Treatment of brain derived neurotrophic factor (BDNF) related diseases by inhibition of natural antisense transcript to BDNF

• US 10,214,745 B2
• Filed: 01/04/2017
• Issued: 02/26/2019
• Est. Priority Date: 03/15/2012
• Status: Active Grant

• Current Assignee: CURNA, Inc. (Opko Health, Inc.), University of Florida Research Foundation, Incorporated (State University System of Florida)
• Original Assignee: CURNA, Inc. (Opko Health, Inc.), The Scripps Research Institute
• Inventors: Faghihi, Mohammad Ali, Coito, Carlos
• Primary Examiner(s): Chong, Kimberly
• Application Number:
  US15/398,630
• Publication Number:
  US 20170211071A1
• Time in Patent Office: 783 Days
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• CPC Class Codes: C12N 15/113 : Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
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• CPC Class Codes: C12N 2310/341 : Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/3521 : Methyl

Abstract

The present invention relates to antisense oligonucleotides that modulate the expression of and/or function of Brain derived neurotrophic factor (BDNF), in particular, by targeting natural antisense polynucleotides of Brain derived neurotrophic factor (BDNF). The invention also relates to the identification of these antisense oligonucleotides and their use in treating diseases and disorders associated with the expression of BDNF.

Claims

1. A method of upregulating the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in a biological system comprising:

contacting said system with at least one antisense oligonucleotide 10 to 30 nucleotides in length wherein said at least one antisense oligonucleotide is at least 90% complementary to and specifically hybridizes to a 10 to 30 nucleotide region of a natural antisense polynucleotide of the BDNF polynucleotide in a 225-nucleotide overlapping region of the natural antisense polynucleotide, wherein said natural antisense polynucleotide consists essentially of a sequence selected from the group consisting of;

nucleotides 1 to 1279 of SEQ ID NO;

3, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527, 1 to 1478 of SEQ ID NO;

4, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 1437 of SEQ ID NO;

5, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 2322 of SEQ ID NO;

6, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 2036 of SEQ ID NO;

7, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527; and

1 to 2364 of SEQ ID NO;

8, comprising the 225-nucleotide overlapping region at nucleotides 402 to 626;

thereby upregulating the expression of the BDNF polynucleotide.

4. The method of claim 1, wherein the expression of the BDNF polynucleotide is upregulated in vivo or in vitro with respect to a mock-transfected control.

13. The method of claim 1, wherein the upregulated expression of the BDNF polynucleotide results in neuronal survival, neuronal proliferation, or both.

15. The method of claim 1, wherein the oligonucleotide is at least 95% complementary to and specifically hybridizes to the 10-30 nucleotide region of the natural antisense polynucleotide.

16. The method of claim 1, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16-31 and 35.

17. The method of claim 16, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16, 19, 20, 23, and 24.

2. A method of upregulating the expression of a BDNF polynucleotide in patient cells or tissues comprising:

contacting said patient cells or tissues with at least one antisense oligonucleotide 10 to 30 nucleotides in length wherein said at least one antisense oligonucleotide is at least 95% complementary to and specifically hybridizes to a 10 to 30 nucleotide region of a natural antisense polynucleotide of BDNF in a 225-nucleotide overlapping region of the natural antisense polynucleotide, wherein said natural antisense polynucleotide consists essentially of a sequence selected from the group consisting of nucleotides 1 to 1279 of SEQ ID NO;

3, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 1478 of SEQ ID NO;

4, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 1437 of SEQ ID NO;

5, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 2322 of SEQ ID NO;

6, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 2036 of SEQ ID NO;

7, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527; and

1 to 2364 of SEQ ID NO;

8, comprising the 225-nucleotide overlapping region at nucleotides 402 to 626;

thereby upregulating the expression of the Brain derived neurotrophic factor (BDNF) polynucleotide.

18. The method of claim 2, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16-31 and 35.

19. The method of claim 18, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16, 19, 20, 23, and 24.

3. A method of upregulating the expression of a BDNF polynucleotide in a patient comprising:

contacting said patient with at least one antisense oligonucleotide of 10 to 30 nucleotides in length that is at least 95% complementary to and specifically hybridizes to a 10 to 30 nucleotide region in a 225-nucleotide overlapping region of a natural antisense polynucleotide of the BDNF polynucleotide, wherein said natural antisense polynucleotide consists essentially of a sequence selected from the group consisting of nucleotides 1 to 1279 of SEQ ID NO;

3, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 1478 of SEQ ID NO;

4, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 1437 of SEQ ID NO;

5, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 2322 of SEQ ID NO;

6, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 2036 of SEQ ID NO;

comprising the 225-nucleotide overlapping region at nucleotides 303 to 527; and

1 to 2364 of SEQ ID NO;

8, comprising the 225-nucleotide overlapping region at nucleotides 402 to 626;

thereby upregulating the function of and/or the expression of the BDNF polynucleotide.

5. The method of claim 3, wherein the at least one antisense oligonucleotide targets a natural antisense polynucleotide that is antisense to coding and/or non-coding nucleic acid sequences of the BDNF polynucleotide.

6. The method of claim 3, wherein the at least one antisense oligonucleotide comprises one or more modifications selected from:

at least one modified sugar moiety, at least one modified internucleoside linkage, at least one modified nucleotide, and combinations thereof.

7. The method of claim 6, wherein the one or more modifications comprise at least one modified sugar moiety selected from:

a 2′

-O-methoxyethyl modified sugar moiety, a 2′

-methoxy modified sugar moiety, a 2′

-O-alkyl modified sugar moiety, a bicyclic sugar moiety, and combinations thereof.

8. The method of claim 6, wherein the one or more modifications comprise at least one modified internucleoside linkage selected from:

a phosphorothioate, alkylphosphonate, phosphorodithioate, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, carboxymethyl ester, and combinations thereof.

9. The method of claim 6, wherein the one or more modifications comprise at least one modified nucleotide selected from:

a peptide nucleic acid (PNA), a locked nucleic acid (LNA), an arabino-nucleic acid (FANA), and combinations thereof.

14. The method of claim 3, wherein the expression of the BDNF polynucleotide is upregulated in the hippocampus, the frontal cortex, or both.

20. The method of claim 3, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16-31 and 35.

21. The method of claim 20, wherein the sequence of the at least one antisense oligonucleotide comprises a sequence selected from the group consisting of the sequences set forth as SEQ ID NOS:

16, 19, 20, 23, and 24.

10. A method of upregulating the expression of a BDNF gene in mammalian cells or tissues in vivo or in vitro comprising:

contacting said cells or tissues with at least one short interfering RNA (siRNA) oligonucleotide 19 to 30 nucleotides in length, said at least one siRNA oligonucleotide being specific for a natural antisense polynucleotide of a BDNF polynucleotide in a 225-nucleotide overlapping region of the natural antisense polynucleotide, and, upregulating the expression of the BDNF gene in mammalian cells or tissues in vivo or in vitro, wherein said natural antisense polynucleotide consists essentially of a sequence selected from the group consisting of;

nucleotides 1 to 1279 of SEQ ID NO;

3, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 1478 of SEQ ID NO;

4, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 1437 of SEQ ID NO;

5, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 2322 of SEQ ID NO;

6, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 2036 of SEQ ID NO;

7, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527; and

1 to 2364 of SEQ ID NO;

8, comprising the 225-nucleotide overlapping region at nucleotides 402 to 626.

11. The method of claim 10, wherein said oligonucleotide has 100% complementarity to said natural antisense polynucleotide.

12. A method for targeting a natural antisense transcript (NAT) of a BDNF polynucleotide with at least one antisense oligonucleotide 10 to 30 nucleotides in length, wherein the at least one antisense oligonucleotide is at least 90% complementary to and specifically hybridizes to a 10 to 30 nucleotide region of the NAT of the BDNF polynucleotide in a 225-nucleotide overlapping region of the NAT, to upregulate the expression of the BDNF polynucleotide, comprising contacting the NAT with the antisense oligonucleotide, wherein the NAT has a sequence consisting essentially of a sequence selected from the group consisting of:

nucleotides 1 to 1279 of SEQ ID NO;

3, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 1478 of SEQ ID NO;

4, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 1437 of SEQ ID NO;

5, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527;

1 to 2322 of SEQ ID NO;

6, comprising the 225-nucleotide overlapping region at nucleotides 372 to 596;

1 to 2036 of SEQ ID NO;

7, comprising the 225-nucleotide overlapping region at nucleotides 303 to 527; and

1 to 2364 of SEQ ID NO;

8, comprising the 225-nucleotide overlapping region at nucleotides 402 to 626.

22. A method of upregulating the expression of a Brain derived neurotrophic factor (BDNF) polynucleotide in a biological system comprising:

contacting the system with at least one antisense oligonucleotide 10 to 30 nucleotides in length wherein the at least one antisense oligonucleotide is at least 90% complementary to and specifically hybridizes to a 10 to 30 nucleotide region of a 225-nucleotide overlapping region of a natural antisense polynucleotide of the BDNF polynucleotide, wherein the 225-nucleotide overlapping region of the natural antisense polynucleotide has at least 95% complementarity to the 225-nucleotide region at nucleotides 1188 to 1412 of the BDNF mRNA of SEQ ID NO;

1, thereby upregulating the expression of the BDNF polynucleotide.

23. The method of claim 22, wherein the 225-nucleotide overlapping region of the natural antisense polynucleotide has 100% complementarity to nucleotides 1188 to 1412 of the BDNF mRNA of SEQ ID NO:

1.