JPWO2018007475A5 - - Google Patents

Description

Example 1 (IN VITRO)
Materials and Methods
AON
Antisense oligonucleotides (AONs) (Table 1, Figures 1-3) had phosphorothioate backbones with 2'-O-methyl monomers and either LNA (SEQ ID NOs: 452, 453, 455 and 456) or 2'-amino-LNA (SEQ ID NO: 456A) or CRN (SEQ ID NOs: 453C and 455C) scaffold modifications. AONs with SEQ ID NO: 452 featured a cytosine, the other SEQ ID NOs featured a 5-methylcytosine. 2'-amino-LNA refers to a scaffold modification sometimes named 2'-amino-2'-deoxyLNA. AONs were synthesized on a 10 μmol scale using an OP-10 synthesizer (GE/AKTA Oligopilot) by standard phosphoramidite protocols. The AONs were cleaved and deprotected in a two-step sequence (diethylamine followed by concentrated _NH4OH treatment), purified by HPLC, dissolved in water, and excess NaCl was added to exchange the ions. After evaporation, the AONs were redissolved in water, desalted by FPLC, and lyophilized. The identity of all AONs was confirmed by mass spectrometry, and purity (determined by UPLC) was found to be acceptable (>80%) for all AONs.

Example 3 (IN VITRO)
AON
Antisense oligonucleotides (AONs) of the invention (Table 3, Figure 4) contained an all phosphorothioate backbone with 2'-O-methyl substitutions in all non-BNA monomers, 5-methylcytosine, and at least one BNA scaffold modification resulting in an LNA monomer (SEQ ID NOs: 455, 459, 4528, 4531, 4532, 4533, 4535, 4542, 4548, and 4568). A control AON with SEQ ID NO: 452 contained a phosphorothioate backbone with 2'-O-methyl monomers, cytosine, and no BNA scaffold modifications. AONs were synthesized on a 5 μmol scale using an OP-10 synthesizer (GE/AKTA Oligopilot) by standard phosphoramidite protocols. The AONs were cleaved and deprotected in a two-step sequence (DEA followed by concentrated _NH4OH treatment), purified by anion exchange chromatography, desalted by size exclusion chromatography, and lyophilized. The identity of all AONs was confirmed by mass spectrometry, and purity (determined by UPLC) was found to be acceptable (>80%) for all AONs.

Example 4 (IN VITRO)
Materials and Methods
AON
Antisense oligonucleotides (AONs) of the invention (Table 4, Figure 5) contained an all phosphorothioate backbone with 2'-O-methyl substitutions, 5-methylcytosines and at least one BNA scaffold modification resulting in an LNA monomer (SEQ ID NOs: 29, 3185 and 863). Control AONs contained an all phosphorothioate backbone with only 2'-O-methyl substituted monomers, 5-methylcytosines but no BNA scaffold modification (SEQ ID NOs: 26, 6049 and 860; for SEQ ID NO: 6049, these modifications are made the same as SEQ ID NO: 6071). AONs were synthesized on a 5 μmol scale using an OP-10 synthesizer (GE/AKTA Oligopilot) by standard phosphoramidite protocols. The AONs were cleaved and deprotected in a two-step sequence (DEA followed by concentrated _NH4OH treatment), purified by anion exchange chromatography, desalted by size exclusion chromatography, and lyophilized. The identity of all AONs was confirmed by mass spectrometry, and purity (determined by UPLC) was found to be acceptable (>80%) for all AONs.

Claims (22)

An oligonucleotide having a length of 16-25 nucleotides and comprising a contiguous stretch of at least 16 nucleotides and up to 25 nucleotides of at least one nucleotide sequence selected from at least one nucleotide sequence selected from SEQ ID NOs: 6065, 6066, 6067, or 6069 ,
i) only 2'-substituted monomers linked by phosphorothioate backbone linkages , and i) at least one monomer comprising a bicyclic nucleic acid (BNA) scaffold modification , wherein at least one BNA scaffold modification is included in a terminal monomer of the oligonucleotide.
Including,
All cytosine bases are replaced with 5-methylcytosine bases,
the oligonucleotide is complementary to, binds to, targets or hybridizes to at least a portion of dystrophin pre-mRNA exon 44 , 45, 51 or 53;
The oligonucleotide, wherein the 2'-substituted monomer is a 2'-substituted RNA monomer, a 2'-F monomer, a 2'-amino monomer, a 2'-O-substituted monomer, a 2'-O-methyl monomer or a 2'-O-(2-methoxyethyl) monomer . The oligonucleotide of claim 1 , wherein the 2'-substituted monomer is a 2'-O-methyl monomer . 3. The oligonucleotide of claim 1 or 2, comprising a contiguous stretch of at least 16 and up to 25 nucleotides of SEQ ID NO:6067. The oligonucleotide of any one of claims 1 to 3 , comprising 1, 2, 3 or 4 monomers that comprise a bicyclic nucleic acid (BNA) scaffold modification . 5. The oligonucleotide of any one of claims 1 to 4, comprising 1, 2, 3 or 4 monomers comprising a bridged nucleic acid scaffold modification. The oligonucleotide of any one of claims 1 to 5 , wherein at least one bicyclic nucleic acid (BNA) scaffold modification is comprised in the 5'-terminal monomer of the oligonucleotide. The oligonucleotide of any one of claims 1 to 6, wherein at least one bicyclic nucleic acid (BNA) scaffold modification is comprised in both terminal monomers of the oligonucleotide. Each occurrence of the bicyclic nucleic acid (BNA) scaffold modification comprises:
and providing monomers independently selected from the group consisting of locked nucleic acid (LNA) monomers, conformationally restricted nucleotide (CRN) monomers, xylo-LNA monomers, α-L-LNA monomers, β-D-LNA monomers, 2'-amino-LNA monomers, 2'-(alkylamino)-LNA monomers, 2'-(acylamino)-LNA monomers, 2'-N-substituted-2'-amino-LNA monomers, (2'-O,4'-C) constrained ethyl (cEt) LNA monomers, (2'-O,4'-C) constrained methoxyethyl (cMOE) BNA monomers, 2',4'-BNA ^NC (N-H) monomers, 2',4'-BNA ^NC (N-Me) monomers, ethylene-bridged nucleic acid (ENA) monomers, 2'-C-bridged bicyclic nucleotide (CBBN) monomers, and derivatives thereof.
The oligonucleotide according to any one of claims 1 to 7 . Each occurrence of the bicyclic nucleic acid (BNA) scaffold modification comprises:
9. The oligonucleotide of any one of claims 1 to 8, which provides monomers independently selected from the group consisting of LNA monomers, ENA monomers, cEt BNA monomers, oxo-CBBN monomers, 2'-amino-LNA monomers and cMOE BNA monomers. Each occurrence of the bicyclic nucleic acid (BNA) scaffold modification comprises:
The oligonucleotide of any one of claims 1 to 9, which provides a monomer independently selected from the group consisting of an LNA monomer, an ENA monomer, a cEt BNA monomer or a cMOE BNA monomer. The oligonucleotide of any one of claims 1 to 10, wherein each occurrence of the bicyclic nucleic acid (BNA) scaffold modification results in a monomer that is an LNA monomer. 12. The oligonucleotide of any one of claims 1 to 11, comprising or consisting of a sequence that is complementary to, binds to, targets or hybridizes to at least a portion of an exon recognition sequence (ERS), an exon splicing silencer (ESS), an intron splicing silencer (ISS), an SR protein binding site , or another splicing element, signal or structure. The oligonucleotide according to any one of claims 1 to 12 , wherein the oligonucleotide induces pre-mRNA splicing regulation. The oligonucleotide of claim 13 , wherein the pre-mRNA splicing modulation alters protein production or composition. The oligonucleotide of claim 14 , wherein the pre-mRNA splicing modulation comprises exon skipping or exon inclusion. The oligonucleotide of claim 15 , wherein the pre-mRNA splicing modulation comprises exon skipping. 17. The oligonucleotide of any one of claims 1 to 16, having improved binding affinity and/or kinetics, exon skipping activity, biostability, (tissue) distribution, cellular uptake and/or transport, and/or immunogenicity compared to a corresponding oligonucleotide not comprising a bicyclic nucleic acid (BNA) scaffold modification. a) at least one monomer is represented by formula I

[Wherein,
B is a nucleobase,
X is F, -NR ¹ R ² or -OR;
R is alkenyl or optionally substituted alkyl, the optional substituents, if present, being halo, OR ¹ , NR ¹ R ² or SR ¹ ;
R ¹ is H, alkyl, cycloalkyl, aryl, heterocycloalkyl, or heteroaryl, each independently optionally further substituted with halo, hydroxy, or alkyl;
^R2 is H or alkyl;

indicates the point of attachment to the remainder of the oligonucleotide.
having
b) at least one monomer comprises a BNA scaffold modification and has formula II

[Wherein,
^B1 is a nucleobase;
Z-Y is a divalent group selected from -(CH ₂ ) _n O-, -C(CH ₂ CH ₂ )O-, -CH ₂ WCH ₂ -, -(CH ₂ ) _n NR ³ -, -CH ₂ S(O _m )-, -CH(CH ₃ )O-, -CH(CH ₂ OCH ₃ )O-, -CH ₂ N(R ³ )O-, -CH ₂ CH ₂ -, -C(O)NR ³ -, -CH═CHO-, -CH ₂ SO ₂ NR ³ -, and -NHC(O)NH-;
n is 1 or 2;
m is 0, 1 or 2;
W is O, S or ^NR3 ;
R ³ is H, —C(O)R ⁴ , —C(═NH)NR ⁵ R ⁵ , benzyl or optionally substituted alkyl, the optional substituents, if present, being selected from halo and alkoxy;
^R4 is alkyl, cycloalkyl or aryl;
^R5 is H or alkyl;

indicates the point of attachment to the remainder of the oligonucleotide.
The oligonucleotide of claim 1 , having the following structure: The oligonucleotide according to any one of claims 1 to 18, comprising or consisting of any of SEQ ID NOs: 29, 453, 455, 456, 459, 863, 3185, 4528, 4531, 4532, 4533, 4535, 4542, 4548 and 4568. A composition comprising the oligonucleotide according to any one of claims 1 to 19 . 21. The composition of claim 20, comprising at least one excipient that may further aid in enhancing targeting and/or delivery of the composition and/or the oligonucleotide to and/or into tissues and/or cells. Use of an oligonucleotide according to any one of claims 1 to 19 or a composition according to claim 20 or 21 for the manufacture of a medicament for treating, preventing and/or delaying Duchenne muscular dystrophy (DMD).