated.

Example 3: Synthesis of Oligonucleotide

Unsubstituted and substituted phosphodiester oligoribonucleotides are synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry with oxidation by iodine.

Phosphorothioate oligonucleotides are synthesized as per the phosphodiester oligonucleotides except the standard oxidation reagent is replaced by 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1,-dioxide in acetonitrile for the stepwise thiation of the phosphite linkage. The thiation wait step is increased to 68 seconds and is followed by the capping step. After cleavage from the column and deblocking in concentrated ammonium hydroxide at 55° C. (18 h), the oligonucleotides are purified by precipitating twice with 2.5 volumes of ethanol from a 0.5 M NaCl solution. Analytical gel electrophoresis is accomplished in 20% acrylamide, 8 M urea, 454 mM Tris-borate buffer, pH 7.

Example 4: Chimeric Phosphorothioate Oligonucleotides

Chimeric oligoribonucleotides having 2′-O-alkyl phophorothioate and 2′-H phosphorothioate oligonucleotide segments are synthesized using an automated DNA synthesizer. Oligonucleotides are synthesized using the automated synthesizer and 5′-dimethoxytrityl-3′-O-phosphoramidite for the unmodified subunits and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for the 3′ terminus and 5′ terminus in addition to internal modified subunits. The standard synthesis cycle is modified by increasing the wait step after the delivery of tetrazole and base. The protecting groups on the exocyclic amines are phenoxyacetyl for adenine and guanine, benzoyl for cytosine, 2′-O-methyl adenine, and 2′-O-methyl cytosine, and isobutyryl for 2′-O-methyl guanine. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Addition of methanolic ammonia at room temperature affords deprotected bases. The resultant is lyophilized to dryness and desalted on a size exclusion column.

Chimeric oligoribonucleotides having 2′-O-alkyl phophorothioate and 2′-OH phosphorothioate oligonucleotide segments are synthesized using an automated DNA synthesizer. Oligonucleotides are synthesized using the automated synthesizer and 5′-dimethoxytrityl-2′-tert-butyldimethylsilyl-3′-O-phosphoramidite for the unmodified subunits and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for the 3′ terminus and 5′ terminus in addition to internal modified subunits. The standard synthesis cycle is modified. The protecting groups on the exocyclic amines are phenoxyacetyl for adenine and guanine, benzoyl for cytosine, 2′-O-methyl adenine, and 2′-O-methyl cytosine, and isobutyryl for 2′-O-methyl guanine. The fully protected oligonucleotide is cleaved from the support and the phosphate group is deprotected in 3:1 ammonia/ethanol at room temperature overnight then lyophilized to dryness. Addition of methanolic ammonia at room temperature affords deprotected bases. Treatment with 1M TBAF in THF for 24 hours at room temperature deprotects the 2′-OH groups. The resultant is lyophilized to dryness and desalted on a size exclusion column.

Example 5: Identifying a Natural Antisense Transcript for ABCA1

In order to identify any potential naturally occurring ABCA1 antisense transcripts, a scan of known gene databases, such as UCSC genome browser and Genebank, was carried out. This yielded a non-coding RNA transcript, approximately 1 Kb in length, within the intergenic region of the ABCA1 gene, which was shown to be transcribed in the opposite, 5′ to 3′ direction (BF133827). Based on this sequence and subsequent, three single-stranded antisense oligonucleotides were demonstrated to be particularly effective in inhibiting expression of this transcript. The BF133827 sequence, containing the specific areas targeted by these antisense oligonucleotides, is presented in FIG. 1.

Example 6: In Vitro Screening of Antisense Oligonucleotides Targeting ABCA1 Antisense Transcript

The effects of antisense oligonucleotides on ABCA1 expression were examined using a RAW264.7 mouse leukaemic macrophage cell line. FIG. 2 depicts the relative expression levels of ABCA1 mRNA observed 48 hours after transfection with each antisense oligonucleotide (50 nM). Of the three active antisense oligonucleotide sequences tested, quantitative RT-PCR analysis revealed a substantial increase of ABCA1 mRNA expression in cells transfected with both CUR1090 and CUR1091, compared to those treated with the control oligonucleotide (CUR586), with the most significant effect observed for CUR1090 (p=0.0004; 1-way ANOVA; n=4). To examine whether this up-regulation of mRNA expression translated to a corresponding increase in ABCA1 protein levels within these cells, western blot analysis was next carried out. After 48 hours following transfection with each antisense oligonucleotide, total cell protein was separated using SDS-PAGE and probed with a commercially available ABCA1 polyclonal antibody. This analysis revealed a band at the expected molecular weight of 220 kDa (FIG. 3a). Densitometric analysis confirmed a three-fold increase of ABCA1 expression in cells treated with CUR1090 compared to those treated with CUR586 (p=0.0024; 1-way ANOVA; n=3) (FIG. 3b).

Real-Time PCR Analysis of ABCA1 mRNA Expression

Total RNA was extracted from cell and tissue samples using Qiagen RNeasy columns. cDNA was prepared from 800 ng of Dnase-treated RNA using a Taqman Reverse Transcription Kit (Applied Biosystems). cDNA from each sample was amplified using a Taqman gene expression assay for mouse ABCA1 (Applied Biosystems, CA, USA). The relative differences between Ct values for ABCA1 and a reference gene (18s RNA) were calculated as ΔΔCt. All real-time PCR was carried out using the 7900HT Fast Real-Time PCR System (Applied Biosystems, CA, USA).

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis and Immunoblotting

Whole-cell and tissue protein was extracted using M-PER protein extraction reagent (Thermo Scientific) and total protein concentrations were determined for each sample using a BCA Protein Assay Kit (Pierce). Protein samples of equal concentrations were separated on a polyacrylamide Criterion gel and electrophoretically transferred to PVDF membranes. The PVDF membrane was blocked using 5% milk, diluted in 1× Tris.-Buffered Saline (containing 2 mM Tris, 500 nM sodium chloride, pH 7.5) for 1 h at room temperature and incubated overnight at 4° C. with a polyclonal rabbit anti-ABCA1 primary antibody (Novus), diluted 1:1000 in blocking buffer. The next day, the PVDF membrane was incubated for 1 h at room temperature with an anti-rabbit, horseradish peroxidase-linked secondary antibody (Cell Signalling), diluted 1:2000 in blocking buffer washed with TBS-T buffer. Protein bands were visualized using chemiluminescence peroxidase substrate and exposed to X-ray film. The X-ray films were scanned and quantitative densitometry of the electrophoretic bands was performed. PVDF membranes were also probed with a primary mouse anti-actin antibody. ABCA1 protein expression was determined by dividing ABCA1 densitometry values by those obtained for the actin loading control.

Example 7: Immunohistochemical Analysis of Macrophage ABCA1 Protein

To further characterize the increase in ABCA1 expression, the cellular distribution of ABCA1 was analyzed using immunohistochemistry. Oligonucleotide-treated RAW264.7 cells were fixed 48 hours after transfection and incubated overnight with a primary ABCA1 polyclonal antibody followed by an Alexa Fluor 488-conjugated secondary antibody. FIG. 4 demonstrates the relative expression levels of ABCA1 in cells treated with each sequence. As can be seen, a significant increase in fluorescent activity was detected in cells transfected with CUR1090, compared with control. Furthermore, in line with its important cellular function, the majority of this ABCA1 expression was evident close to or along the macrophage cell membrane.

Immunostaining of ABCA1 in RAW264.7 Cells

At 48 hours post-transfection, cells were fixed in culture with paraformaldehyde (4%) for 20 minutes, followed by 2-3 washes in PBS. The cells were then permeabilized with ethanol (95):acetic acid (5) for 20 minutes at −20° C., followed by 2-3 washes in PBS (5% FBS). A polyclonal rabbit anti-ABCA1 primary antibody (Novus), diluted 1:1000 in PBS (5% FBS), was added and samples were maintained overnight at 4° C. The next day, cell samples were blocked with PBS (5% FBS) for 20 minutes, followed by a 40 minute incubation at room temperature with an Alexa Fluor 488-conjugated secondary antibody. Following 2-3 washes with PBS, the coverslip was mounted using aqueous mounting medium, containing antifade. Nuclear staining was carried out using Hoechst blue, diluted in PBS (5% FBS). Fluorescence was analyzed by con-focal microscopy.

Example 8: In Vitro Screening of Antisense Oligonucleotides Targeting ABCA1 Antisense Transcript

The effects of antisense oligonucleotides on ABCA1 expression were assessed using primary astrocytes. As can be seen in FIG. 5, quantitative real-time PCR analysis demonstrated a statistically significant up-regulation of ABCA1 mRNA expression in primary astrocytes transfected with CUR 1090 and CUR1091 compared to those treated with the control oligonucleotide (p=0.0003; 1-way ANOVA; n=3). A three-fold increase in ABCA1 transcription was observed for cells treated with CUR1090. In addition, western blot analysis confirmed a corresponding four-fold increase in protein levels within these cells (p=0.0007; 1-way ANOVA; n=3) (FIGS. 6a and 6b).

Example 9: AntagoNATs Targeting ABCA1 Antisense Transcript

CUR1090 was selected for further chemical modification. Two different 2′-O-methyl modified versions of CUR1090, based on CURNA's antagoNAT construct were synthesized, and tested in macrophage RAW264.7 cells. The sequences of these modified oligonucleotides are presented in Table 1. As control samples, RAW264.7 cells were also transfected with scrambled versions of both the original and modified active oligonucleotides (CUR 1461 and CUR1575).


TABLE 1
Sequence
Sequence ID
Name
Sequence
Sequences of 2′-Unmodified and 2′-Modified ABCA1-AS Antisense Oligonucleotides
SEQ ID NO: 13
CUR-1087
C*A*T*G*T*C*T*C*C*T*G*C*C*T*T*T*C*C*T*G*T
SEQ ID NO: 14
CUR-1090
G*G*A*C*A*G*G*G*T*A*G*C*A*A*C*G*C*C*A*T*T
SEQ ID NO: 15
CUR-1091
C*C*A*C*C*T*C*A*G*T*T*G*C*A*C*G*G*A*A
Chemically Modified Oligonucleotide Sequences
SEQ ID NO: 16
CUR-1457
mG*mG*mA*mC*mA*G*G*G*T*A*G*C*A*A*C*G*mC*mC*mA*mU*mU
SEQ ID NO: 17
CUR-1463
mG*mG*mA*C*A*G*G*G*mU*A*G*mC*A*A*mC*G*mC*C*mA*mU*mU
Control Oligonucleotide Sequences
SEQ ID NO: 18
CUR-586
C*T*G*A*C*T* A*C*C*T*C*T*T*G*A
SEQ ID NO: 19
CUR-1458
mA*mC*mC*mA*mU*G*G*T*G*C*G*C*G*A*A*A*mU*mG*mG*mC*mA
SEQ ID NO: 20
CUR-1461
A*C*C*A*T*G*G*T*G*C*G*C*G*A*A*A*T*G*G*C*A
SEQ ID NO: 21
CUR-1575
mA*mC*mC*A*mU*G*G*mU*G*C*G*C*mG*A*A*A*mU*G*mG*mC*mA

Example 10: In Vitro Screening of AntagoNAT Targeting ABCA1 Antisense Transcript in Macrophages

Treatment of macrophages with both CUR1575 and CUR1463 led to an increase in ABCA1 protein levels relative to the two scrambled sequence control samples (FIG. 7). Importantly, a further increase in ABCA1 protein expression was observed in cells transfected with CUR1463, compared to those treated with the unmodified, active CUR1090, indicating that the 2′-O-methyl antagoNAT modifications of this oligonucleotide increased its efficacy for up-regulating ABCA1 at the protein level. FIG. 7 shows ABCA1 protein expression in macrophages, at 48 hours following treatment with chemically modified ABCA1-AS antisense oligonucleotides.

Example 11: In Vitro Screening of AntagoNAT Targeting ABCA1 Antisense Transcript in Mouse NIH 3T3 Cells

Treatment of mouse NIH 3T3 cells with CUR1090, CUR1575 and CUR1463 led to increased ABCA1 mRNA levels in mouse NIH 3T3 cells compared to scrambled sequence control sample as assessed by RT-PCR analysis (FIG. 8). The antagoNAT CUR1463 with additional 2′-O-methyl modifications showed much lower toxicity than the same oligonucleotide sequence with phosphorothioate backbone modifications (CUR-1090).

Treatment of mouse NIH 3T3 cells with CUR1090, CUR1457 and CUR1463 led to an increase in ABCA1 protein levels relative to the two scrambled sequence control samples as assessed by Western immunoblot analysis (FIG. 9). The antagoNAT CUR1463 with internal sugar 2′-O-methyl modifications shows a greater increase in ABCA1 protein than CUR1457 which have 2′-O-methyl modifications only on each end.

Example 12: In-Vivo Testing of Active Antisense Oligonucleotide CUR1463 on ABCA1 Expression

In Vivo Administration of ABCA1-AS Antisense Oligonucleotides

In vivo studies were carried out using adult, four-month-old male C57BL6 mice. CUR1463 and CUR1575 were diluted in sterile PBS, and injecting intraperitoneally at a concentration of 5 or 50 mg/kg, in a final volume of 100 cc. Separate groups of mice were treated with either CUR1463 (5 or 50 mg/kg), CUR1575 (5 or 50 mg/kg) or a saline vehicle control twice a week, for four weeks. These mice were then sacrificed 24 hours after the final injection, and various peripheral organs, isolated brain regions, and serum samples were collected.

Analysis of mRNA Expression

ABCA1 has been found to play a key role in liver cholesterol homeostasis and studies using transgenic mice have suggested hepatic ABCA1 expression as a major source of HDL cholesterol in plasma. For this reason, ABCA1 mRNA expression was analyzed from liver samples collected from each treatment group. In line with previous in vitro results, treatment of mice with 5 mg/kg of CUR1463 led to a statistically significant increase in liver ABCA1 mRNA expression, compared to treatment with either saline or an equivalent dose of the CUR1575 control (p=0.0075; 1-way ANOVA; 4≤n≤9) (FIG. 10). This experiment was repeated, with a separate cohort of animals receiving 5 mg/kg CUR1463 twice a week for four weeks. On both occasions, treatment with CUR1463 led to a statistically significant increase in liver ABCA1 levels, relative to a control. In addition to inducing an increase in mRNA expression, this 5 mg/kg dose of CUR1463 also showed an increase in ABCA1 protein expression within the liver, compared to vehicle or scrambled oligonucleotide controls (p=0.0459; 1-way ANOVA; 5≤n≤6) (FIG. 11).

Quantification of HDL and LDL Cholesterol

To examine the functional significance of this increase in hepatic ABCA1 expression, total serum cholesterol, LDL cholesterol and HDL cholesterol levels were measured for all three treatment groups. Serum was isolated from blood samples by high-speed centrifugation for 5 minutes. Serum HDL and LDL cholesterol levels were determined using a HDL/LDL Cholesterol Quantification Kit (Biovision). HDL and LDL fractions were separated by adding a 2× Precipitation Buffer, followed by centrifugation. 50 μl of a reaction mixture (containing 44 μl cholesterol assay buffer, 2 μl cholesterol probe, 2 μl enzyme mix and 2 μl cholesterol esterase) was then added to each unknown sample, and all reactions were incubated, in the dark, for 1 hour at 37° C. Optical density was measured at 570 nm in a micro-titer plate reader. Serum HDL and LDL cholesterol levels were determined using a standard curve generated from samples of known cholesterol concentration.

As can be seen in FIGS. 12a and 12b, mice injected with 5 mg/kg CUR1463 showed not only a statistically significant reduction of total serum cholesterol, compared with either saline or the CUR1575 control (p=0.0281; 1-way ANOVA; n=5), but also a 50% reduction of serum LDL cholesterol (p=0.0175; 1-way ANOVA; 5≤n≤10). Furthermore, these mice also showed an increase in the ratio of HDL cholesterol to LDL cholesterol (p=0.0093; 1-way ANOVA; 5≤n≤10) (FIG. 13).

Quantification of Serum Triglyceride Content

Serum triglyceride levels were monitored using a Triglyceride assay kit (Cayman). This analysis was based on the enzymatic hydrolysis of the triglycerides by lipase to glycerol, the release of which could be measured, in the form of absorbance, by a coupled enzymatic reaction system. First, standards of known triglyceride concentrations were prepared from a provided triglyceride stock. 150 μl of a diluted enzyme buffer solution was added to 10 μl of each standard or unknown sample, followed by incubation for 15 minutes at room temperature. Absorbance was read at 540 nm. Triglyceride concentration (mg/dl) was calculated according to the manufacturer's recommendations.

Importantly, treatment with CUR1463, while lowering serum LDL cholesterol, did not decrease atheroprotective HDL levels, nor did it lead to any changes in serum triglyceride content (FIGS. 14a and 14b).

Measuring Serum Alanine Transaminase (ALT) Activity

ALT activity was determined using the Alanine Transaminase Activity Assay Kit (Cayman). This analysis was achieved by monitoring the rate of NADH oxidation in a coupled reaction system employing lactate dehydrogenase (LDH), which was accompanied by a decrease in absorbance at 340 nm. This decrease was directly proportional to ALT activity. 190 μl of a reaction mixture (containing 150 μl ALT substrate, 20 μl ALT cofactor and 20 μl of either test or positive control samples) was incubated for 15 minutes at 37° C., following which 20 μl of ALT indicator was added. Each reaction was read at 340 nm once every minute for a period of 5 minutes. ALT activity (U/ml) was calculated according to the manufacturer's recommendations.

To examine for any potential toxicity of these antisense oligonucleotides, serum alanine transaminase (ALT) activity was also measured for all treatment groups. Serum ALT activity, which is used as a way of screening for liver damage, is represented in FIG. 15. As demonstrated, treatment with the effective dose (5 mg/kg) of CUR1463 led to significantly less ALT activity than the higher, apparently more hepatotoxic dose (50 mg/kg). More importantly, no differences in toxicity were found between the 5 mg/kg effective dose and the saline control, indicating that the effective treatment dose had no adverse liver effects.

Example 13: In Vitro Screening of Antisense Oligonucleotides Targeting the Human ABCA1 Antisense Transcript

A human ABCA1 antisense transcript (AK311445) was identified using the UCSC genome browser. Initially two siRNAs were designed against this transcript to determine if this transcript regulates ABCA1 expression. siRNAs were transfected into human melanoma (518A2) cells at 20 nM. FIG. 16 shows that one of the siRNAs (CUR0521) increased ABCA1 mRNA expression by 3 fold compared to vehicle control (P=0.05). The siRNA CUR0519 also increased ABCA1 mRNA expression, but was not statistically significant (P=0.06).

Single stranded 2′-O-methyl modified antagoNATs were designed to target the human ABCA1 antisense transcript. The sequence of an antagoNAT and control oligonucleotides are presented in Table 2.


TABLE 2
Sequences of AntagoNAT and Control Oligonucleotides Targeted
to ABCA1-AS Antisense Oligonucleotides
SEQ ID NO: 22
CUR-1745
+T*+C*T*C*T*C*T*G*G*+G*+A*+C
SEQ ID NO: 23
CUR-1746
+T*+T*A*C*C*T*T*C*A*+T*+A*+C
SEQ ID NO: 24
CUR-1747
+A*+A*+T*C*A*C*T*T*A*G*C*C*+A*+C*+T
SEQ ID NO: 25
CUR-1716
mG*mC*mC*T*C*T*T*C*T*A*T*G*G*G*T*C*T*mG*mU*mC
SEQ ID NO: 26
CUR-1719
mA*mA*mU*C*A*A*mU*G*G*C*mU*G*T*T*mC*T*C*mU*C*U*C*mU*G*G
*mG*mA*mC

FIG. 17 shows that the ABCA1 mRNA expression is increased in human hepatocellular carcinoma (HepG2) cells treated with the antagoNAT CUR1719 compared to vehicle treated cells, as assessed by RT-PCR analysis of ABCA1 mRNA levels. 2′-Bicyclic modified gapmer configuration oligonucleotides (CUR1745 and CUR1747) and 2′-O-methyl gapmer configuration oligonucleotide (CUR1716) did not significantly elevate ABCA1 mRNA expression.

Human hepatocellular carcinoma (HepG2) cells were treated with the antagoNATs at 20 nM concentration and incubated for 48 hours. FIG. 18 shows that antagoNAT CUR1719 yielded a 1.6-fold increase in ABCA1 mRNA expression compared to vehicle control (P=0.05). A second chemically modified oligonucleotide (CUR-1716) also increased ABCA1 expression, but was not statistically significant (P=0.1).

Human epithelial colorectal adenocarcinoma (CaCo2) cells were treated with antagoNATs and incubated for 48 hours. FIGS. 19a and 19b shows the relative ABCA1 protein expression of these cells, as assessed by Western immunoblot analysis. Cells treated with 50 nM CUR1719 demonstrated a 2.5-fold increase in ABCA1 protein expression in these human cells (p=0.0053; 1-way ANOVA; n=3), compared to vehicle treated cells and treatment with a control oligonucleotide. A similar increase in protein levels was also seen following treatment with CUR1716, but this result failed to reach significance.

Example 14: In Vitro Screening of Antisense Oligonucleotides Targeting the Human SCN1A Antisense Transcript

The methods described for the design and analysis of ABCA1 antagoNATs were applied to the design and analysis of antagoNATs targeted to a human SCN1A antisense transcript. A human SCN1A antisense transcript was identified using the UCSC genome browser. Single stranded 2′-O-methyl modified antagoNATs were designed to target the human SCN1A antisense transcript. The sequences of antagoNATs and control oligonucleotides are presented in Table 3.


TABLE 3
Sequences of Chemically Modified Oligonucleotides Targeted
to SCN1A-AS Antisense Oligonucleotide
SEQ ID NO: 27
CUR-1763
+G*+T*G*G*T*A*+T*A*G*G*A*A*+C*+T*+G
SEQ ID NO: 28
CUR-1764
mG*mU*mG*G*mU*A*mU*A*G*G*A*A*mC*T*G*G*mC*A*mG*mC*mA
SEQ ID NO: 29
CUR-1770
mG*mC*mC*A*G*T*mC*A*C*A*A*A*mU*T*mC*A*G*A*mU*mC*mA

Human hepatocellular carcinoma (HepG2) cells were treated with antagoNATs targeted to the human SCN1A antisense transcript and incubated for 48 hours. FIG. 20 shows the relative SCN1A protein expression of these treated cells, as assessed by RT-PCR analysis of SCN1A mRNA levels. SCN1A mRNA expression is increased in HepG2 cells treated with the antagoNAT CUR-1764 compared to vehicle treated cells.

Example 15: In Vitro Screening of Antisense Oligonucleotides Targeting the Human SIRT1 Antisense Transcript

The methods described for the design and analysis of ABCA1 antagoNATs were applied to the design and analysis of antagoNATs targeted to a SIRT1 antisense transcript. A SIRT1 antisense transcript was identified using the UCSC genome browser. Single stranded 2′-O-methyl modified antagoNATs were designed to target the SIRT1 antisense transcript. The sequences of antagoNATs and control oligonucleotides are presented in Table 4.


TABLE 4
Sequences of AntagoNAT and Control Oligonucleotides Targeted
to SIRT1 Antisense Oligonucleotides
SEQ ID NO: 30
CUR-1099
+A*+C*C*C*T*C*C*T*T*C*C*T*+C*+C*+C
SEQ ID NO: 31
CUR-1654
mC*mA*mG*A*A*mU*T*T*mC*A*T*G*mG*mU*mA
SEQ ID NO: 32
CUR-1655
mA*mC*mA*G*G*mU*G*C*mU*C*A*G*mA*mA*mU
SEQ ID NO: 33
CUR-1656
mA*mC*mA*G*G*mU*G*C*T*mC*A*G*A*A*mU*T*T*mC*A*mU*G*mG
*mU*mA
SEQ ID NO: 34
CUR-1657
+C*+A*G*A*A*+T*T*T*+C*A*T*G*+G*+T*+A
SEQ ID NO: 35
CUR-1658
+A*+C*A*G*G*+T*G*C*+T*C*A*G*+A*+A*+T

Mouse NIH 3T3 cells were treated with antagoNATs targeted to a SIRT1 antisense transcript and incubated for 48 hours. FIG. 21 shows the relative SIRT1 protein expression of these treated cells, as assessed by RT-PCR analysis of SIRT1 mRNA levels.

Example 16: Upregulation of Mouse Sirt1 mRNA in NIH3T3 Cell Line by Treatment with Antisense Oligonucleotides Targeting Mouse Sirt1-Specific Natural Antisense Transcript

In this Example, antisense oligonucleotides of different chemistries targeting mouse Sirt1-specific natural antisense transcript were screened in NIH3T3 cell line at a final concentration of 20 nM. This is a mouse cell cell line. The data below confirms that upregulation of Sirt1 mRNA through modulation of the function of the mouse Sirt1-specific natural antisense transcript.

Materials and Methods

3T3 mouse embryonic fibroblast cells from ATCC (cat# CRL-1658) were grown in Growth Media (Dulbecco's Modified Eagle's Medium (Cellgrow 10-013-CV)+10% Fetal Calf Serum (Cellgrow 35-22-CV)+penicillin/streptomycin (Mediatech cat# MT30-002-CI)) at 37° C. and 5% CO2. The cells were treated with antisense oligonucleotides using the following method. The cells were replated at the density of approximately 105/well into 6 well plates in Growth Media, dosed with 20 nM antisense oligonucleotides and incubated at 37° C. and 5% CO2 overnight. Next day, the media in the 6 well plates was changed to fresh Growth Media (1.5 ml/well). All antisense oligonucleotides were manufactured by IDT Inc. (Coralville, Iowa) or Exiqon (Vedbaek, Denmark). The sequences for all oligonucleotides are listed in Table 5. Stock solutions of oligonucleotides were diluted to the concentration of 20 M in DNAse/RNAse-free sterile water. To dose one well, 2 μl of this solution was incubated with 400 μl of Opti-MEM media (Gibco cat#31985-070) and 4 μl of Lipofectamine 2000 (Invitrogen cat#11668019) at room temperature for 20 min and applied dropwise to one well of a 6 well plate with cells. Similar mixture including 2 μl of water instead of the oligonucleotide solution was used for the mock-transfected controls. Additionally an inactive oligonucleotide CUR-1462 at the same concentration was used as control. After about 18 h of incubation at 37° C. and 5% CO2 the media was changed to fresh Growth Media. Forty eight hours after addition of antisense oligonucleotides the media was removed and RNA was extracted from the cells using SV Total RNA Isolation System from Promega (cat # Z3105) following the manufacturers' instructions. Six hundred nanograms of purified total RNA was added to the reverse transcription reaction performed using SuperScript VILO cDNA Synthesis Kit from Invitrogen (cat#11754-250) as described in the manufacturer's protocol. The cDNA from this reverse transcription reaction was used to monitor gene expression by real time PCR using ABI Taqman Gene Expression Mix (cat#4369510) and primers/probes designed by ABI (assays Mm01168521_ml for mouse Sirt1). The following PCR cycle was used: 50° C. for 2 min, 95° C. for 10 min, 40 cycles of (95° C. for 15 seconds, 60° C. for 1 min) using StepOne Plus Real Time PCR system (Applied Biosystems). The assay for 18S was manufactured by ABI (cat#4319413E). Fold change in gene expression after treatment with antisense oligonucleotides was calculated based on the difference in 18S-normalized dCt values between treated and mock-transfected samples.


TABLE 5
Sequences of AntagoNAT and Control Oligonucleotides Targeted
to SIRT1 Antisense Oligonucleotides
SEQ ID NO: 30
CUR-1099
+A*+C*C*C*T*C*C*T*T*C*C*T*+C*+C*+C
SEQ ID NO: 31
CUR-1654
mC*mA*mG*A*A*mU*T*T*mC*A*T*G*mG*mU*mA
SEQ ID NO: 36
CUR-1578
mA*mC*mA*mG*mG*mU*G*C*T*C*A*G*A*A*T*T*T*C*mA*mU*mG*m
G*mU*mA
SEQ ID NO: 37
CUR-1748
+A*+C*A*G*G*T*G*C*T*C*A*G*+A*+A*+T
SEQ ID NO: 38
CUR-1749 
+A*+C*A*G*G*mU*G*C*T*mC*A*G*+A*+A*+T
SEQ ID NO: 39
CUR-1750
+C*+C*A*C*G*C*G*C*G*A*G*T*+A*+C*+A

Results:

Mouse Sirt1 mRNA levels in NIH3T3 cells after treatment with 20 nM of antisense oligonucleotides compared to mock-transfected control are shown in FIG. 22. As seen from the data some of the oligonucleotides (CUR-1099, CUR-1578, CUR-1748) when applied at 20 nM were active at upregulating the levels of mouse Sirt1 mRNA. Some of the oligonucleotides (CUR-1658, CUR-1749) designed against the mouse Sirt1 natural antisense sequence did not affect the Sirt1 mRNA levels in NIH3T3 cells. The mouse Sirt1 levels in NIH3T3 cells treated with an oligonucleotide with no homology to the mouse Sirt-1 natural antisense sequence but of similar LNA chemistry (CUR-1750) did not show any significant regulation.

Conclusions:

These differences are in agreement with literature data which indicates that binding of oligonucleotides may depend on the secondary and tertiary structures of the oligonuclotide's target sequence. The result with CUR-1750 confirms that the effects of CUR-1099, CUR-1578, CUR-1748 are specific and do not depend on the non-specific toxicity of these molecules.

<160> NUMBER OF SEQ ID NOS: 40

<210> SEQ ID NO: 1

<211> LENGTH: 10515

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 1

ggaggaggga gagcacaggc tttgaccgat agtaacctct gcgctcggtg cagccgaatc 60

tataaaagga actagtcccg gcaaaaaccc cgtaattgcg agcgagagtg agtggggccg 120

ggacccgcag agccgagccg acccttctct cccgggctgc ggcagggcag ggcggggagc 180

tccgcgcacc aacagagccg gttctcaggg cgctttgctc cttgtttttt ccccggttct 240

gttttctccc cttctccgga aggcttgtca aggggtagga gaaagagacg caaacacaaa 300

agtggaaaac agttaatgac cagccacggc gtccctgctg tgagctctgg ccgctgcctt 360

ccagggctcc cgagccacac gctgggggtg ctggctgagg gaacatggct tgttggcctc 420

agctgaggtt gctgctgtgg aagaacctca ctttcagaag aagacaaaca tgtcagctgc 480

tgctggaagt ggcctggcct ctatttatct tcctgatcct gatctctgtt cggctgagct 540

acccacccta tgaacaacat gaatgccatt ttccaaataa agccatgccc tctgcaggaa 600

cacttccttg ggttcagggg attatctgta atgccaacaa cccctgtttc cgttacccga 660

ctcctgggga ggctcccgga gttgttggaa actttaacaa atccattgtg gctcgcctgt 720

tctcagatgc tcggaggctt cttttataca gccagaaaga caccagcatg aaggacatgc 780

gcaaagttct gagaacatta cagcagatca agaaatccag ctcaaacttg aagcttcaag 840

atttcctggt ggacaatgaa accttctctg ggttcctgta tcacaacctc tctctcccaa 900

agtctactgt ggacaagatg ctgagggctg atgtcattct ccacaaggta tttttgcaag 960

gctaccagtt acatttgaca agtctgtgca atggatcaaa atcagaagag atgattcaac 1020

ttggtgacca agaagtttct gagctttgtg gcctaccaag ggagaaactg gctgcagcag 1080

agcgagtact tcgttccaac atggacatcc tgaagccaat cctgagaaca ctaaactcta 1140

catctccctt cccgagcaag gagctggctg aagccacaaa aacattgctg catagtcttg 1200

ggactctggc ccaggagctg ttcagcatga gaagctggag tgacatgcga caggaggtga 1260

tgtttctgac caatgtgaac agctccagct cctccaccca aatctaccag gctgtgtctc 1320

gtattgtctg cgggcatccc gagggagggg ggctgaagat caagtctctc aactggtatg 1380

aggacaacaa ctacaaagcc ctctttggag gcaatggcac tgaggaagat gctgaaacct 1440

tctatgacaa ctctacaact ccttactgca atgatttgat gaagaatttg gagtctagtc 1500

ctctttcccg cattatctgg aaagctctga agccgctgct cgttgggaag atcctgtata 1560

cacctgacac tccagccaca aggcaggtca tggctgaggt gaacaagacc ttccaggaac 1620

tggctgtgtt ccatgatctg gaaggcatgt gggaggaact cagccccaag atctggacct 1680

tcatggagaa cagccaagaa atggaccttg tccggatgct gttggacagc agggacaatg 1740

accacttttg ggaacagcag ttggatggct tagattggac agcccaagac atcgtggcgt 1800

ttttggccaa gcacccagag gatgtccagt ccagtaatgg ttctgtgtac acctggagag 1860

aagctttcaa cgagactaac caggcaatcc ggaccatatc tcgcttcatg gagtgtgtca 1920

acctgaacaa gctagaaccc atagcaacag aagtctggct catcaacaag tccatggagc 1980

tgctggatga gaggaagttc tgggctggta ttgtgttcac tggaattact ccaggcagca 2040

ttgagctgcc ccatcatgtc aagtacaaga tccgaatgga cattgacaat gtggagagga 2100

caaataaaat caaggatggg tactgggacc ctggtcctcg agctgacccc tttgaggaca 2160

tgcggtacgt ctgggggggc ttcgcctact tgcaggatgt ggtggagcag gcaatcatca 2220

gggtgctgac gggcaccgag aagaaaactg gtgtctatat gcaacagatg ccctatccct 2280

gttacgttga tgacatcttt ctgcgggtga tgagccggtc aatgcccctc ttcatgacgc 2340

tggcctggat ttactcagtg gctgtgatca tcaagggcat cgtgtatgag aaggaggcac 2400

ggctgaaaga gaccatgcgg atcatgggcc tggacaacag catcctctgg tttagctggt 2460

tcattagtag cctcattcct cttcttgtga gcgctggcct gctagtggtc atcctgaagt 2520

taggaaacct gctgccctac agtgatccca gcgtggtgtt tgtcttcctg tccgtgtttg 2580

ctgtggtgac aatcctgcag tgcttcctga ttagcacact cttctccaga gccaacctgg 2640

cagcagcctg tgggggcatc atctacttca cgctgtacct gccctacgtc ctgtgtgtgg 2700

catggcagga ctacgtgggc ttcacactca agatcttcgc tagcctgctg tctcctgtgg 2760

cttttgggtt tggctgtgag tactttgccc tttttgagga gcagggcatt ggagtgcagt 2820

gggacaacct gtttgagagt cctgtggagg aagatggctt caatctcacc acttcggtct 2880

ccatgatgct gtttgacacc ttcctctatg gggtgatgac ctggtacatt gaggctgtct 2940

ttccaggcca gtacggaatt cccaggccct ggtattttcc ttgcaccaag tcctactggt 3000

ttggcgagga aagtgatgag aagagccacc ctggttccaa ccagaagaga atatcagaaa 3060

tctgcatgga ggaggaaccc acccacttga agctgggcgt gtccattcag aacctggtaa 3120

aagtctaccg agatgggatg aaggtggctg tcgatggcct ggcactgaat ttttatgagg 3180

gccagatcac ctccttcctg ggccacaatg gagcggggaa gacgaccacc atgtcaatcc 3240

tgaccgggtt gttccccccg acctcgggca ccgcctacat cctgggaaaa gacattcgct 3300

ctgagatgag caccatccgg cagaacctgg gggtctgtcc ccagcataac gtgctgtttg 3360

acatgctgac tgtcgaagaa cacatctggt tctatgcccg cttgaaaggg ctctctgaga 3420

agcacgtgaa ggcggagatg gagcagatgg ccctggatgt tggtttgcca tcaagcaagc 3480

tgaaaagcaa aacaagccag ctgtcaggtg gaatgcagag aaagctatct gtggccttgg 3540

cctttgtcgg gggatctaag gttgtcattc tggatgaacc cacagctggt gtggaccctt 3600

actcccgcag gggaatatgg gagctgctgc tgaaataccg acaaggccgc accattattc 3660

tctctacaca ccacatggat gaagcggacg tcctggggga caggattgcc atcatctccc 3720

atgggaagct gtgctgtgtg ggctcctccc tgtttctgaa gaaccagctg ggaacaggct 3780

actacctgac cttggtcaag aaagatgtgg aatcctccct cagttcctgc agaaacagta 3840

gtagcactgt gtcatacctg aaaaaggagg acagtgtttc tcagagcagt tctgatgctg 3900

gcctgggcag cgaccatgag agtgacacgc tgaccatcga tgtctctgct atctccaacc 3960

tcatcaggaa gcatgtgtct gaagcccggc tggtggaaga catagggcat gagctgacct 4020

atgtgctgcc atatgaagct gctaaggagg gagcctttgt ggaactcttt catgagattg 4080

atgaccggct ctcagacctg ggcatttcta gttatggcat ctcagagacg accctggaag 4140

aaatattcct caaggtggcc gaagagagtg gggtggatgc tgagacctca gatggtacct 4200

tgccagcaag acgaaacagg cgggccttcg gggacaagca gagctgtctt cgcccgttca 4260

ctgaagatga tgctgctgat ccaaatgatt ctgacataga cccagaatcc agagagacag 4320

acttgctcag tgggatggat ggcaaagggt cctaccaggt gaaaggctgg aaacttacac 4380

agcaacagtt tgtggccctt ttgtggaaga gactgctaat tgccagacgg agtcggaaag 4440

gattttttgc tcagattgtc ttgccagctg tgtttgtctg cattgccctt gtgttcagcc 4500

tgatcgtgcc accctttggc aagtacccca gcctggaact tcagccctgg atgtacaacg 4560

aacagtacac atttgtcagc aatgatgctc ctgaggacac gggaaccctg gaactcttaa 4620

acgccctcac caaagaccct ggcttcggga cccgctgtat ggaaggaaac ccaatcccag 4680

acacgccctg ccaggcaggg gaggaagagt ggaccactgc cccagttccc cagaccatca 4740

tggacctctt ccagaatggg aactggacaa tgcagaaccc ttcacctgca tgccagtgta 4800

gcagcgacaa aatcaagaag atgctgcctg tgtgtccccc aggggcaggg gggctgcctc 4860

ctccacaaag aaaacaaaac actgcagata tccttcagga cctgacagga agaaacattt 4920

cggattatct ggtgaagacg tatgtgcaga tcatagccaa aagcttaaag aacaagatct 4980

gggtgaatga gtttaggtat ggcggctttt ccctgggtgt cagtaatact caagcacttc 5040

ctccgagtca agaagttaat gatgccatca aacaaatgaa gaaacaccta aagctggcca 5100

aggacagttc tgcagatcga tttctcaaca gcttgggaag atttatgaca ggactggaca 5160

ccaaaaataa tgtcaaggtg tggttcaata acaagggctg gcatgcaatc agctctttcc 5220

tgaatgtcat caacaatgcc attctccggg ccaacctgca aaagggagag aaccctagcc 5280

attatggaat tactgctttc aatcatcccc tgaatctcac caagcagcag ctctcagagg 5340

tggctctgat gaccacatca gtggatgtcc ttgtgtccat ctgtgtcatc tttgcaatgt 5400

ccttcgtccc agccagcttt gtcgtattcc tgatccagga gcgggtcagc aaagcaaaac 5460

acctgcagtt catcagtgga gtgaagcctg tcatctactg gctctctaat tttgtctggg 5520

atatgtgcaa ttacgttgtc cctgccacac tggtcattat catcttcatc tgcttccagc 5580

agaagtccta tgtgtcctcc accaatctgc ctgtgctagc ccttctactt ttgctgtatg 5640

ggtggtcaat cacacctctc atgtacccag cctcctttgt gttcaagatc cccagcacag 5700

cctatgtggt gctcaccagc gtgaacctct tcattggcat taatggcagc gtggccacct 5760

ttgtgctgga gctgttcacc gacaataagc tgaataatat caatgatatc ctgaagtccg 5820

tgttcttgat cttcccacat ttttgcctgg gacgagggct catcgacatg gtgaaaaacc 5880

aggcaatggc tgatgccctg gaaaggtttg gggagaatcg ctttgtgtca ccattatctt 5940

gggacttggt gggacgaaac ctcttcgcca tggccgtgga aggggtggtg ttcttcctca 6000

ttactgttct gatccagtac agattcttca tcaggcccag acctgtaaat gcaaagctat 6060

ctcctctgaa tgatgaagat gaagatgtga ggcgggaaag acagagaatt cttgatggtg 6120

gaggccagaa tgacatctta gaaatcaagg agttgacgaa gatatataga aggaagcgga 6180

agcctgctgt tgacaggatt tgcgtgggca ttcctcctgg tgagtgcttt gggctcctgg 6240

gagttaatgg ggctggaaaa tcatcaactt tcaagatgtt aacaggagat accactgtta 6300

ccagaggaga tgctttcctt aacaaaaata gtatcttatc aaacatccat gaagtacatc 6360

agaacatggg ctactgccct cagtttgatg ccatcacaga gctgttgact gggagagaac 6420

acgtggagtt ctttgccctt ttgagaggag tcccagagaa agaagttggc aaggttggtg 6480

agtgggcgat tcggaaactg ggcctcgtga agtatggaga aaaatatgct ggtaactata 6540

gtggaggcaa caaacgcaag ctctctacag ccatggcttt gatcggcggg cctcctgtgg 6600

tgtttctgga tgaacccacc acaggcatgg atcccaaagc ccggcggttc ttgtggaatt 6660

gtgccctaag tgttgtcaag gaggggagat cagtagtgct tacatctcat agtatggaag 6720

aatgtgaagc tctttgcact aggatggcaa tcatggtcaa tggaaggttc aggtgccttg 6780

gcagtgtcca gcatctaaaa aataggtttg gagatggtta tacaatagtt gtacgaatag 6840

cagggtccaa cccggacctg aagcctgtcc aggatttctt tggacttgca tttcctggaa 6900

gtgttctaaa agagaaacac cggaacatgc tacaatacca gcttccatct tcattatctt 6960

ctctggccag gatattcagc atcctctccc agagcaaaaa gcgactccac atagaagact 7020

actctgtttc tcagacaaca cttgaccaag tatttgtgaa ctttgccaag gaccaaagtg 7080

atgatgacca cttaaaagac ctctcattac acaaaaacca gacagtagtg gacgttgcag 7140

ttctcacatc ttttctacag gatgagaaag tgaaagaaag ctatgtatga agaatcctgt 7200

tcatacgggg tggctgaaag taaagaggaa ctagactttc ctttgcacca tgtgaagtgt 7260

tgtggagaaa agagccagaa gttgatgtgg gaagaagtaa actggatact gtactgatac 7320

tattcaatgc aatgcaattc aatgcaatga aaacaaaatt ccattacagg ggcagtgcct 7380

ttgtagccta tgtcttgtat ggctctcaag tgaaagactt gaatttagtt ttttacctat 7440

acctatgtga aactctatta tggaacccaa tggacatatg ggtttgaact cacacttttt 7500

tttttttttt tgttcctgtg tattctcatt ggggttgcaa caataattca tcaagtaatc 7560

atggccagcg attattgatc aaaatcaaaa ggtaatgcac atcctcattc actaagccat 7620

gccatgccca ggagactggt ttcccggtga cacatccatt gctggcaatg agtgtgccag 7680

agttattagt gccaagtttt tcagaaagtt tgaagcacca tggtgtgtca tgctcacttt 7740

tgtgaaagct gctctgctca gagtctatca acattgaata tcagttgaca gaatggtgcc 7800

atgcgtggct aacatcctgc tttgattccc tctgataagc tgttctggtg gcagtaacat 7860

gcaacaaaaa tgtgggtgtc tccaggcacg ggaaacttgg ttccattgtt atattgtcct 7920

atgcttcgag ccatgggtct acagggtcat ccttatgaga ctcttaaata tacttagatc 7980

ctggtaagag gcaaagaatc aacagccaaa ctgctggggc tgcaagctgc tgaagccagg 8040

gcatgggatt aaagagattg tgcgttcaaa cctagggaag cctgtgccca tttgtcctga 8100

ctgtctgcta acatggtaca ctgcatctca agatgtttat ctgacacaag tgtattattt 8160

ctggcttttt gaattaatct agaaaatgaa aagatggagt tgtattttga caaaaatgtt 8220

tgtacttttt aatgttattt ggaattttaa gttctatcag tgacttctga atccttagaa 8280

tggcctcttt gtagaaccct gtggtataga ggagtatggc cactgcccca ctatttttat 8340

tttcttatgt aagtttgcat atcagtcatg actagtgcct agaaagcaat gtgatggtca 8400

ggatctcatg acattatatt tgagtttctt tcagatcatt taggatactc ttaatctcac 8460

ttcatcaatc aaatattttt tgagtgtatg ctgtagctga aagagtatgt acgtacgtat 8520

aagactagag agatattaag tctcagtaca cttcctgtgc catgttattc agctcactgg 8580

tttacaaata taggttgtct tgtggttgta ggagcccact gtaacaatac tgggcagcct 8640

tttttttttt ttttttaatt gcaacaatgc aaaagccaag aaagtataag ggtcacaagt 8700

ctaaacaatg aattcttcaa cagggaaaac agctagcttg aaaacttgct gaaaaacaca 8760

acttgtgttt atggcattta gtaccttcaa ataattggct ttgcagatat tggatacccc 8820

attaaatctg acagtctcaa atttttcatc tcttcaatca ctagtcaaga aaaatataaa 8880

aacaacaaat acttccatat ggagcatttt tcagagtttt ctaacccagt cttatttttc 8940

tagtcagtaa acatttgtaa aaatactgtt tcactaatac ttactgttaa ctgtcttgag 9000

agaaaagaaa aatatgagag aactattgtt tggggaagtt caagtgatct ttcaatatca 9060

ttactaactt cttccacttt ttccagaatt tgaatattaa cgctaaaggt gtaagacttc 9120

agatttcaaa ttaatctttc tatatttttt aaatttacag aatattatat aacccactgc 9180

tgaaaaagaa aaaaatgatt gttttagaag ttaaagtcaa tattgatttt aaatataagt 9240

aatgaaggca tatttccaat aactagtgat atggcatcgt tgcattttac agtatcttca 9300

aaaatacaga atttatagaa taatttctcc tcatttaata tttttcaaaa tcaaagttat 9360

ggtttcctca ttttactaaa atcgtattct aattcttcat tatagtaaat ctatgagcaa 9420

ctccttactt cggttcctct gatttcaagg ccatatttta aaaaatcaaa aggcactgtg 9480

aactattttg aagaaaacac aacattttaa tacagattga aaggacctct tctgaagcta 9540

gaaacaatct atagttatac atcttcatta atactgtgtt accttttaaa atagtaattt 9600

tttacatttt cctgtgtaaa cctaattgtg gtagaaattt ttaccaactc tatactcaat 9660

caagcaaaat ttctgtatat tccctgtgga atgtacctat gtgagtttca gaaattctca 9720

aaatacgtgt tcaaaaattt ctgcttttgc atctttggga cacctcagaa aacttattaa 9780

caactgtgaa tatgagaaat acagaagaaa ataataagcc ctctatacat aaatgcccag 9840

cacaattcat tgttaaaaaa caaccaaacc tcacactact gtatttcatt atctgtactg 9900

aaagcaaatg ctttgtgact attaaatgtt gcacatcatt cattcactgt atagtaatca 9960

ttgactaaag ccatttgtct gtgttttctt cttgtggttg tatatatcag gtaaaatatt 10020

ttccaaagag ccatgtgtca tgtaatactg aaccactttg atattgagac attaatttgt 10080

acccttgtta ttatctacta gtaataatgt aatactgtag aaatattgct ctaattcttt 10140

tcaaaattgt tgcatccccc ttagaatgtt tctatttcca taaggattta ggtatgctat 10200

tatcccttct tataccctaa gatgaagctg tttttgtgct ctttgttcat cattggccct 10260

cattccaagc actttacgct gtctgtaatg ggatctattt ttgcactgga atatctgaga 10320

attgcaaaac tagacaaaag tttcacaaca gatttctaag ttaaatcatt ttcattaaaa 10380

ggaaaaaaga aaaaaaattt tgtatgtcaa taactttata tgaagtatta aaatgcatat 10440

ttctatgttg taatataatg agtcacaaaa taaagctgtg acagttctgt tggtctacag 10500

aaaaaaaaaa aaaaa 10515

<210> SEQ ID NO: 2

<211> LENGTH: 8133

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 2

aatgtgcagg atgacaagat ggagcaaaca gtgcttgtac caccaggacc tgacagcttc 60

aacttcttca ccagagaatc tcttgcggct attgaaagac gcattgcaga agaaaaggca 120

aagaatccca aaccagacaa aaaagatgac gacgaaaatg gcccaaagcc aaatagtgac 180

ttggaagctg gaaagaacct tccatttatt tatggagaca ttcctccaga gatggtgtca 240

gagcccctgg aggacctgga cccctactat atcaataaga aaacttttat agtattgaat 300

aaagggaagg ccatcttccg gttcagtgcc acctctgccc tgtacatttt aactcccttc 360

aatcctctta ggaaaatagc tattaagatt ttggtacatt cattattcag catgctaatt 420

atgtgcacta ttttgacaaa ctgtgtgttt atgacaatga gtaaccctcc tgattggaca 480

aagaatgtag aatacacctt cacaggaata tatacttttg aatcacttat aaaaattatt 540

gcaaggggat tctgtttaga agattttact ttccttcggg atccatggaa ctggctcgat 600

ttcactgtca ttacatttgc gtacgtcaca gagtttgtgg acctgggcaa tgtctcggca 660

ttgagaacat tcagagttct ccgagcattg aagacgattt cagtcattcc aggcctgaaa 720

accattgtgg gagccctgat ccagtctgtg aagaagctct cagatgtaat gatcctgact 780

gtgttctgtc tgagcgtatt tgctctaatt gggctgcagc tgttcatggg caacctgagg 840

aataaatgta tacaatggcc tcccaccaat gcttccttgg aggaacatag tatagaaaag 900

aatataactg tgaattataa tggtacactt ataaatgaaa ctgtctttga gtttgactgg 960

aagtcatata ttcaagattc aagatatcat tatttcctgg agggtttttt agatgcacta 1020

ctatgtggaa atagctctga tgcaggccaa tgtccagagg gatatatgtg tgtgaaagct 1080

ggtagaaatc ccaattatgg ctacacaagc tttgatacct tcagttgggc ttttttgtcc 1140

ttgtttcgac taatgactca ggacttctgg gaaaatcttt atcaactgac attacgtgct 1200

gctgggaaaa cgtacatgat attttttgta ttggtcattt tcttgggctc attctaccta 1260

ataaatttga tcctggctgt ggtggccatg gcctacgagg aacagaatca ggccaccttg 1320

gaagaagcag aacagaaaga ggccgaattt cagcagatga ttgaacagct taaaaagcaa 1380

caggaggcag ctcagcaggc agcaacggca actgcctcag aacattccag agagcccagt 1440

gcagcaggca ggctctcaga cagctcatct gaagcctcta agttgagttc caagagtgct 1500

aaggaaagaa gaaatcggag gaagaaaaga aaacagaaag agcagtctgg tggggaagag 1560

aaagatgagg atgaattcca aaaatctgaa tctgaggaca gcatcaggag gaaaggtttt 1620

cgcttctcca ttgaagggaa ccgattgaca tatgaaaaga ggtactcctc cccacaccag 1680

tctttgttga gcatccgtgg ctccctattt tcaccaaggc gaaatagcag aacaagcctt 1740

ttcagcttta gagggcgagc aaaggatgtg ggatctgaga acgacttcgc agatgatgag 1800

cacagcacct ttgaggataa cgagagccgt agagattcct tgtttgtgcc ccgacgacac 1860

ggagagagac gcaacagcaa cctgagtcag accagtaggt catcccggat gctggcagtg 1920

tttccagcga atgggaagat gcacagcact gtggattgca atggtgtggt ttccttggtt 1980

ggtggacctt cagttcctac atcgcctgtt ggacagcttc tgccagaggt gataatagat 2040

aagccagcta ctgatgacaa tggaacaacc actgaaactg aaatgagaaa gagaaggtca 2100

agttctttcc acgtttccat ggactttcta gaagatcctt cccaaaggca acgagcaatg 2160

agtatagcca gcattctaac aaatacagta gaagaacttg aagaatccag gcagaaatgc 2220

ccaccctgtt ggtataaatt ttccaacata ttcttaatct gggactgttc tccatattgg 2280

ttaaaagtga aacatgttgt caacctggtt gtgatggacc catttgttga cctggccatc 2340

accatctgta ttgtcttaaa tactcttttc atggccatgg agcactatcc aatgacggac 2400

catttcaata atgtgcttac agtaggaaac ttggttttca ctgggatctt tacagcagaa 2460

atgtttctga aaattattgc catggatcct tactattatt tccaagaagg ctggaatatc 2520

tttgacggtt ttattgtgac gcttagcctg gtagaacttg gactcgccaa tgtggaagga 2580

ttatctgttc tccgttcatt tcgattgctg cgagttttca agttggcaaa atcttggcca 2640

acgttaaata tgctaataaa gatcatcggc aattccgtgg gggctctggg aaatttaacc 2700

ctcgtcttgg ccatcatcgt cttcattttt gccgtggtcg gcatgcagct ctttggtaaa 2760

agctacaaag attgtgtctg caagatcgcc agtgattgtc aactcccacg ctggcacatg 2820

aatgacttct tccactcctt cctgattgtg ttccgcgtgc tgtgtgggga gtggatagag 2880

accatgtggg actgtatgga ggttgctggt caagccatgt gccttactgt cttcatgatg 2940

gtcatggtga ttggaaacct agtggtcctg aatctctttc tggccttgct tctgagctca 3000

tttagtgcag acaaccttgc agccactgat gatgataatg aaatgaataa tctccaaatt 3060

gctgtggata ggatgcacaa aggagtagct tatgtgaaaa gaaaaatata tgaatttatt 3120

caacagtcct tcattaggaa acaaaagatt ttagatgaaa ttaaaccact tgatgatcta 3180

aacaacaaga aagacagttg tatgtccaat catacagcag aaattgggaa agatcttgac 3240

tatcttaaag atgtaaatgg aactacaagt ggtataggaa ctggcagcag tgttgaaaaa 3300

tacattattg atgaaagtga ttacatgtca ttcataaaca accccagtct tactgtgact 3360

gtaccaattg ctgtaggaga atctgacttt gaaaatttaa acacggaaga ctttagtagt 3420

gaatcggatc tggaagaaag caaagagaaa ctgaatgaaa gcagtagctc atcagaaggt 3480

agcactgtgg acatcggcgc acctgtagaa gaacagcccg tagtggaacc tgaagaaact 3540

cttgaaccag aagcttgttt cactgaaggc tgtgtacaaa gattcaagtg ttgtcaaatc 3600

aatgtggaag aaggcagagg aaaacaatgg tggaacctga gaaggacgtg tttccgaata 3660

gttgaacata actggtttga gaccttcatt gttttcatga ttctccttag tagtggtgct 3720

ctggcatttg aagatatata tattgatcag cgaaagacga ttaagacgat gttggaatat 3780

gctgacaagg ttttcactta cattttcatt ctggaaatgc ttctaaaatg ggtggcatat 3840

ggctatcaaa catatttcac caatgcctgg tgttggctgg acttcttaat tgttgatgtt 3900

tcattggtca gtttaacagc aaatgccttg ggttactcag aacttggagc catcaaatct 3960

ctcaggacac taagagctct gagacctcta agagccttat ctcgatttga agggatgagg 4020

gtggttgtga atgccctttt aggagcaatt ccatccatca tgaatgtgct tctggtttgt 4080

cttatattct ggctaatttt cagcatcatg ggcgtaaatt tgtttgctgg caaattctac 4140

cactgtatta acaccacaac tggtgacagg tttgacatcg aagacgtgaa taatcatact 4200

gattgcctaa aactaataga aagaaatgag actgctcgat ggaaaaatgt gaaagtaaac 4260

tttgataatg taggatttgg gtatctctct ttgcttcaag ttgccacatt caaaggatgg 4320

atggatataa tgtatgcagc agttgattcc agaaatgtgg aactccagcc taagtatgaa 4380

gaaagtctgt acatgtatct ttactttgtt attttcatca tctttgggtc cttcttcacc 4440

ttgaacctgt ttattggtgt catcatagat aatttcaacc agcagaaaaa gaagtttgga 4500

ggtcaagaca tctttatgac agaagaacag aagaaatact ataatgcaat gaaaaaatta 4560

ggatcgaaaa aaccgcaaaa gcctatacct cgaccaggaa acaaatttca aggaatggtc 4620

tttgacttcg taaccagaca agtttttgac ataagcatca tgattctcat ctgtcttaac 4680

atggtcacaa tgatggtgga aacagatgac cagagtgaat atgtgactac cattttgtca 4740

cgcatcaatc tggtgttcat tgtgctattt actggagagt gtgtactgaa actcatctct 4800

ctacgccatt attattttac cattggatgg aatatttttg attttgtggt tgtcattctc 4860

tccattgtag gtatgtttct tgccgagctg atagaaaagt atttcgtgtc ccctaccctg 4920

ttccgagtga tccgtcttgc taggattggc cgaatcctac gtctgatcaa aggagcaaag 4980

gggatccgca cgctgctctt tgctttgatg atgtcccttc ctgcgttgtt taacatcggc 5040

ctcctactct tcctagtcat gttcatctac gccatctttg ggatgtccaa ctttgcctat 5100

gttaagaggg aagttgggat cgatgacatg ttcaactttg agacctttgg caacagcatg 5160

atctgcctat tccaaattac aacctctgct ggctgggatg gattgctagc acccattctc 5220

aacagtaagc cacccgactg tgaccctaat aaagttaacc ctggaagctc agttaaggga 5280

gactgtggga acccatctgt tggaattttc ttttttgtca gttacatcat catatccttc 5340

ctggttgtgg tgaacatgta catcgcggtc atcctggaga acttcagtgt tgctactgaa 5400

gaaagtgcag agcctctgag tgaggatgac tttgagatgt tctatgaggt ttgggagaag 5460

tttgatcccg atgcaactca gttcatggaa tttgaaaaat tatctcagtt tgcagctgcg 5520

cttgaaccgc ctctcaatct gccacaacca aacaaactcc agctcattgc catggatttg 5580

cccatggtga gtggtgaccg gatccactgt cttgatatct tatttgcttt tacaaagcgg 5640

gttctaggag agagtggaga gatggatgct ctacgaatac agatggaaga gcgattcatg 5700

gcttccaatc cttccaaggt ctcctatcag ccaatcacta ctactttaaa acgaaaacaa 5760

gaggaagtat ctgctgtcat tattcagcgt gcttacagac gccacctttt aaagcgaact 5820

gtaaaacaag cttcctttac gtacaataaa aacaaaatca aaggtggggc taatcttctt 5880

ataaaagaag acatgataat tgacagaata aatgaaaact ctattacaga aaaaactgat 5940

ctgaccatgt ccactgcagc ttgtccacct tcctatgacc gggtgacaaa gccaattgtg 6000

gaaaaacatg agcaagaagg caaagatgaa aaagccaaag ggaaataaat gaaaataaat 6060

aaaaataatt gggtgacaaa ttgtttacag cctgtgaagg tgatgtattt ttatcaacag 6120

gactccttta ggaggtcaat gccaaactga ctgtttttac acaaatctcc ttaaggtcag 6180

tgcctacaat aagacagtga ccccttgtca gcaaactgtg actctgtgta aaggggagat 6240

gaccttgaca ggaggttact gttctcacta ccagctgaca ctgctgaaga taagatgcac 6300

aatggctagt cagactgtag ggaccagttt caaggggtgc aaacctgtga ttttggggtt 6360

gtttaacatg aaacacttta gtgtagtaat tgtatccact gtttgcattt caactgccac 6420

atttgtcaca tttttatgga atctgttagt ggattcatct ttttgttaat ccatgtgttt 6480

attatatgtg actatttttg taaacgaagt ttctgttgag aaataggcta aggacctcta 6540

taacaggtat gccacctggg gggtatggca accacatggc cctcccagct acacaaagtc 6600

gtggtttgca tgagggcatg ctgcacttag agatcatgca tgagaaaaag tcacaagaaa 6660

aacaaattct taaatttcac catatttctg ggaggggtaa ttgggtgata agtggaggtg 6720

ctttgttgat cttgttttgc gaaatccagc ccctagacca agtagattat ttgtgggtag 6780

gccagtaaat cttagcaggt gcaaacttca ttcaaatgtt tggagtcata aatgttatgt 6840

ttctttttgt tgtattaaaa aaaaaacctg aatagtgaat attgcccctc accctccacc 6900

gccagaagac tgaattgacc aaaattactc tttataaatt tctgcttttt cctgcacttt 6960

gtttagccat cttcggctct cagcaaggtt gacactgtat atgttaatga aatgctattt 7020

attatgtaaa tagtcatttt accctgtggt gcacgtttga gcaaacaaat aatgacctaa 7080

gcacagtatt tattgcatca aatatgtacc acaagaaatg tagagtgcaa gctttacaca 7140

ggtaataaaa tgtattctgt accatttata gatagtttgg atgctatcaa tgcatgttta 7200

tattaccatg ctgctgtatc tggtttctct cactgctcag aatctcattt atgagaaacc 7260

atatgtcagt ggtaaagtca aggaaattgt tcaacagatc tcatttattt aagtcattaa 7320

gcaatagttt gcagcacttt aacagctttt tggttatttt tacattttaa gtggataaca 7380

tatggtatat agccagactg tacagacatg tttaaaaaaa cacactgctt aacctattaa 7440

atatgtgttt agaattttat aagcaaatat aaatactgta aaaagtcact ttattttatt 7500

tttcagcatt atgtacataa atatgaagag gaaattatct tcaggttgat atcacaatca 7560

cttttcttac tttctgtcca tagtactttt tcatgaaaga aatttgctaa ataagacatg 7620

aaaacaagac tgggtagttg tagatttctg ctttttaaat tacatttgct aattttagat 7680

tatttcacaa ttttaaggag caaaataggt tcacgattca tatccaaatt atgctttgca 7740

attggaaaag ggtttaaaat tttatttata tttctggtag tacctgcact aactgaattg 7800

aaggtagtgc ttatgttatt tttgttcttt ttttctgact tcggtttatg ttttcatttc 7860

tttggagtaa tgctgctcta gattgttcta aatagaatgt gggcttcata attttttttt 7920

ccacaaaaac agagtagtca acttatatag tcaattacat caggacattt tgtgtttctt 7980

acagaagcaa accataggct cctcttttcc ttaaaactac ttagataaac tgtattcgtg 8040

aactgcatgc tggaaaatgc tactattatg ctaaataatg ctaaccaaca tttaaaatgt 8100

gcaaaactaa taaagattac attttttatt tta 8133

<210> SEQ ID NO: 3

<211> LENGTH: 8100

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 3

aatgtgcagg atgacaagat ggagcaaaca gtgcttgtac caccaggacc tgacagcttc 60

aacttcttca ccagagaatc tcttgcggct attgaaagac gcattgcaga agaaaaggca 120

aagaatccca aaccagacaa aaaagatgac gacgaaaatg gcccaaagcc aaatagtgac 180

ttggaagctg gaaagaacct tccatttatt tatggagaca ttcctccaga gatggtgtca 240

gagcccctgg aggacctgga cccctactat atcaataaga aaacttttat agtattgaat 300

aaagggaagg ccatcttccg gttcagtgcc acctctgccc tgtacatttt aactcccttc 360

aatcctctta ggaaaatagc tattaagatt ttggtacatt cattattcag catgctaatt 420

atgtgcacta ttttgacaaa ctgtgtgttt atgacaatga gtaaccctcc tgattggaca 480

aagaatgtag aatacacctt cacaggaata tatacttttg aatcacttat aaaaattatt 540

gcaaggggat tctgtttaga agattttact ttccttcggg atccatggaa ctggctcgat 600

ttcactgtca ttacatttgc gtacgtcaca gagtttgtgg acctgggcaa tgtctcggca 660

ttgagaacat tcagagttct ccgagcattg aagacgattt cagtcattcc aggcctgaaa 720

accattgtgg gagccctgat ccagtctgtg aagaagctct cagatgtaat gatcctgact 780

gtgttctgtc tgagcgtatt tgctctaatt gggctgcagc tgttcatggg caacctgagg 840

aataaatgta tacaatggcc tcccaccaat gcttccttgg aggaacatag tatagaaaag 900

aatataactg tgaattataa tggtacactt ataaatgaaa ctgtctttga gtttgactgg 960

aagtcatata ttcaagattc aagatatcat tatttcctgg agggtttttt agatgcacta 1020

ctatgtggaa atagctctga tgcaggccaa tgtccagagg gatatatgtg tgtgaaagct 1080

ggtagaaatc ccaattatgg ctacacaagc tttgatacct tcagttgggc ttttttgtcc 1140

ttgtttcgac taatgactca ggacttctgg gaaaatcttt atcaactgac attacgtgct 1200

gctgggaaaa cgtacatgat attttttgta ttggtcattt tcttgggctc attctaccta 1260

ataaatttga tcctggctgt ggtggccatg gcctacgagg aacagaatca ggccaccttg 1320

gaagaagcag aacagaaaga ggccgaattt cagcagatga ttgaacagct taaaaagcaa 1380

caggaggcag ctcagcaggc agcaacggca actgcctcag aacattccag agagcccagt 1440

gcagcaggca ggctctcaga cagctcatct gaagcctcta agttgagttc caagagtgct 1500

aaggaaagaa gaaatcggag gaagaaaaga aaacagaaag agcagtctgg tggggaagag 1560

aaagatgagg atgaattcca aaaatctgaa tctgaggaca gcatcaggag gaaaggtttt 1620

cgcttctcca ttgaagggaa ccgattgaca tatgaaaaga ggtactcctc cccacaccag 1680

tctttgttga gcatccgtgg ctccctattt tcaccaaggc gaaatagcag aacaagcctt 1740

ttcagcttta gagggcgagc aaaggatgtg ggatctgaga acgacttcgc agatgatgag 1800

cacagcacct ttgaggataa cgagagccgt agagattcct tgtttgtgcc ccgacgacac 1860

ggagagagac gcaacagcaa cctgagtcag accagtaggt catcccggat gctggcagtg 1920

tttccagcga atgggaagat gcacagcact gtggattgca atggtgtggt ttccttggtt 1980

ggtggacctt cagttcctac atcgcctgtt ggacagcttc tgccagaggg aacaaccact 2040

gaaactgaaa tgagaaagag aaggtcaagt tctttccacg tttccatgga ctttctagaa 2100

gatccttccc aaaggcaacg agcaatgagt atagccagca ttctaacaaa tacagtagaa 2160

gaacttgaag aatccaggca gaaatgccca ccctgttggt ataaattttc caacatattc 2220

ttaatctggg actgttctcc atattggtta aaagtgaaac atgttgtcaa cctggttgtg 2280

atggacccat ttgttgacct ggccatcacc atctgtattg tcttaaatac tcttttcatg 2340

gccatggagc actatccaat gacggaccat ttcaataatg tgcttacagt aggaaacttg 2400

gttttcactg ggatctttac agcagaaatg tttctgaaaa ttattgccat ggatccttac 2460

tattatttcc aagaaggctg gaatatcttt gacggtttta ttgtgacgct tagcctggta 2520

gaacttggac tcgccaatgt ggaaggatta tctgttctcc gttcatttcg attgctgcga 2580

gttttcaagt tggcaaaatc ttggccaacg ttaaatatgc taataaagat catcggcaat 2640

tccgtggggg ctctgggaaa tttaaccctc gtcttggcca tcatcgtctt catttttgcc 2700

gtggtcggca tgcagctctt tggtaaaagc tacaaagatt gtgtctgcaa gatcgccagt 2760

gattgtcaac tcccacgctg gcacatgaat gacttcttcc actccttcct gattgtgttc 2820

cgcgtgctgt gtggggagtg gatagagacc atgtgggact gtatggaggt tgctggtcaa 2880

gccatgtgcc ttactgtctt catgatggtc atggtgattg gaaacctagt ggtcctgaat 2940

ctctttctgg ccttgcttct gagctcattt agtgcagaca accttgcagc cactgatgat 3000

gataatgaaa tgaataatct ccaaattgct gtggatagga tgcacaaagg agtagcttat 3060

gtgaaaagaa aaatatatga atttattcaa cagtccttca ttaggaaaca aaagatttta 3120

gatgaaatta aaccacttga tgatctaaac aacaagaaag acagttgtat gtccaatcat 3180

acagcagaaa ttgggaaaga tcttgactat cttaaagatg taaatggaac tacaagtggt 3240

ataggaactg gcagcagtgt tgaaaaatac attattgatg aaagtgatta catgtcattc 3300

ataaacaacc ccagtcttac tgtgactgta ccaattgctg taggagaatc tgactttgaa 3360

aatttaaaca cggaagactt tagtagtgaa tcggatctgg aagaaagcaa agagaaactg 3420

aatgaaagca gtagctcatc agaaggtagc actgtggaca tcggcgcacc tgtagaagaa 3480

cagcccgtag tggaacctga agaaactctt gaaccagaag cttgtttcac tgaaggctgt 3540

gtacaaagat tcaagtgttg tcaaatcaat gtggaagaag gcagaggaaa acaatggtgg 3600

aacctgagaa ggacgtgttt ccgaatagtt gaacataact ggtttgagac cttcattgtt 3660

ttcatgattc tccttagtag tggtgctctg gcatttgaag atatatatat tgatcagcga 3720

aagacgatta agacgatgtt ggaatatgct gacaaggttt tcacttacat tttcattctg 3780

gaaatgcttc taaaatgggt ggcatatggc tatcaaacat atttcaccaa tgcctggtgt 3840

tggctggact tcttaattgt tgatgtttca ttggtcagtt taacagcaaa tgccttgggt 3900

tactcagaac ttggagccat caaatctctc aggacactaa gagctctgag acctctaaga 3960

gccttatctc gatttgaagg gatgagggtg gttgtgaatg cccttttagg agcaattcca 4020

tccatcatga atgtgcttct ggtttgtctt atattctggc taattttcag catcatgggc 4080

gtaaatttgt ttgctggcaa attctaccac tgtattaaca ccacaactgg tgacaggttt 4140

gacatcgaag acgtgaataa tcatactgat tgcctaaaac taatagaaag aaatgagact 4200

gctcgatgga aaaatgtgaa agtaaacttt gataatgtag gatttgggta tctctctttg 4260

cttcaagttg ccacattcaa aggatggatg gatataatgt atgcagcagt tgattccaga 4320

aatgtggaac tccagcctaa gtatgaagaa agtctgtaca tgtatcttta ctttgttatt 4380

ttcatcatct ttgggtcctt cttcaccttg aacctgttta ttggtgtcat catagataat 4440

ttcaaccagc agaaaaagaa gtttggaggt caagacatct ttatgacaga agaacagaag 4500

aaatactata atgcaatgaa aaaattagga tcgaaaaaac cgcaaaagcc tatacctcga 4560

ccaggaaaca aatttcaagg aatggtcttt gacttcgtaa ccagacaagt ttttgacata 4620

agcatcatga ttctcatctg tcttaacatg gtcacaatga tggtggaaac agatgaccag 4680

agtgaatatg tgactaccat tttgtcacgc atcaatctgg tgttcattgt gctatttact 4740

ggagagtgtg tactgaaact catctctcta cgccattatt attttaccat tggatggaat 4800

atttttgatt ttgtggttgt cattctctcc attgtaggta tgtttcttgc cgagctgata 4860

gaaaagtatt tcgtgtcccc taccctgttc cgagtgatcc gtcttgctag gattggccga 4920

atcctacgtc tgatcaaagg agcaaagggg atccgcacgc tgctctttgc tttgatgatg 4980

tcccttcctg cgttgtttaa catcggcctc ctactcttcc tagtcatgtt catctacgcc 5040

atctttggga tgtccaactt tgcctatgtt aagagggaag ttgggatcga tgacatgttc 5100

aactttgaga cctttggcaa cagcatgatc tgcctattcc aaattacaac ctctgctggc 5160

tgggatggat tgctagcacc cattctcaac agtaagccac ccgactgtga ccctaataaa 5220

gttaaccctg gaagctcagt taagggagac tgtgggaacc catctgttgg aattttcttt 5280

tttgtcagtt acatcatcat atccttcctg gttgtggtga acatgtacat cgcggtcatc 5340

ctggagaact tcagtgttgc tactgaagaa agtgcagagc ctctgagtga ggatgacttt 5400

gagatgttct atgaggtttg ggagaagttt gatcccgatg caactcagtt catggaattt 5460

gaaaaattat ctcagtttgc agctgcgctt gaaccgcctc tcaatctgcc acaaccaaac 5520

aaactccagc tcattgccat ggatttgccc atggtgagtg gtgaccggat ccactgtctt 5580

gatatcttat ttgcttttac aaagcgggtt ctaggagaga gtggagagat ggatgctcta 5640

cgaatacaga tggaagagcg attcatggct tccaatcctt ccaaggtctc ctatcagcca 5700

atcactacta ctttaaaacg aaaacaagag gaagtatctg ctgtcattat tcagcgtgct 5760

tacagacgcc accttttaaa gcgaactgta aaacaagctt cctttacgta caataaaaac 5820

aaaatcaaag gtggggctaa tcttcttata aaagaagaca tgataattga cagaataaat 5880

gaaaactcta ttacagaaaa aactgatctg accatgtcca ctgcagcttg tccaccttcc 5940

tatgaccggg tgacaaagcc aattgtggaa aaacatgagc aagaaggcaa agatgaaaaa 6000

gccaaaggga aataaatgaa aataaataaa aataattggg tgacaaattg tttacagcct 6060

gtgaaggtga tgtattttta tcaacaggac tcctttagga ggtcaatgcc aaactgactg 6120

tttttacaca aatctcctta aggtcagtgc ctacaataag acagtgaccc cttgtcagca 6180

aactgtgact ctgtgtaaag gggagatgac cttgacagga ggttactgtt ctcactacca 6240

gctgacactg ctgaagataa gatgcacaat ggctagtcag actgtaggga ccagtttcaa 6300

ggggtgcaaa cctgtgattt tggggttgtt taacatgaaa cactttagtg tagtaattgt 6360

atccactgtt tgcatttcaa ctgccacatt tgtcacattt ttatggaatc tgttagtgga 6420

ttcatctttt tgttaatcca tgtgtttatt atatgtgact atttttgtaa acgaagtttc 6480

tgttgagaaa taggctaagg acctctataa caggtatgcc acctgggggg tatggcaacc 6540

acatggccct cccagctaca caaagtcgtg gtttgcatga gggcatgctg cacttagaga 6600

tcatgcatga gaaaaagtca caagaaaaac aaattcttaa atttcaccat atttctggga 6660

ggggtaattg ggtgataagt ggaggtgctt tgttgatctt gttttgcgaa atccagcccc 6720

tagaccaagt agattatttg tgggtaggcc agtaaatctt agcaggtgca aacttcattc 6780

aaatgtttgg agtcataaat gttatgtttc tttttgttgt attaaaaaaa aaacctgaat 6840

agtgaatatt gcccctcacc ctccaccgcc agaagactga attgaccaaa attactcttt 6900

ataaatttct gctttttcct gcactttgtt tagccatctt cggctctcag caaggttgac 6960

actgtatatg ttaatgaaat gctatttatt atgtaaatag tcattttacc ctgtggtgca 7020

cgtttgagca aacaaataat gacctaagca cagtatttat tgcatcaaat atgtaccaca 7080

agaaatgtag agtgcaagct ttacacaggt aataaaatgt attctgtacc atttatagat 7140

agtttggatg ctatcaatgc atgtttatat taccatgctg ctgtatctgg tttctctcac 7200

tgctcagaat ctcatttatg agaaaccata tgtcagtggt aaagtcaagg aaattgttca 7260

acagatctca tttatttaag tcattaagca atagtttgca gcactttaac agctttttgg 7320

ttatttttac attttaagtg gataacatat ggtatatagc cagactgtac agacatgttt 7380

aaaaaaacac actgcttaac ctattaaata tgtgtttaga attttataag caaatataaa 7440

tactgtaaaa agtcacttta ttttattttt cagcattatg tacataaata tgaagaggaa 7500

attatcttca ggttgatatc acaatcactt ttcttacttt ctgtccatag tactttttca 7560

tgaaagaaat ttgctaaata agacatgaaa acaagactgg gtagttgtag atttctgctt 7620

tttaaattac atttgctaat tttagattat ttcacaattt taaggagcaa aataggttca 7680

cgattcatat ccaaattatg ctttgcaatt ggaaaagggt ttaaaatttt atttatattt 7740

ctggtagtac ctgcactaac tgaattgaag gtagtgctta tgttattttt gttctttttt 7800

tctgacttcg gtttatgttt tcatttcttt ggagtaatgc tgctctagat tgttctaaat 7860

agaatgtggg cttcataatt tttttttcca caaaaacaga gtagtcaact tatatagtca 7920

attacatcag gacattttgt gtttcttaca gaagcaaacc ataggctcct cttttcctta 7980

aaactactta gataaactgt attcgtgaac tgcatgctgg aaaatgctac tattatgcta 8040

aataatgcta accaacattt aaaatgtgca aaactaataa agattacatt ttttatttta 8100

<210> SEQ ID NO: 4

<211> LENGTH: 8049

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 4

aatgtgcagg atgacaagat ggagcaaaca gtgcttgtac caccaggacc tgacagcttc 60

aacttcttca ccagagaatc tcttgcggct attgaaagac gcattgcaga agaaaaggca 120

aagaatccca aaccagacaa aaaagatgac gacgaaaatg gcccaaagcc aaatagtgac 180

ttggaagctg gaaagaacct tccatttatt tatggagaca ttcctccaga gatggtgtca 240

gagcccctgg aggacctgga cccctactat atcaataaga aaacttttat agtattgaat 300

aaagggaagg ccatcttccg gttcagtgcc acctctgccc tgtacatttt aactcccttc 360

aatcctctta ggaaaatagc tattaagatt ttggtacatt cattattcag catgctaatt 420

atgtgcacta ttttgacaaa ctgtgtgttt atgacaatga gtaaccctcc tgattggaca 480

aagaatgtag aatacacctt cacaggaata tatacttttg aatcacttat aaaaattatt 540

gcaaggggat tctgtttaga agattttact ttccttcggg atccatggaa ctggctcgat 600

ttcactgtca ttacatttgc gtacgtcaca gagtttgtgg acctgggcaa tgtctcggca 660

ttgagaacat tcagagttct ccgagcattg aagacgattt cagtcattcc aggcctgaaa 720

accattgtgg gagccctgat ccagtctgtg aagaagctct cagatgtaat gatcctgact 780

gtgttctgtc tgagcgtatt tgctctaatt gggctgcagc tgttcatggg caacctgagg 840

aataaatgta tacaatggcc tcccaccaat gcttccttgg aggaacatag tatagaaaag 900

aatataactg tgaattataa tggtacactt ataaatgaaa ctgtctttga gtttgactgg 960

aagtcatata ttcaagattc aagatatcat tatttcctgg agggtttttt agatgcacta 1020

ctatgtggaa atagctctga tgcaggccaa tgtccagagg gatatatgtg tgtgaaagct 1080

ggtagaaatc ccaattatgg ctacacaagc tttgatacct tcagttgggc ttttttgtcc 1140

ttgtttcgac taatgactca ggacttctgg gaaaatcttt atcaactgac attacgtgct 1200

gctgggaaaa cgtacatgat attttttgta ttggtcattt tcttgggctc attctaccta 1260

ataaatttga tcctggctgt ggtggccatg gcctacgagg aacagaatca ggccaccttg 1320

gaagaagcag aacagaaaga ggccgaattt cagcagatga ttgaacagct taaaaagcaa 1380

caggaggcag ctcagcaggc agcaacggca actgcctcag aacattccag agagcccagt 1440

gcagcaggca ggctctcaga cagctcatct gaagcctcta agttgagttc caagagtgct 1500

aaggaaagaa gaaatcggag gaagaaaaga aaacagaaag agcagtctgg tggggaagag 1560

aaagatgagg atgaattcca aaaatctgaa tctgaggaca gcatcaggag gaaaggtttt 1620

cgcttctcca ttgaagggaa ccgattgaca tatgaaaaga ggtactcctc cccacaccag 1680

tctttgttga gcatccgtgg ctccctattt tcaccaaggc gaaatagcag aacaagcctt 1740

ttcagcttta gagggcgagc aaaggatgtg ggatctgaga acgacttcgc agatgatgag 1800

cacagcacct ttgaggataa cgagagccgt agagattcct tgtttgtgcc ccgacgacac 1860

ggagagagac gcaacagcaa cctgagtcag accagtaggt catcccggat gctggcagtg 1920

tttccagcga atgggaagat gcacagcact gtggattgca atggtgtggt ttccttggga 1980

acaaccactg aaactgaaat gagaaagaga aggtcaagtt ctttccacgt ttccatggac 2040

tttctagaag atccttccca aaggcaacga gcaatgagta tagccagcat tctaacaaat 2100

acagtagaag aacttgaaga atccaggcag aaatgcccac cctgttggta taaattttcc 2160

aacatattct taatctggga ctgttctcca tattggttaa aagtgaaaca tgttgtcaac 2220

ctggttgtga tggacccatt tgttgacctg gccatcacca tctgtattgt cttaaatact 2280

cttttcatgg ccatggagca ctatccaatg acggaccatt tcaataatgt gcttacagta 2340

ggaaacttgg ttttcactgg gatctttaca gcagaaatgt ttctgaaaat tattgccatg 2400

gatccttact attatttcca agaaggctgg aatatctttg acggttttat tgtgacgctt 2460

agcctggtag aacttggact cgccaatgtg gaaggattat ctgttctccg ttcatttcga 2520

ttgctgcgag ttttcaagtt ggcaaaatct tggccaacgt taaatatgct aataaagatc 2580

atcggcaatt ccgtgggggc tctgggaaat ttaaccctcg tcttggccat catcgtcttc 2640

atttttgccg tggtcggcat gcagctcttt ggtaaaagct acaaagattg tgtctgcaag 2700

atcgccagtg attgtcaact cccacgctgg cacatgaatg acttcttcca ctccttcctg 2760

attgtgttcc gcgtgctgtg tggggagtgg atagagacca tgtgggactg tatggaggtt 2820

gctggtcaag ccatgtgcct tactgtcttc atgatggtca tggtgattgg aaacctagtg 2880

gtcctgaatc tctttctggc cttgcttctg agctcattta gtgcagacaa ccttgcagcc 2940

actgatgatg ataatgaaat gaataatctc caaattgctg tggataggat gcacaaagga 3000

gtagcttatg tgaaaagaaa aatatatgaa tttattcaac agtccttcat taggaaacaa 3060

aagattttag atgaaattaa accacttgat gatctaaaca acaagaaaga cagttgtatg 3120

tccaatcata cagcagaaat tgggaaagat cttgactatc ttaaagatgt aaatggaact 3180

acaagtggta taggaactgg cagcagtgtt gaaaaataca ttattgatga aagtgattac 3240

atgtcattca taaacaaccc cagtcttact gtgactgtac caattgctgt aggagaatct 3300

gactttgaaa atttaaacac ggaagacttt agtagtgaat cggatctgga agaaagcaaa 3360

gagaaactga atgaaagcag tagctcatca gaaggtagca ctgtggacat cggcgcacct 3420

gtagaagaac agcccgtagt ggaacctgaa gaaactcttg aaccagaagc ttgtttcact 3480

gaaggctgtg tacaaagatt caagtgttgt caaatcaatg tggaagaagg cagaggaaaa 3540

caatggtgga acctgagaag gacgtgtttc cgaatagttg aacataactg gtttgagacc 3600

ttcattgttt tcatgattct ccttagtagt ggtgctctgg catttgaaga tatatatatt 3660

gatcagcgaa agacgattaa gacgatgttg gaatatgctg acaaggtttt cacttacatt 3720

ttcattctgg aaatgcttct aaaatgggtg gcatatggct atcaaacata tttcaccaat 3780

gcctggtgtt ggctggactt cttaattgtt gatgtttcat tggtcagttt aacagcaaat 3840

gccttgggtt actcagaact tggagccatc aaatctctca ggacactaag agctctgaga 3900

cctctaagag ccttatctcg atttgaaggg atgagggtgg ttgtgaatgc ccttttagga 3960

gcaattccat ccatcatgaa tgtgcttctg gtttgtctta tattctggct aattttcagc 4020

atcatgggcg taaatttgtt tgctggcaaa ttctaccact gtattaacac cacaactggt 4080

gacaggtttg acatcgaaga cgtgaataat catactgatt gcctaaaact aatagaaaga 4140

aatgagactg ctcgatggaa aaatgtgaaa gtaaactttg ataatgtagg atttgggtat 4200

ctctctttgc ttcaagttgc cacattcaaa ggatggatgg atataatgta tgcagcagtt 4260

gattccagaa atgtggaact ccagcctaag tatgaagaaa gtctgtacat gtatctttac 4320

tttgttattt tcatcatctt tgggtccttc ttcaccttga acctgtttat tggtgtcatc 4380

atagataatt tcaaccagca gaaaaagaag tttggaggtc aagacatctt tatgacagaa 4440

gaacagaaga aatactataa tgcaatgaaa aaattaggat cgaaaaaacc gcaaaagcct 4500

atacctcgac caggaaacaa atttcaagga atggtctttg acttcgtaac cagacaagtt 4560

tttgacataa gcatcatgat tctcatctgt cttaacatgg tcacaatgat ggtggaaaca 4620

gatgaccaga gtgaatatgt gactaccatt ttgtcacgca tcaatctggt gttcattgtg 4680

ctatttactg gagagtgtgt actgaaactc atctctctac gccattatta ttttaccatt 4740

ggatggaata tttttgattt tgtggttgtc attctctcca ttgtaggtat gtttcttgcc 4800

gagctgatag aaaagtattt cgtgtcccct accctgttcc gagtgatccg tcttgctagg 4860

attggccgaa tcctacgtct gatcaaagga gcaaagggga tccgcacgct gctctttgct 4920

ttgatgatgt cccttcctgc gttgtttaac atcggcctcc tactcttcct agtcatgttc 4980

atctacgcca tctttgggat gtccaacttt gcctatgtta agagggaagt tgggatcgat 5040

gacatgttca actttgagac ctttggcaac agcatgatct gcctattcca aattacaacc 5100

tctgctggct gggatggatt gctagcaccc attctcaaca gtaagccacc cgactgtgac 5160

cctaataaag ttaaccctgg aagctcagtt aagggagact gtgggaaccc atctgttgga 5220

attttctttt ttgtcagtta catcatcata tccttcctgg ttgtggtgaa catgtacatc 5280

gcggtcatcc tggagaactt cagtgttgct actgaagaaa gtgcagagcc tctgagtgag 5340

gatgactttg agatgttcta tgaggtttgg gagaagtttg atcccgatgc aactcagttc 5400

atggaatttg aaaaattatc tcagtttgca gctgcgcttg aaccgcctct caatctgcca 5460

caaccaaaca aactccagct cattgccatg gatttgccca tggtgagtgg tgaccggatc 5520

cactgtcttg atatcttatt tgcttttaca aagcgggttc taggagagag tggagagatg 5580

gatgctctac gaatacagat ggaagagcga ttcatggctt ccaatccttc caaggtctcc 5640

tatcagccaa tcactactac tttaaaacga aaacaagagg aagtatctgc tgtcattatt 5700

cagcgtgctt acagacgcca ccttttaaag cgaactgtaa aacaagcttc ctttacgtac 5760

aataaaaaca aaatcaaagg tggggctaat cttcttataa aagaagacat gataattgac 5820

agaataaatg aaaactctat tacagaaaaa actgatctga ccatgtccac tgcagcttgt 5880

ccaccttcct atgaccgggt gacaaagcca attgtggaaa aacatgagca agaaggcaaa 5940

gatgaaaaag ccaaagggaa ataaatgaaa ataaataaaa ataattgggt gacaaattgt 6000

ttacagcctg tgaaggtgat gtatttttat caacaggact cctttaggag gtcaatgcca 6060

aactgactgt ttttacacaa atctccttaa ggtcagtgcc tacaataaga cagtgacccc 6120

ttgtcagcaa actgtgactc tgtgtaaagg ggagatgacc ttgacaggag gttactgttc 6180

tcactaccag ctgacactgc tgaagataag atgcacaatg gctagtcaga ctgtagggac 6240

cagtttcaag gggtgcaaac ctgtgatttt ggggttgttt aacatgaaac actttagtgt 6300

agtaattgta tccactgttt gcatttcaac tgccacattt gtcacatttt tatggaatct 6360

gttagtggat tcatcttttt gttaatccat gtgtttatta tatgtgacta tttttgtaaa 6420

cgaagtttct gttgagaaat aggctaagga cctctataac aggtatgcca cctggggggt 6480

atggcaacca catggccctc ccagctacac aaagtcgtgg tttgcatgag ggcatgctgc 6540

acttagagat catgcatgag aaaaagtcac aagaaaaaca aattcttaaa tttcaccata 6600

tttctgggag gggtaattgg gtgataagtg gaggtgcttt gttgatcttg ttttgcgaaa 6660

tccagcccct agaccaagta gattatttgt gggtaggcca gtaaatctta gcaggtgcaa 6720

acttcattca aatgtttgga gtcataaatg ttatgtttct ttttgttgta ttaaaaaaaa 6780

aacctgaata gtgaatattg cccctcaccc tccaccgcca gaagactgaa ttgaccaaaa 6840

ttactcttta taaatttctg ctttttcctg cactttgttt agccatcttc ggctctcagc 6900

aaggttgaca ctgtatatgt taatgaaatg ctatttatta tgtaaatagt cattttaccc 6960

tgtggtgcac gtttgagcaa acaaataatg acctaagcac agtatttatt gcatcaaata 7020

tgtaccacaa gaaatgtaga gtgcaagctt tacacaggta ataaaatgta ttctgtacca 7080

tttatagata gtttggatgc tatcaatgca tgtttatatt accatgctgc tgtatctggt 7140

ttctctcact gctcagaatc tcatttatga gaaaccatat gtcagtggta aagtcaagga 7200

aattgttcaa cagatctcat ttatttaagt cattaagcaa tagtttgcag cactttaaca 7260

gctttttggt tatttttaca ttttaagtgg ataacatatg gtatatagcc agactgtaca 7320

gacatgttta aaaaaacaca ctgcttaacc tattaaatat gtgtttagaa ttttataagc 7380

aaatataaat actgtaaaaa gtcactttat tttatttttc agcattatgt acataaatat 7440

gaagaggaaa ttatcttcag gttgatatca caatcacttt tcttactttc tgtccatagt 7500

actttttcat gaaagaaatt tgctaaataa gacatgaaaa caagactggg tagttgtaga 7560

tttctgcttt ttaaattaca tttgctaatt ttagattatt tcacaatttt aaggagcaaa 7620

ataggttcac gattcatatc caaattatgc tttgcaattg gaaaagggtt taaaatttta 7680

tttatatttc tggtagtacc tgcactaact gaattgaagg tagtgcttat gttatttttg 7740

ttcttttttt ctgacttcgg tttatgtttt catttctttg gagtaatgct gctctagatt 7800

gttctaaata gaatgtgggc ttcataattt ttttttccac aaaaacagag tagtcaactt 7860

atatagtcaa ttacatcagg acattttgtg tttcttacag aagcaaacca taggctcctc 7920

ttttccttaa aactacttag ataaactgta ttcgtgaact gcatgctgga aaatgctact 7980

attatgctaa ataatgctaa ccaacattta aaatgtgcaa aactaataaa gattacattt 8040

tttatttta 8049

<210> SEQ ID NO: 5

<211> LENGTH: 8342

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 5

gaaactcatg gaactgttcc tccagattaa cacttcaggg gttatggaag ctggaggaag 60

ctgagctttt actacatctt ttgggggttt gggcaattat gaataaggct gctgtataca 120

tccgtgtgca ggattttgtg tggacataag ttttcaactc ctttggttaa atcctaagga 180

atttcatatg cagaataaat ggtaattaaa atgtgcagga tgacaagatg gagcaaacag 240

tgcttgtacc accaggacct gacagcttca acttcttcac cagagaatct cttgcggcta 300

ttgaaagacg cattgcagaa gaaaaggcaa agaatcccaa accagacaaa aaagatgacg 360

acgaaaatgg cccaaagcca aatagtgact tggaagctgg aaagaacctt ccatttattt 420

atggagacat tcctccagag atggtgtcag agcccctgga ggacctggac ccctactata 480

tcaataagaa aacttttata gtattgaata aagggaaggc catcttccgg ttcagtgcca 540

cctctgccct gtacatttta actcccttca atcctcttag gaaaatagct attaagattt 600

tggtacattc attattcagc atgctaatta tgtgcactat tttgacaaac tgtgtgttta 660

tgacaatgag taaccctcct gattggacaa agaatgtaga atacaccttc acaggaatat 720

atacttttga atcacttata aaaattattg caaggggatt ctgtttagaa gattttactt 780

tccttcggga tccatggaac tggctcgatt tcactgtcat tacatttgcg tacgtcacag 840

agtttgtgga cctgggcaat gtctcggcat tgagaacatt cagagttctc cgagcattga 900

agacgatttc agtcattcca ggcctgaaaa ccattgtggg agccctgatc cagtctgtga 960

agaagctctc agatgtaatg atcctgactg tgttctgtct gagcgtattt gctctaattg 1020

ggctgcagct gttcatgggc aacctgagga ataaatgtat acaatggcct cccaccaatg 1080

cttccttgga ggaacatagt atagaaaaga atataactgt gaattataat ggtacactta 1140

taaatgaaac tgtctttgag tttgactgga agtcatatat tcaagattca agatatcatt 1200

atttcctgga gggtttttta gatgcactac tatgtggaaa tagctctgat gcaggccaat 1260

gtccagaggg atatatgtgt gtgaaagctg gtagaaatcc caattatggc tacacaagct 1320

ttgatacctt cagttgggct tttttgtcct tgtttcgact aatgactcag gacttctggg 1380

aaaatcttta tcaactgaca ttacgtgctg ctgggaaaac gtacatgata ttttttgtat 1440

tggtcatttt cttgggctca ttctacctaa taaatttgat cctggctgtg gtggccatgg 1500

cctacgagga acagaatcag gccaccttgg aagaagcaga acagaaagag gccgaatttc 1560

agcagatgat tgaacagctt aaaaagcaac aggaggcagc tcagcaggca gcaacggcaa 1620

ctgcctcaga acattccaga gagcccagtg cagcaggcag gctctcagac agctcatctg 1680

aagcctctaa gttgagttcc aagagtgcta aggaaagaag aaatcggagg aagaaaagaa 1740

aacagaaaga gcagtctggt ggggaagaga aagatgagga tgaattccaa aaatctgaat 1800

ctgaggacag catcaggagg aaaggttttc gcttctccat tgaagggaac cgattgacat 1860

atgaaaagag gtactcctcc ccacaccagt ctttgttgag catccgtggc tccctatttt 1920

caccaaggcg aaatagcaga acaagccttt tcagctttag agggcgagca aaggatgtgg 1980

gatctgagaa cgacttcgca gatgatgagc acagcacctt tgaggataac gagagccgta 2040

gagattcctt gtttgtgccc cgacgacacg gagagagacg caacagcaac ctgagtcaga 2100

ccagtaggtc atcccggatg ctggcagtgt ttccagcgaa tgggaagatg cacagcactg 2160

tggattgcaa tggtgtggtt tccttggttg gtggaccttc agttcctaca tcgcctgttg 2220

gacagcttct gccagaggtg ataatagata agccagctac tgatgacaat ggaacaacca 2280

ctgaaactga aatgagaaag agaaggtcaa gttctttcca cgtttccatg gactttctag 2340

aagatccttc ccaaaggcaa cgagcaatga gtatagccag cattctaaca aatacagtag 2400

aagaacttga agaatccagg cagaaatgcc caccctgttg gtataaattt tccaacatat 2460

tcttaatctg ggactgttct ccatattggt taaaagtgaa acatgttgtc aacctggttg 2520

tgatggaccc atttgttgac ctggccatca ccatctgtat tgtcttaaat actcttttca 2580

tggccatgga gcactatcca atgacggacc atttcaataa tgtgcttaca gtaggaaact 2640

tggttttcac tgggatcttt acagcagaaa tgtttctgaa aattattgcc atggatcctt 2700

actattattt ccaagaaggc tggaatatct ttgacggttt tattgtgacg cttagcctgg 2760

tagaacttgg actcgccaat gtggaaggat tatctgttct ccgttcattt cgattgctgc 2820

gagttttcaa gttggcaaaa tcttggccaa cgttaaatat gctaataaag atcatcggca 2880

attccgtggg ggctctggga aatttaaccc tcgtcttggc catcatcgtc ttcatttttg 2940

ccgtggtcgg catgcagctc tttggtaaaa gctacaaaga ttgtgtctgc aagatcgcca 3000

gtgattgtca actcccacgc tggcacatga atgacttctt ccactccttc ctgattgtgt 3060

tccgcgtgct gtgtggggag tggatagaga ccatgtggga ctgtatggag gttgctggtc 3120

aagccatgtg ccttactgtc ttcatgatgg tcatggtgat tggaaaccta gtggtcctga 3180

atctctttct ggccttgctt ctgagctcat ttagtgcaga caaccttgca gccactgatg 3240

atgataatga aatgaataat ctccaaattg ctgtggatag gatgcacaaa ggagtagctt 3300

atgtgaaaag aaaaatatat gaatttattc aacagtcctt cattaggaaa caaaagattt 3360

tagatgaaat taaaccactt gatgatctaa acaacaagaa agacagttgt atgtccaatc 3420

atacagcaga aattgggaaa gatcttgact atcttaaaga tgtaaatgga actacaagtg 3480

gtataggaac tggcagcagt gttgaaaaat acattattga tgaaagtgat tacatgtcat 3540

tcataaacaa ccccagtctt actgtgactg taccaattgc tgtaggagaa tctgactttg 3600

aaaatttaaa cacggaagac tttagtagtg aatcggatct ggaagaaagc aaagagaaac 3660

tgaatgaaag cagtagctca tcagaaggta gcactgtgga catcggcgca cctgtagaag 3720

aacagcccgt agtggaacct gaagaaactc ttgaaccaga agcttgtttc actgaaggct 3780

gtgtacaaag attcaagtgt tgtcaaatca atgtggaaga aggcagagga aaacaatggt 3840

ggaacctgag aaggacgtgt ttccgaatag ttgaacataa ctggtttgag accttcattg 3900

ttttcatgat tctccttagt agtggtgctc tggcatttga agatatatat attgatcagc 3960

gaaagacgat taagacgatg ttggaatatg ctgacaaggt tttcacttac attttcattc 4020

tggaaatgct tctaaaatgg gtggcatatg gctatcaaac atatttcacc aatgcctggt 4080

gttggctgga cttcttaatt gttgatgttt cattggtcag tttaacagca aatgccttgg 4140

gttactcaga acttggagcc atcaaatctc tcaggacact aagagctctg agacctctaa 4200

gagccttatc tcgatttgaa gggatgaggg tggttgtgaa tgccctttta ggagcaattc 4260

catccatcat gaatgtgctt ctggtttgtc ttatattctg gctaattttc agcatcatgg 4320

gcgtaaattt gtttgctggc aaattctacc actgtattaa caccacaact ggtgacaggt 4380

ttgacatcga agacgtgaat aatcatactg attgcctaaa actaatagaa agaaatgaga 4440

ctgctcgatg gaaaaatgtg aaagtaaact ttgataatgt aggatttggg tatctctctt 4500

tgcttcaagt tgccacattc aaaggatgga tggatataat gtatgcagca gttgattcca 4560

gaaatgtgga actccagcct aagtatgaag aaagtctgta catgtatctt tactttgtta 4620

ttttcatcat ctttgggtcc ttcttcacct tgaacctgtt tattggtgtc atcatagata 4680

atttcaacca gcagaaaaag aagtttggag gtcaagacat ctttatgaca gaagaacaga 4740

agaaatacta taatgcaatg aaaaaattag gatcgaaaaa accgcaaaag cctatacctc 4800

gaccaggaaa caaatttcaa ggaatggtct ttgacttcgt aaccagacaa gtttttgaca 4860

taagcatcat gattctcatc tgtcttaaca tggtcacaat gatggtggaa acagatgacc 4920

agagtgaata tgtgactacc attttgtcac gcatcaatct ggtgttcatt gtgctattta 4980

ctggagagtg tgtactgaaa ctcatctctc tacgccatta ttattttacc attggatgga 5040

atatttttga ttttgtggtt gtcattctct ccattgtagg tatgtttctt gccgagctga 5100

tagaaaagta tttcgtgtcc cctaccctgt tccgagtgat ccgtcttgct aggattggcc 5160

gaatcctacg tctgatcaaa ggagcaaagg ggatccgcac gctgctcttt gctttgatga 5220

tgtcccttcc tgcgttgttt aacatcggcc tcctactctt cctagtcatg ttcatctacg 5280

ccatctttgg gatgtccaac tttgcctatg ttaagaggga agttgggatc gatgacatgt 5340

tcaactttga gacctttggc aacagcatga tctgcctatt ccaaattaca acctctgctg 5400

gctgggatgg attgctagca cccattctca acagtaagcc acccgactgt gaccctaata 5460

aagttaaccc tggaagctca gttaagggag actgtgggaa cccatctgtt ggaattttct 5520

tttttgtcag ttacatcatc atatccttcc tggttgtggt gaacatgtac atcgcggtca 5580

tcctggagaa cttcagtgtt gctactgaag aaagtgcaga gcctctgagt gaggatgact 5640

ttgagatgtt ctatgaggtt tgggagaagt ttgatcccga tgcaactcag ttcatggaat 5700

ttgaaaaatt atctcagttt gcagctgcgc ttgaaccgcc tctcaatctg ccacaaccaa 5760

acaaactcca gctcattgcc atggatttgc ccatggtgag tggtgaccgg atccactgtc 5820

ttgatatctt atttgctttt acaaagcggg ttctaggaga gagtggagag atggatgctc 5880

tacgaataca gatggaagag cgattcatgg cttccaatcc ttccaaggtc tcctatcagc 5940

caatcactac tactttaaaa cgaaaacaag aggaagtatc tgctgtcatt attcagcgtg 6000

cttacagacg ccacctttta aagcgaactg taaaacaagc ttcctttacg tacaataaaa 6060

acaaaatcaa aggtggggct aatcttctta taaaagaaga catgataatt gacagaataa 6120

atgaaaactc tattacagaa aaaactgatc tgaccatgtc cactgcagct tgtccacctt 6180

cctatgaccg ggtgacaaag ccaattgtgg aaaaacatga gcaagaaggc aaagatgaaa 6240

aagccaaagg gaaataaatg aaaataaata aaaataattg ggtgacaaat tgtttacagc 6300

ctgtgaaggt gatgtatttt tatcaacagg actcctttag gaggtcaatg ccaaactgac 6360

tgtttttaca caaatctcct taaggtcagt gcctacaata agacagtgac cccttgtcag 6420

caaactgtga ctctgtgtaa aggggagatg accttgacag gaggttactg ttctcactac 6480

cagctgacac tgctgaagat aagatgcaca atggctagtc agactgtagg gaccagtttc 6540

aaggggtgca aacctgtgat tttggggttg tttaacatga aacactttag tgtagtaatt 6600

gtatccactg tttgcatttc aactgccaca tttgtcacat ttttatggaa tctgttagtg 6660

gattcatctt tttgttaatc catgtgttta ttatatgtga ctatttttgt aaacgaagtt 6720

tctgttgaga aataggctaa ggacctctat aacaggtatg ccacctgggg ggtatggcaa 6780

ccacatggcc ctcccagcta cacaaagtcg tggtttgcat gagggcatgc tgcacttaga 6840

gatcatgcat gagaaaaagt cacaagaaaa acaaattctt aaatttcacc atatttctgg 6900

gaggggtaat tgggtgataa gtggaggtgc tttgttgatc ttgttttgcg aaatccagcc 6960

cctagaccaa gtagattatt tgtgggtagg ccagtaaatc ttagcaggtg caaacttcat 7020

tcaaatgttt ggagtcataa atgttatgtt tctttttgtt gtattaaaaa aaaaacctga 7080

atagtgaata ttgcccctca ccctccaccg ccagaagact gaattgacca aaattactct 7140

ttataaattt ctgctttttc ctgcactttg tttagccatc ttcggctctc agcaaggttg 7200

acactgtata tgttaatgaa atgctattta ttatgtaaat agtcatttta ccctgtggtg 7260

cacgtttgag caaacaaata atgacctaag cacagtattt attgcatcaa atatgtacca 7320

caagaaatgt agagtgcaag ctttacacag gtaataaaat gtattctgta ccatttatag 7380

atagtttgga tgctatcaat gcatgtttat attaccatgc tgctgtatct ggtttctctc 7440

actgctcaga atctcattta tgagaaacca tatgtcagtg gtaaagtcaa ggaaattgtt 7500

caacagatct catttattta agtcattaag caatagtttg cagcacttta acagcttttt 7560

ggttattttt acattttaag tggataacat atggtatata gccagactgt acagacatgt 7620

ttaaaaaaac acactgctta acctattaaa tatgtgttta gaattttata agcaaatata 7680

aatactgtaa aaagtcactt tattttattt ttcagcatta tgtacataaa tatgaagagg 7740

aaattatctt caggttgata tcacaatcac ttttcttact ttctgtccat agtacttttt 7800

catgaaagaa atttgctaaa taagacatga aaacaagact gggtagttgt agatttctgc 7860

tttttaaatt acatttgcta attttagatt atttcacaat tttaaggagc aaaataggtt 7920

cacgattcat atccaaatta tgctttgcaa ttggaaaagg gtttaaaatt ttatttatat 7980

ttctggtagt acctgcacta actgaattga aggtagtgct tatgttattt ttgttctttt 8040

tttctgactt cggtttatgt tttcatttct ttggagtaat gctgctctag attgttctaa 8100

atagaatgtg ggcttcataa tttttttttc cacaaaaaca gagtagtcaa cttatatagt 8160

caattacatc aggacatttt gtgtttctta cagaagcaaa ccataggctc ctcttttcct 8220

taaaactact tagataaact gtattcgtga actgcatgct ggaaaatgct actattatgc 8280

taaataatgc taaccaacat ttaaaatgtg caaaactaat aaagattaca ttttttattt 8340

ta 8342

<210> SEQ ID NO: 6

<211> LENGTH: 3806

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 6

gccagtgccg cgcgtcgagc ggagcagagg aggcgagggc ggagggccag agaggcagtt 60

ggaagatggc ggacgaggtg gcgctcgccc ttcaggccgc cggctcccct tccgcggcgg 120

ccgccatgga ggccgcgtcg cagccggcgg acgagccgct ccgcaagagg ccccgccgag 180

acgggcctgg cctcgggcgc agcccgggcg agccgagcgc agcagtggcg ccggcggccg 240

cggggtgtga ggcggcgagc gccgcggccc cggcggcgct gtggcgggag gcggcagggg 300

cggcggcgag cgcggagcgg gaggccccgg cgacggccgt ggccggggac ggagacaatg 360

ggtccggcct gcggcgggag ccgagggcgg ctgacgactt cgacgacgac gagggcgagg 420

aggaggacga ggcggcggcg gcagcggcgg cggcagcgat cggctaccga ggtccatata 480

cttttgttca gcaacatctc atgattggca ccgatcctcg aacaattctt aaagatttat 540

taccagaaac aattcctcca cctgagctgg atgatatgac gctgtggcag attgttatta 600

atatcctttc agaaccacca aagcggaaaa aaagaaaaga tatcaataca attgaagatg 660

ctgtgaagtt actgcaggag tgtaaaaaga taatagttct gactggagct ggggtttctg 720

tctcctgtgg gattcctgac ttcagatcaa gagacggtat ctatgctcgc cttgcggtgg 780

acttcccaga cctcccagac cctcaagcca tgtttgatat tgagtatttt agaaaagacc 840

caagaccatt cttcaagttt gcaaaggaaa tatatcccgg acagttccag ccgtctctgt 900

gtcacaaatt catagctttg tcagataagg aaggaaaact acttcgaaat tatactcaaa 960

atatagatac cttggagcag gttgcaggaa tccaaaggat ccttcagtgt catggttcct 1020

ttgcaacagc atcttgcctg atttgtaaat acaaagttga ttgtgaagct gttcgtggag 1080

acatttttaa tcaggtagtt cctcggtgcc ctaggtgccc agctgatgag ccacttgcca 1140

tcatgaagcc agagattgtc ttctttggtg aaaacttacc agaacagttt catagagcca 1200

tgaagtatga caaagatgaa gttgacctcc tcattgttat tggatcttct ctgaaagtga 1260

gaccagtagc actaattcca agttctatac cccatgaagt gcctcaaata ttaataaata 1320

gggaaccttt gcctcatcta cattttgatg tagagctcct tggagactgc gatgttataa 1380

ttaatgagtt gtgtcatagg ctaggtggtg aatatgccaa actttgttgt aaccctgtaa 1440

agctttcaga aattactgaa aaacctccac gcccacaaaa ggaattggtt catttatcag 1500

agttgccacc aacacctctt catatttcgg aagactcaag ttcacctgaa agaactgtac 1560

cacaagactc ttctgtgatt gctacacttg tagaccaagc aacaaacaac aatgttaatg 1620

atttagaagt atctgaatca agttgtgtgg aagaaaaacc acaagaagta cagactagta 1680

ggaatgttga gaacattaat gtggaaaatc cagattttaa ggctgttggt tccagtactg 1740

cagacaaaaa tgaaagaact tcagttgcag aaacagtgag aaaatgctgg cctaatagac 1800

ttgcaaagga gcagattagt aagcggcttg agggtaatca atacctgttt gtaccaccaa 1860

atcgttacat attccacggt gctgaggtat actcagactc tgaagatgac gtcttgtcct 1920

ctagttcctg tggcagtaac agtgacagtg gcacatgcca gagtccaagt ttagaagaac 1980

ccttggaaga tgaaagtgaa attgaagaat tctacaatgg cttggaagat gatacggaga 2040

ggcccgaatg tgctggagga tctggatttg gagctgatgg aggggatcaa gaggttgtta 2100

atgaagctat agctacaaga caggaattga cagatgtaaa ctatccatca gacaaatcat 2160

aacactattg aagctgtccg gattcaggaa ttgctccacc agcattggga actttagcat 2220

gtcaaaaaat gaatgtttac ttgtgaactt gaacaaggaa atctgaaaga tgtattattt 2280

atagactgga aaatagattg tcttcttgga taatttctaa agttccatca tttctgtttg 2340

tacttgtaca ttcaacactg ttggttgact tcatcttcct ttcaaggttc atttgtatga 2400

tacattcgta tgtatgtata attttgtttt ttgcctaatg agtttcaacc ttttaaagtt 2460

ttcaaaagcc attggaatgt taatgtaaag ggaacagctt atctagacca aagaatggta 2520

tttcacactt ttttgtttgt aacattgaat agtttaaagc cctcaatttc tgttctgctg 2580

aacttttatt tttaggacag ttaacttttt aaacactggc attttccaaa acttgtggca 2640

gctaactttt taaaatcaca gatgacttgt aatgtgagga gtcagcaccg tgtctggagc 2700

actcaaaact tggtgctcag tgtgtgaagc gtacttactg catcgttttt gtacttgctg 2760

cagacgtggt aatgtccaaa caggcccctg agactaatct gataaatgat ttggaaatgt 2820

gtttcagttg ttctagaaac aatagtgcct gtctatatag gtccccttag tttgaatatt 2880

tgccattgtt taattaaata cctatcactg tggtagagcc tgcatagatc ttcaccacaa 2940

atactgccaa gatgtgaata tgcaaagcct ttctgaatct aataatggta cttctactgg 3000

ggagagtgta atattttgga ctgctgtttt tccattaatg aggaaagcaa taggcctctt 3060

aattaaagtc ccaaagtcat aagataaatt gtagctcaac cagaaagtac actgttgcct 3120

gttgaggatt tggtgtaatg tatcccaagg tgttagcctt gtattatgga gatgaataca 3180

gatccaatag tcaaatgaaa ctagttctta gttatttaaa agcttagctt gccttaaaac 3240

tagggatcaa ttttctcaac tgcagaaact tttagccttt caaacagttc acacctcaga 3300

aagtcagtat ttattttaca gacttctttg gaacattgcc cccaaattta aatattcatg 3360

tgggtttagt atttattaca aaaaaatgat ttgaaatata gctgttcttt atgcataaaa 3420

tacccagtta ggaccattac tgccagagga gaaaagtatt aagtagctca tttccctacc 3480

taaaagataa ctgaatttat ttggctacac taaagaatgc agtatattta gttttccatt 3540

tgcatgatgt gtttgtgcta tagacaatat tttaaattga aaaatttgtt ttaaattatt 3600

tttacagtga agactgtttt cagctctttt tatattgtac atagactttt atgtaatctg 3660

gcatatgttt tgtagaccgt ttaatgactg gattatcttc ctccaacttt tgaaatacaa 3720

aaacagtgtt ttatacttgt atcttgtttt aaagtcttat attaaaattg tcatttgact 3780

tttttcccgt taaaaaaaaa aaaaaa 3806

<210> SEQ ID NO: 7

<211> LENGTH: 3920

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 7

agtgccgcgc gtcgagcgga gcagaggagg cgagggcgga gggccagaga ggcagttgga 60

agatggcgga cgaggtggcg ctcgcccttc aggccgccgg ctccccttcc gcggcggccg 120

ccatggaggc cgcgtcgcag ccggcggacg agccgctccg caagaggccc cgccgagacg 180

ggcctggcct cgggcgcagc ccgggcgagc cgagcgcagc agtggcgccg gcggccgcgg 240

ggtgtgaggc ggcgagcgcc gcggccccgg cggcgctgtg gcgggaggcg gcaggggcgg 300

cggcgagcgc ggagcgggag gccccggcga cggccgtggc cggggacgga gacaatgggt 360

ccggcctgcg gcgggagccg agggcggctg acgacttcga cgacgacgag ggcgaggagg 420

aggacgaggc ggcggcggca gcggcggcgg cagcgatcgg ctaccgagac aacctcctgt 480

tgaccgatgg actcctcact aatggctttc attcctgtga aagtgatgac gatgacagaa 540

cgtcacacgc cagctctagt gactggactc cgcggccgcg gataggtcca tatacttttg 600

ttcagcaaca tctcatgatt ggcaccgatc ctcgaacaat tcttaaagat ttattaccag 660

aaacaattcc tccacctgag ctggatgata tgacgctgtg gcagattgtt attaatatcc 720

tttcagaacc accaaagcgg aaaaaaagaa aagatatcaa tacaattgaa gatgctgtga 780

agttactgca ggagtgtaaa aagataatag ttctgactgg agctggggtt tctgtctcct 840

gtgggattcc tgacttcaga tcaagagacg gtatctatgc tcgccttgcg gtggacttcc 900

cagacctccc agaccctcaa gccatgtttg atattgagta ttttagaaaa gacccaagac 960

cattcttcaa gtttgcaaag gaaatatatc ccggacagtt ccagccgtct ctgtgtcaca 1020

aattcatagc tttgtcagat aaggaaggaa aactacttcg aaattatact caaaatatag 1080

ataccttgga gcaggttgca ggaatccaaa ggatccttca gtgtcatggt tcctttgcaa 1140

cagcatcttg cctgatttgt aaatacaaag ttgattgtga agctgttcgt ggagacattt 1200

ttaatcaggt agttcctcgg tgccctaggt gcccagctga tgagccactt gccatcatga 1260

agccagagat tgtcttcttt ggtgaaaact taccagaaca gtttcataga gccatgaagt 1320

atgacaaaga tgaagttgac ctcctcattg ttattggatc ttctctgaaa gtgagaccag 1380

tagcactaat tccaagttct ataccccatg aagtgcctca aatattaata aatagggaac 1440

ctttgcctca tctacatttt gatgtagagc tccttggaga ctgcgatgtt ataattaatg 1500

agttgtgtca taggctaggt ggtgaatatg ccaaactttg ttgtaaccct gtaaagcttt 1560

cagaaattac tgaaaaacct ccacgcccac aaaaggaatt ggttcattta tcagagttgc 1620

caccaacacc tcttcatatt tcggaagact caagttcacc tgaaagaact gtaccacaag 1680

actcttctgt gattgctaca cttgtagacc aagcaacaaa caacaatgtt aatgatttag 1740

aagtatctga atcaagttgt gtggaagaaa aaccacaaga agtacagact agtaggaatg 1800

ttgagaacat taatgtggaa aatccagatt ttaaggctgt tggttccagt actgcagaca 1860

aaaatgaaag aacttcagtt gcagaaacag tgagaaaatg ctggcctaat agacttgcaa 1920

aggagcagat tagtaagcgg cttgagggta atcaatacct gtttgtacca ccaaatcgtt 1980

acatattcca cggtgctgag gtatactcag actctgaaga tgacgtcttg tcctctagtt 2040

cctgtggcag taacagtgac agtggcacat gccagagtcc aagtttagaa gaacccttgg 2100

aagatgaaag tgaaattgaa gaattctaca atggcttgga agatgatacg gagaggcccg 2160

aatgtgctgg aggatctgga tttggagctg atggagggga tcaagaggtt gttaatgaag 2220

ctatagctac aagacaggaa ttgacagatg taaactatcc atcagacaaa tcataacact 2280

attgaagctg tccggattca ggaattgctc caccagcatt gggaacttta gcatgtcaaa 2340

aaatgaatgt ttacttgtga acttgaacaa ggaaatctga aagatgtatt atttatagac 2400

tggaaaatag attgtcttct tggataattt ctaaagttcc atcatttctg tttgtacttg 2460

tacattcaac actgttggtt gacttcatct tcctttcaag gttcatttgt atgatacatt 2520

cgtatgtatg tataattttg ttttttgcct aatgagtttc aaccttttaa agttttcaaa 2580

agccattgga atgttaatgt aaagggaaca gcttatctag accaaagaat ggtatttcac 2640

acttttttgt ttgtaacatt gaatagttta aagccctcaa tttctgttct gctgaacttt 2700

tatttttagg acagttaact ttttaaacac tggcattttc caaaacttgt ggcagctaac 2760

tttttaaaat cacagatgac ttgtaatgtg aggagtcagc accgtgtctg gagcactcaa 2820

aacttggtgc tcagtgtgtg aagcgtactt actgcatcgt ttttgtactt gctgcagacg 2880

tggtaatgtc caaacaggcc cctgagacta atctgataaa tgatttggaa atgtgtttca 2940

gttgttctag aaacaatagt gcctgtctat ataggtcccc ttagtttgaa tatttgccat 3000

tgtttaatta aatacctatc actgtggtag agcctgcata gatcttcacc acaaatactg 3060

ccaagatgtg aatatgcaaa gcctttctga atctaataat ggtacttcta ctggggagag 3120

tgtaatattt tggactgctg tttttccatt aatgaggaaa gcaataggcc tcttaattaa 3180

agtcccaaag tcataagata aattgtagct caaccagaaa gtacactgtt gcctgttgag 3240

gatttggtgt aatgtatccc aaggtgttag ccttgtatta tggagatgaa tacagatcca 3300

atagtcaaat gaaactagtt cttagttatt taaaagctta gcttgcctta aaactaggga 3360

tcaattttct caactgcaga aacttttagc ctttcaaaca gttcacacct cagaaagtca 3420

gtatttattt tacagacttc tttggaacat tgcccccaaa tttaaatatt catgtgggtt 3480

tagtatttat tacaaaaaaa tgatttgaaa tatagctgtt ctttatgcat aaaataccca 3540

gttaggacca ttactgccag aggagaaaag tattaagtag ctcatttccc tacctaaaag 3600

ataactgaat ttatttggct acactaaaga atgcagtata tttagttttc catttgcatg 3660

atgtgtttgt gctatagaca atattttaaa ttgaaaaatt tgttttaaat tatttttaca 3720

gtgaagactg ttttcagctc tttttatatt gtacatagac ttttatgtaa tctggcatat 3780

gttttgtaga ccgtttaatg actggattat cttcctccaa cttttgaaat acaaaaacag 3840

tgttttatac ttgtatcttg ttttaaagtc ttatattaaa attgtcattt gacttttttc 3900

ccgttaaaaa aaaaaaaaaa 3920

<210> SEQ ID NO: 8

<211> LENGTH: 3400

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 8

ggagggccag agaggcagtt ggaagatggc ggcggcagcg gcggcggcag cgatcggcta 60

ccgaggtcca tatacttttg ttcagcaaca tctcatgatt ggcaccgatc ctcgaacaat 120

tcttaaagat ttattaccag aaacaattcc tccacctgag ctggatgata tgacgctgtg 180

gcagattgtt attaatatcc tttcagaacc accaaagcgg aaaaaaagaa aagatatcaa 240

tacaattgaa gatgctgtga agttactgca ggagtgtaaa aagataatag ttctgactgg 300

agctggggtt tctgtctcct gtgggattcc tgacttcaga tcaagagacg gtatctatgc 360

tcgccttgcg gtggacttcc cagacctccc agaccctcaa gccatgtttg atattgagta 420

ttttagaaaa gacccaagac cattcttcaa gtttgcaaag gaaatatatc ccggacagtt 480

ccagccgtct ctgtgtcaca aattcatagc tttgtcagat aaggaaggaa aactacttcg 540

aaattatact caaaatatag ataccttgga gcaggttgca ggaatccaaa ggatccttca 600

gtgtcatggt tcctttgcaa cagcatcttg cctgatttgt aaatacaaag ttgattgtga 660

agctgttcgt ggagacattt ttaatcaggt agttcctcgg tgccctaggt gcccagctga 720

tgagccactt gccatcatga agccagagat tgtcttcttt ggtgaaaact taccagaaca 780

gtttcataga gccatgaagt atgacaaaga tgaagttgac ctcctcattg ttattggatc 840

ttctctgaaa gtgagaccag tagcactaat tccaagttct ataccccatg aagtgcctca 900

aatattaata aatagggaac ctttgcctca tctacatttt gatgtagagc tccttggaga 960

ctgcgatgtt ataattaatg agttgtgtca taggctaggt ggtgaatatg ccaaactttg 1020

ttgtaaccct gtaaagcttt cagaaattac tgaaaaacct ccacgcccac aaaaggaatt 1080

ggttcattta tcagagttgc caccaacacc tcttcatatt tcggaagact caagttcacc 1140

tgaaagaact gtaccacaag actcttctgt gattgctaca cttgtagacc aagcaacaaa 1200

caacaatgtt aatgatttag aagtatctga atcaagttgt gtggaagaaa aaccacaaga 1260

agtacagact agtaggaatg ttgagaacat taatgtggaa aatccagatt ttaaggctgt 1320

tggttccagt actgcagaca aaaatgaaag aacttcagtt gcagaaacag tgagaaaatg 1380

ctggcctaat agacttgcaa aggagcagat tagtaagcgg cttgagggta atcaatacct 1440

gtttgtacca ccaaatcgtt acatattcca cggtgctgag gtatactcag actctgaaga 1500

tgacgtcttg tcctctagtt cctgtggcag taacagtgac agtggcacat gccagagtcc 1560

aagtttagaa gaacccttgg aagatgaaag tgaaattgaa gaattctaca atggcttgga 1620

agatgatacg gagaggcccg aatgtgctgg aggatctgga tttggagctg atggagggga 1680

tcaagaggtt gttaatgaag ctatagctac aagacaggaa ttgacagatg taaactatcc 1740

atcagacaaa tcataacact attgaagctg tccggattca ggaattgctc caccagcatt 1800

gggaacttta gcatgtcaaa aaatgaatgt ttacttgtga acttgaacaa ggaaatctga 1860

aagatgtatt atttatagac tggaaaatag attgtcttct tggataattt ctaaagttcc 1920

atcatttctg tttgtacttg tacattcaac actgttggtt gacttcatct tcctttcaag 1980

gttcatttgt atgatacatt cgtatgtatg tataattttg ttttttgcct aatgagtttc 2040

aaccttttaa agttttcaaa agccattgga atgttaatgt aaagggaaca gcttatctag 2100

accaaagaat ggtatttcac acttttttgt ttgtaacatt gaatagttta aagccctcaa 2160

tttctgttct gctgaacttt tatttttagg acagttaact ttttaaacac tggcattttc 2220

caaaacttgt ggcagctaac tttttaaaat cacagatgac ttgtaatgtg aggagtcagc 2280

accgtgtctg gagcactcaa aacttggtgc tcagtgtgtg aagcgtactt actgcatcgt 2340

ttttgtactt gctgcagacg tggtaatgtc caaacaggcc cctgagacta atctgataaa 2400

tgatttggaa atgtgtttca gttgttctag aaacaatagt gcctgtctat ataggtcccc 2460

ttagtttgaa tatttgccat tgtttaatta aatacctatc actgtggtag agcctgcata 2520

gatcttcacc acaaatactg ccaagatgtg aatatgcaaa gcctttctga atctaataat 2580

ggtacttcta ctggggagag tgtaatattt tggactgctg tttttccatt aatgaggaaa 2640

gcaataggcc tcttaattaa agtcccaaag tcataagata aattgtagct caaccagaaa 2700

gtacactgtt gcctgttgag gatttggtgt aatgtatccc aaggtgttag ccttgtatta 2760

tggagatgaa tacagatcca atagtcaaat gaaactagtt cttagttatt taaaagctta 2820

gcttgcctta aaactaggga tcaattttct caactgcaga aacttttagc ctttcaaaca 2880

gttcacacct cagaaagtca gtatttattt tacagacttc tttggaacat tgcccccaaa 2940

tttaaatatt catgtgggtt tagtatttat tacaaaaaaa tgatttgaaa tatagctgtt 3000

ctttatgcat aaaataccca gttaggacca ttactgccag aggagaaaag tattaagtag 3060

ctcatttccc tacctaaaag ataactgaat ttatttggct acactaaaga atgcagtata 3120

tttagttttc catttgcatg atgtgtttgt gctatagaca atattttaaa ttgaaaaatt 3180

tgttttaaat tatttttaca gtgaagactg ttttcagctc tttttatatt gtacatagac 3240

ttttatgtaa tctggcatat gttttgtaga ccgtttaatg actggattat cttcctccaa 3300

cttttgaaat acaaaaacag tgttttatac ttgtatcttg ttttaaagtc ttatattaaa 3360

attgtcattt gacttttttc ccgttaaaaa aaaaaaaaaa 3400

<210> SEQ ID NO: 9

<211> LENGTH: 1299

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 9

acgcccggga acccccgacc cctctgagcc cggggtactg cgcccgggtc tccacgccca 60

gagatgctcc ccggtctcca ccgtcgggca agccccaagc gcagcagcgc agagtcctgg 120

ggtcaccaga gctcgtacta ggacatcgtc tccccattta acaccgcctc cggtcccatc 180

tgagttgcaa gtggtgggga tgtggggctc cggatcaaag tccccgaaac cgagcacttc 240

ccgaagcctc cttggcctcg aaacaaaaca ataacgccca actccatcat attccagaac 300

tcccaccacc tgcatacaga cattcagctg cacaagcccc ctccatgcta cagtcaacag 360

gatctccagg ccacggctca agcccaggta ctcacatcag tggttctatc aacactcagg 420

acagacccat agaagaggcc caagcaggcc ctggaagtgc atgtggaggc caccaggcaa 480

ggaattctgg agtcccaggt actcataact ctgggtggca tggccccttt gcaccatgga 540

ctgtttgccc ttagaaaggg atggatctga gctgggcgca gtggctcatg cctgtaatcc 600

cagcactttg ggaggccaag gtgggcagct cacctcaggt caggttggtc tcaaactcct 660

gacctcaggc gatccacctc agcctctcaa agtgctggaa ttataggtgt gagccactgt 720

gcccagccca aaatcattct ttttggaatt ttgaagcata taattccaaa aggtatgaag 780

gtaatcactt agattgctct aataagggaa tgggaacagt taagtcctat acaaataaga 840

caaagataag atactacaaa aaggggatga gcccaagaaa aaaatcaaag tcccagagag 900

agaacagcca ttgattctaa atacacaagt ctatggcccc aacccaaact tgtttcacta 960

agaacaacct gtggtttcga gaatctggtc atcccccaca gtgaatacat gaacacattg 1020

taatgtttga aatgtttatt tttcttgttg atttcttact gttagaagag ctaagtgatt 1080

tggcccaaag tggctaagtg attcggccag tttgtacaca gggatataag tttgctgaca 1140

ccaagctcat actttacaaa tgtaatatct tcataaaaca aaaatactgg gccgggcgcg 1200

gtggctcacg cctgtaatcc cagcattttg ggaggccgag gcgggcggat catgagatca 1260

ggagatcgag accatcctgg ctagcagggt gaagccccg 1299

<210> SEQ ID NO: 10

<211> LENGTH: 720

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 10

agcggggggt tcccgcccgc gcctctccct ccacacctcc ccgcaagcag agggagccgg 60

ctccggcctt ggccagccca gagacaggct cccacagtgc agcggcgggc tgaagggctc 120

ctcaagcact gccagagtgg acaccgaggc cgaggagacg ccaagagcga tgaaacaagc 180

ttctggttca agagtttctt caggttccac tacgggctgt tcttctacag gtgcgccgat 240

gtccacagtg ctaccttctg atgagctact gctttcattc agtttctgct gacttaatat 300

gtgagctgac agaatgcaga gaccacgttg tatatgaagc atccactttg ccttgtacac 360

cagggcattc aataaccact taataactac aaccctgatg atccgattca ctactaaagt 420

cttccgtgtt taaattttca aagtcagatt ctcctacagc aattggtaca gtcacagtaa 480

gactggggtt gtttatgaat gacatgtaat cactttcatc aataatgtat ttttcaacac 540

tgctgccagt tcctatacca cttgtagttc catttacatc tttaagatag tcaagatctt 600

tcccaatttc tgctgtatga ttggacatac aactgtcttt cttgttgttt agatcatcaa 660

gtggtttaat ttcatctaaa atcttttgtt tcctaatgaa ggactgttga ataaattcat 720

<210> SEQ ID NO: 11

<211> LENGTH: 1123

<212> TYPE: DNA

<213> ORGANISM: Homo sapiens

<400> SEQENCE: 11

agcggggggt tcccgcccgc gcctctccct ccacacctcc ccgcaagcag agggagccgg 60

ctccggcctt ggccagccca gagacaggct cccacagtgc agcggcgggc tgaagggctc 120

ctcaagcact gccagagtgg acaccgaggc cgaggagacg ccaagagcga tgaaacaagc 180

ttctggttca agagtttctt caggttccac tacgggctgt tcttctacag gtgcgccgat 240

gtccacagtg ctaccttctg atgagctact gctttcattc agtttctgct gacttaatat 300

gtgagctgac agaatgcaga gaccacgttg tatatgaagc atccactttg ccttgtacac 360

cagggcattc aataaccact taataactac aaccctgatg atccgattca ctactaaagt 420

cttccgtgtt taaattttca aagtcagatt ctcctacagc aattggtaca gtcacagtaa 480

gactggggtt gtttatgaat gacatgtaat cactttcatc aataatgtat ttttcaacac 540

tgctgccagt tcctatacca cttgtagttc catttacatc tttaagatag tcaagatctt 600

tcccaatttc tgctgtatga ttggacatac aactgtcttt cttgttgttt agatcatcaa 660

gtggtttaat ttcatctaaa atcttttgtt tcctaatgaa ggactgttga ataaattcat 720

atatttttct tttcacataa gctactcctt tgtgcatcct atccacagca atttggagat 780

tattcatttc attatcatca tcagtggctg caaggttgtc tgcactaaat gagctcagaa 840

gcaaggccag aaagagattc aggaccttaa aaacaacaaa aacatgatta taattttaca 900

ccaatgtagg gaagagcaga ttacaatcac ttattctttc ttttaagtgt ggaaaaaact 960

ctaagttcta aaacttgatg agaaggaaac accacagcat agtgattaga agatgggtga 1020

tctgaatttg tgactggctc aatagcacat ccttggacaa agacatgatt tctgttgctc 1080

tcaagttctc ccattcgtaa agtgaaattg aatgagctaa tct 1123

<210> SEQ ID NO: 12

<211> LENGTH: 1713

<212> TYPE: DNA

<213> ORGANISM: Mus musculus

<400> SEQENCE: 12

gggctcccct cagcggcctc tggcgcctcc cgcccgcccg acccgttcgc tcgctcgctc 60

gctcgctcgc ttgctcgtcc gggatcgccg cggtggttca agtttgcgat ggcgccgcca 120

cttcccacct gggcctcacg cgtgcacctt gcctgcctgc gcctcttcgc ctcaagtcgg 180

cttttacctc aggggctctg gagagcccaa cctggccgac gccggccttc ctgaggagaa 240

ctcctccacc tgccttgccc ttgctctgtg acagctcttc ctcaggttac ccctgtggtc 300

tctcctcagg aagtttgcgc tctctcccaa tctcccttct caagtgcaat ggaatgccca 360

agccagccct cggggcctgt tgccctcctg gaaagatctg gcgattgagg acccgcccta 420

tctgctctct ggacccacca ggtcctctgt acctcgcttt agtctttggt aaaattcatc 480

tcttggggca gcaagagaga ggacagaagg gagagtggtt ggttctccac aaacttctgt 540

gttaagagtc agattgggcc tgggctcttg tgacttgggc gattgactga accttttcta 600

agcccagttt ttaatcatct ctaaaatgac agggccagga ccgaaagaga ctgtagctca 660

gttgtaaagt cacgcttgcc agacaacccc gaagccctag agagagggag gaaggagggt 720

aagttgaagg taatctccaa ctacttagga agttcaaaaa aggcctggaa tacataagac 780

ctcgtctcaa aaacgaaatt taaaacgata gaccatgaga aatcagctag tcaggtttaa 840

agtaaatgac attagtttta aaatcctagg cagttgatgg tggcacaggc ctttaatccc 900

agcaagctgg aggagacagg aggaggttca ctaggacagc caaggctaca caaagaaacc 960

ctgtctcgaa aaaataatct tacttctaga attgtagaaa tggctctgta gttaacagca 1020

cttgttgctc ctgcagaggc cctaggtttg actcccatca tccacatgac agctcatacc 1080

ttcagatctg acacctgctt ttggtaaaca cagacatgta tggagccaaa agacccaaac 1140

acataaaaat cctctttgtt gttgttttat gagttagggt ttctctgtgt agccctggct 1200

gtccaggaac tctgtagatc aggctgtcct tgaactcaga ggccacctgc ctctgcttct 1260

tgaactgctg ggattaaaga tgtacaccag caagcccagc ataaaaatac atatttaaat 1320

aattttttaa ataatcctta gttccttcac aactctaagc cccttcactt tctagttacc 1380

atgaaattct gagcacctgt atccatttgg atcattaggg ctcaattgca catggttcaa 1440

ttacagtggg gtttccccag attttagagt tagaggcagc aggatcagaa aattaaatcc 1500

atttgcacta ggtaataaat ttgatcccac cctatctcaa aaacaaaaca ctagccacac 1560

gtggcagcac acacctttta caacaggact caggagcctg gcatgatggg acagaccttt 1620

actccctgca cttgaggcag atgcaggcaa atctaggcat cctggtgtac atatgaagtt 1680

caggcaagcc agggccacgt aggctcaaag acg 1713

<210> SEQ ID NO: 13

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 13

catgtctcct gcctttcctg t 21

<210> SEQ ID NO: 14

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 14

ggacagggta gcaacgccat t 21

<210> SEQ ID NO: 15

<211> LENGTH: 19

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 15

ccacctcagt tgcacggaa 19

<210> SEQ ID NO: 16

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 16

ggacagggta gcaacgccau u 21

<210> SEQ ID NO: 17

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 17

ggacagggua gcaacgccau u 21

<210> SEQ ID NO: 18

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 18

ctgactacct cttga 15

<210> SEQ ID NO: 19

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 19

accauggtgc gcgaaauggc a 21

<210> SEQ ID NO: 20

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 20

accatggtgc gcgaaatggc a 21

<210> SEQ ID NO: 21

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 21

accauggugc gcgaaauggc a 21

<210> SEQ ID NO: 22

<211> LENGTH: 12

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 22

tctctctggg ac 12

<210> SEQ ID NO: 23

<211> LENGTH: 12

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 23

ttaccttcat ac 12

<210> SEQ ID NO: 24

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 24

aatcacttag ccact 15

<210> SEQ ID NO: 25

<211> LENGTH: 20

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 25

gcctcttcta tgggtctguc 20

<210> SEQ ID NO: 26

<211> LENGTH: 27

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 26

aaucaauggc ugttctcucu cugggac 27

<210> SEQ ID NO: 27

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 27

gtggtatagg aactg 15

<210> SEQ ID NO: 28

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 28

gugguauagg aactggcagc a 21

<210> SEQ ID NO: 29

<211> LENGTH: 21

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 29

gccagtcaca aautcagauc a 21

<210> SEQ ID NO: 30

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 30

accctccttc ctccc 15

<210> SEQ ID NO: 31

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 31

cagaauttca tggua 15

<210> SEQ ID NO: 32

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 32

acaggugcuc agaau 15

<210> SEQ ID NO: 33

<211> LENGTH: 23

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 33

acaggugctc agaauttcau ggu 23

<210> SEQ ID NO: 34

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 34

cagaatttca tggta 15

<210> SEQ ID NO: 35

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 35

acaggtgctc agaat 15

<210> SEQ ID NO: 36

<211> LENGTH: 24

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 36

acaggugctc agaatttcau ggua 24

<210> SEQ ID NO: 37

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 37

acaggtgctc agaat 15

<210> SEQ ID NO: 38

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 38

acaggugctc agaat 15

<210> SEQ ID NO: 39

<211> LENGTH: 15

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 39

ccacgcgcga gtaca 15

<210> SEQ ID NO: 40

<211> LENGTH: 676

<212> TYPE: DNA

<213> ORGANISM: Artificial Sequence

<220> FEATURE:

<223> OTHER INFORMATION: Antisense oligonucleotide

<400> SEQENCE: 40

caaaatggcg tgctaccctg tccaaccttg tctgtagaca gagtcaattg aacactgtct 60

ttggacttcc gtgcaactga ggtgggcggg cttgaagcac aaagctttca gggagaacca 120

aactttatgc ccaagctgct ctctgccacc cacagggtaa atgaatctca tacaggaaag 180

gcaagagaca tgtgacactg ttgttctgat ggtcacaagt caagcttttt aaaaagcagc 240

ctgatattgt gagctaacat ggctttctgt aattgaatgc aatgtatttt ctatgcttgt 300

ctgggtaaag ttgaccttgg tttgatttag ctcaagcaat atttcaacag tgcactgggg 360

ctctgtcccc tgactactgt ttgactagag ccaggctctg ccctggatgg caaccaacag 420

cccaggctct ggggcacagc cgggctttga caggtctggg gaaatgttca ccggagatga 480

aaggtttcaa actatgaaac tctaaaatct caagtcaaaa cttttgacaa gcacacacag 540

gacatgaatt acaatcaccc gaagattttt acaggcttct caattttaat gacatgctga 600

cacgtgtcat cagatctcac aacaagatga cacatgggtg tcaggtatgg cgcagaagac 660

tagagtcggg gtgtaa 676

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Patent Valuation

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24.64/100 Score

Market Attractiveness

It shows from an IP point of view how many competitors are active and innovations are made in the different technical fields of the company. On a company level, the market attractiveness is often also an indicator of how diversified a company is. Here we look into the commercial relevance of the market.

30.0/100 Score

Market Coverage

It shows the sizes of the market that is covered with the IP and in how many countries the IP guarantees protection. It reflects a market size that is potentially addressable with the invented technology/formulation with a legal protection which also includes a freedom to operate. Here we look into the size of the impacted market.

72.84/100 Score

Technology Quality

It shows the degree of innovation that can be derived from a company’s IP. Here we look into ease of detection, ability to design around and significance of the patented feature to the product/service.

59.0/100 Score

Assignee Score

It takes the R&D behavior of the company itself into account that results in IP. During the invention phase, larger companies are considered to assign a higher R&D budget on a certain technology field, these companies have a better influence on their market, on what is marketable and what might lead to a standard.

25.0/100 Score

Legal Score

It shows the legal strength of IP in terms of its degree of protecting effect. Here we look into claim scope, claim breadth, claim quality, stability and priority.

Citation

Patents Cited in This Cited by
Title Current Assignee Application Date Publication Date
Methods and compositions for the treatment of cancer THE JOHNS HOPKINS UNIVERSITY 06 April 2008 16 October 2008
Modulation of hypoxia-inducible factor 1 alpha expression ISIS PHARMACEUTICALS INC. 23 November 2002 27 May 2004
Antisense modulation of HMG-CoA reductase expression ISIS PHARMACEUTICALS INC. 02 July 2002 08 January 2004
Genetically engineered plant cells and plants, as well as recombinant DNA suitable therefor VERENIGING VOOR CHRISTELIJK WETENSCHAPPELIJK ONDERWIJS 22 March 1989 04 October 1989
Use of resveratrol to regulate expression of apolipoprotein A1 RESVERLOGIX, INC. 15 August 2002 19 February 2004
See full citation <>

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