Compositions and methods for modulating MECP2 expression

• US 10,655,128 B2
• Filed: 10/19/2017
• Issued: 05/19/2020
• Est. Priority Date: 05/16/2012
• Status: Active Grant

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: Translate Bio MA Inc. (Sanofi )
• Inventors: Krieg, Arthur M., Subramanian, Romesh, McSwiggen, James, Lee, Jeannie T.
• Primary Examiner(s): Vivlemore, Tracy
• Application Number:
  US15/787,876
• Publication Number:
  US 20180223282A1
• Time in Patent Office: 943 Days
• Field of Search: None
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: A61K 47/555 pre-targeting systems involving an organic compound, other than a peptide, protein or antibody, for targeting specific cells
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/6455 Polycationic oligopeptides, polypeptides or polyamino acids, e.g. for complexing nucleic acids
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• 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
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/3231 having an additional ring, e.g. LNA, ENA
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

Aspects of the invention provide single stranded oligonucleotides for activating or enhancing expression of MECP2. Further aspects provide compositions and kits comprising single stranded oligonucleotides for activating or enhancing expression of MECP2. Methods for modulating expression of MECP2 using the single stranded oligonucleotides are also provided. Further aspects of the invention provide methods for selecting a candidate oligonucleotide for activating or enhancing expression of MECP2.

Claims

1. A single stranded oligonucleotide produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) has a sequence 5′

-X-Y-Z, wherein X is any nucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a seed sequence of a human microRNA, and Z is a nucleotide sequence of 3-23 nucleotides in length,

(b) is complementary with at least 10 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, wherein the PRC2-associated region is a region of the MECP2 gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the MECP2 gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody; and

and

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue.

2. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.

3. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide is 8 to 30 nucleotides in length.

4. The single stranded oligonucleotide of claim 1, wherein the at least one nucleotide analogue results in an increase in Tm of the oligonucleotide in a range of 1 to 5°

C. compared with an oligonucleotide that does not have the at least one nucleotide analogue.

5. The single stranded oligonucleotide of claim 1, wherein at least one nucleotide of the oligonucleotide comprises a 2′

O-methyl.

6. The single stranded oligonucleotide of claim 1, wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.

7. The single strand oligonucleotide of claim 6, wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.

8. The single stranded oligonucleotide of claim 1, further comprising phosphorothioate internucleotide linkages between at least two nucleotides.

9. The single stranded oligonucleotide of claim 8, further comprising phosphorothioate internucleotide linkages between all nucleotides.

10. The single stranded oligonucleotide of claim 1, wherein the nucleotide at the 3′

position of the oligonucleotide has a 3′

hydroxyl group.

12. A composition comprising a single stranded oligonucleotide of claim 1 and a carrier.

13. A composition comprising a single stranded oligonucleotide of claim 1 in a buffered solution.

14. A pharmaceutical composition comprising a composition of claim 12 and a pharmaceutically acceptable carrier.

15. A kit comprising a container housing the composition of claim 12.

16. A method for preparing a pharmaceutical composition comprising providing the single stranded oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.

11. A single stranded oligonucleotide produced by a process comprising:

synthesizing a single stranded oligonucleotide that;

(a) comprises a region of complementarity that is complementary with at least 10 consecutive nucleotides of a PRC2-associated region of a MECP2 gene, wherein the PRC2-associated region is a region of the MECP2 gene that has a sequence that occurs at a higher frequency in a sequencing reaction of products of an RNA-immunoprecipitation assay that employs an antibody that targets Ezh2 to immunoprecipitate RNA-associated PRC2 complexes from cells comprising the MECP2 gene compared to a control sequencing reaction of products of a control RNA-immunoprecipitation assay that employs a control antibody;

(b) has at least one of;

(i) a sequence that is 5′

X-Y-Z, wherein X is any nucleotide and wherein X is anchored at the 5′

end of the oligonucleotide, Y is a nucleotide sequence of 6 nucleotides in length that is not a human seed sequence of a microRNA, and Z is a nucleotide sequence of 3 to 23 nucleotides in length;

(ii) a sequence that does not comprise three or more consecutive guanosine nucleotides;

(iii) a sequence that is complementary to a PRC2-associated region that encodes an RNA that forms a secondary structure comprising at least two single stranded loops; and

/or

(iv) a sequence that has greater than 60% G-C content,

wherein, during the synthesis, at least one nucleotide incorporated into the oligonucleotide is a nucleotide analogue.