WO2021158810A1 - Oligonucléotides pour la modulation d'épissage de camk2d - Google Patents

Oligonucléotides pour la modulation d'épissage de camk2d Download PDF

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WO2021158810A1
WO2021158810A1 PCT/US2021/016646 US2021016646W WO2021158810A1 WO 2021158810 A1 WO2021158810 A1 WO 2021158810A1 US 2021016646 W US2021016646 W US 2021016646W WO 2021158810 A1 WO2021158810 A1 WO 2021158810A1
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aso
seq
camk2d
expression
aspects
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PCT/US2021/016646
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English (en)
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Marianne L. JENSEN
Jonas VIKESAA
Jesper Worm
Peter Hagedorn
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Bristol-Myers Squibb Company
Roche Innovation Center Copenhagen A/S
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Publication of WO2021158810A1 publication Critical patent/WO2021158810A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
    • C12N15/1137—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 against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11017—Ca2+/Calmodulin-dependent protein kinase (2.7.11.17)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/33—Alteration of splicing

Definitions

  • the present disclosure relates to splice modulating oligonucleotides, e.g., antisense oligonucleotides (ASOs), that are complementary to the CAMK2D pre-mRNA, which reduce the incorporation of a CAMK2D exon 14 in CAMK2D mRNA transcripts.
  • ASOs antisense oligonucleotides
  • the ASOs of the disclosure are useful in the treatment of a range of medical disorders, such as cardiovascular- related diseases or disorders.
  • CaMKs Calcium/calmodulin (Ca 2+ /CaM)-dependent serine/threonine kinases (CaMKs) constitute a family of 81 proteins in the human proteasome that play a central role in cellular signaling by transmitting Ca 2+ signals.
  • CaMKII isozymes a, b, g, and d
  • splice variants are expressed in humans. Braun, A.P., et al. , Annual Review of Physiology 57:417-445 (1995).
  • CAMK2D CaMKII5
  • ECC excitation-contraction coupling
  • CAMK2D transcripts which comprise exon 14 have been shown to be highly expressed in muscle and heart tissue.
  • CAMK2D activity has also been described as being important in the recovery process after certain heart related injury (e.g, ischemia-reperfusion injury). Said M., et al, Am J Physiol Heart Circ Physiol 285 :H1198-205 (2003).
  • the present disclosure is directed to an antisense oligonucleotide (ASO) comprising a contiguous nucleotide sequence of at least 10 nucleotides that has at least about 90% complementarity to nucleotides 258,637 - 259,107 of SEQ ID NO: 1 (i.e., SEQ ID NO: 70), wherein the ASO has a length of 10 to 40 nucleotides and is capable of selectively reducing the expression of CAMK2D mRNA transcript comprising CAMK2D exon 14 ( CAMK2D-E14 ) in a cell which is expressing a CAMK2D pre-mRNA transcript, as compared to the expression of CAMK2D mRNA transcript which does not comprise CAMK2D exon 14 ( CAMK2DA14 ).
  • ASO antisense oligonucleotide
  • the ASO is capable of selectively reducing the expression of
  • the ASO is also capable of increasing the expression of CAMK2DA14 in the cell.
  • the ASO is not capable of, or essentially not capable of, recruiting an RNase H. In some aspects, the ASO does not comprise a region of more than 3 contiguous DNA nucleosides.
  • the ASO is not a gapmer. In some aspects, the ASO is a mixmer.
  • the ASO is a totalmer.
  • the ASO is a splice modulating ASO which is capable of decreasing the incorporation of CAMK2D exon 14 into CAMK2D mRNA transcript.
  • the ASO is capable of decreasing the level of protein encoded by the CAMK2D-E14 in the cell.
  • the ASO is capable of decreasing the level of protein encoded by the CAMK2D-E14 in the cell but does not significantly reduce the expression level of protein encoded by CAMK2-D14.
  • the CAMK2D-E14, or the protein encoded thereof is reduced by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASO.
  • the CAMK2DA14, or the protein encoded thereof is increased by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASO.
  • the ASO is capable of changing a ratio of CAMK2DA14 expression to CAMK2D-E14 expression in the cell.
  • the ratio of CAMK2DA14 expression to CAMK2D-E14 expression in the cell is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20- fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least about 50-fold, compared to the ratio of CAMK2DA14 expression to CAMK2D-E14 expression in the cell prior to being treated with the ASO.
  • the CAMK2DA14 expression and the CAMK2D-E14 expression are measured as described in Example 2.
  • the cell that can be treated with an ASO of the present disclosure e.g ., to reduce the expression of CAMK2D-E14 and/or to enhance the expression of CAMK2DA14 , comprises cardiomyocytes (e.g, human cardiomyocytes derived from pluripotent stem cells).
  • the ASO described herein has a length of 10 to 25 nucleotides. In certain aspects, the ASO has a length of 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, or 20 nucleotides.
  • the contiguous nucleotide sequence of an ASO described herein has 100% complementarity to a region within nucleotides 258,637 - 259,107 of SEQ ID NO 1 (i.e., SEQ ID NO: 70).
  • the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 10.
  • the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 6.
  • the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 9.
  • the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 7. In some aspects, the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 8. In some aspects, the contiguous nucleotide sequence is complementary, e.g, fully complementary, to a region within SEQ ID NO: 71. In certain aspects, the contiguous nucleotide sequence is complementary, e.g, fully complementary, to any one of the nucleic acid sequences set forth in SEQ ID NOs: 41 to 69.
  • the contiguous nucleotide sequence of an ASO disclosed herein comprises any one of the nucleotide sequences set forth in SEQ ID NOs: 12 to 40. [0018] In some aspects, the contiguous nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO:
  • SEQ ID NO: 22 SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
  • SEQ ID NO: 33 SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or any combination thereof.
  • the contiguous nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO:
  • SEQ ID NO: 24 SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, or any combination thereof
  • the contiguous nucleotide sequence comprises the nucleotide sequence set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO:
  • SEQ ID NO: 34 SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 39, or any combination thereof.
  • the contiguous nucleotide sequence comprises any one of the nucleotide sequences set forth in SEQ ID NOs: 12 to 40 with one mismatch.
  • the ASO, or contiguous nucleotide sequence thereof comprises one or more nucleoside analogs.
  • the one or more nucleoside analogs comprise a 2'-0-alkyl-RNA; 2'-0-methyl RNA (2'-OMe); 2'-alkoxy-RNA; 2'-0-methoxyethyl-RNA (2'- MOE); 2'-amino-DNA; 2'-fluro-RNA; 2'-fluoro-DNA; arabino nucleic acid (ANA); 2'-fluoro- ANA; bicyclic nucleoside analog (LNA); or any combination thereof.
  • one or more of the nucleoside analogs comprise a bicyclic sugar.
  • one or more of the nucleoside analogs are a sugar modified nucleoside.
  • the sugar modified nucleoside is an affinity enhancing 2' sugar modified nucleoside.
  • the affinity enhancing 2' sugar modified nucleoside is an LNA.
  • the LNA comprises constrained ethyl nucleoside (cEt), 2',4'-constrained 2'-0- methoxyethyl (cMOE), a-L-oxy-LNA, b-D-oxy-LNA, 2'-0,4'-C-ethylene-bridged nucleic acids (ENA), amino-LNA, thio-LNA, or any combination thereof.
  • the ASO, or contiguous nucleotide sequence thereof comprises one or more 5'-methyl-cytosine nucleobases.
  • one or more of the intemucleoside linkages are modified. In certain aspects, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% of the intemucleoside linkages are modified. In some aspects, the one or more modified intemucleoside linkages comprise a phosphorothioate linkage.
  • the ASO comprises an oligonucleotide provided herein, such as a compound selected from the group consisting of compound ID NO #12 1 - #40 2.
  • the present disclosure also provides a conjugate comprising an ASO disclosed herein, and at least one conjugate moiety covalently attached to the ASO.
  • the conjugate moiety comprises a protein, a fatty acid chain, a sugar residue, a glycoprotein, a polymer, or any combinations thereof.
  • the salt comprises a sodium salt, a potassium salt, an ammonium salt, or any combination thereof.
  • the present disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising an ASO or conjugate described herein, and a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the pharmaceutical composition further comprises at least one additional therapeutic agent.
  • the additional therapeutic agent comprises a CAMK2D antagonist.
  • the CAMK2D antagonist is an anti- CAMK2D antibody or fragment thereof.
  • kits comprising the ASO, conjugate, pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure, and instructions for use.
  • a diagnostic kit comprising the ASO, conjugate, pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure, and instructions for use.
  • the present disclosure provides a method of modulating the splicing of a
  • CAMK2D pre-mRNA in a target cell expressing the CAMK2D pre-mRNA comprising administering the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure to the target cell.
  • an in vitro method for modulating the splicing of a CAMK2D pre-mRNA in a target cell which is expressing CAMK2D pre-mRNA comprising contacting the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition described herein to the target cell.
  • the administering or the contacting of the ASO results in reduced expression of a CAMK2D-E14 , or the protein encoded thereof, in the target cell.
  • the expression of the CAMK2D-E14 , or the protein encoded thereof is reduced by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASO.
  • the administering or the contacting of the ASO results in enhanced expression of a CAMK2DA14 , or the protein encoded thereof, in the target cell.
  • the expression of CAMK2DA14 , or the protein encoded thereof is increased by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASO.
  • an ASO that can be used in a method described herein e.g ., method of modulating the splicing of a CAMK2D pre-mRNA in a target cell expressing the CAMK2D pre-mRNA
  • a ratio of CAMK2DA14 expression to CAMK2D- E14 expression in the target cell is capable of changing a ratio of CAMK2DA14 expression to CAMK2D- E14 expression in the target cell.
  • the ratio of CAMK2DA14 expression to CAMK2D-E14 expression in the target cell is increased by at least about 2-fold, at least about 3- fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, or at least 50-fold, compared to the ratio of CAMK2DA14 expression to CAMK2D- E14 expression in the target cell prior to being treated with the ASO.
  • the CAMK2DA14 expression and/or the CAMK2D-E14 expression in a method described herein are measured as described in Example 2.
  • the target cell (i.e., expressing the CAMK2D pre-mRNA) comprises a human cell or a mammalian cell.
  • the target cell comprises a cardiomyocyte (e.g, human cardiomyocytes derived from pluripotent stem cells).
  • the CAMK2D is a human CAMK2D.
  • the present disclosure provides a method for treating or preventing a cardiovascular disease in a subject in need thereof, comprising administering to the subject a therapeutically or prophylactically effective amount of the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure.
  • the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure is for use as a medicament.
  • the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure is for use in the treatment of a cardiovascular disease.
  • the use of the ASO, the conjugate, the pharmaceutically acceptable salt, or the pharmaceutical composition of the present disclosure for the preparation of a medicament for the treatment or preventing of a cardiovascular disease.
  • the cardiovascular disease comprises a coronary artery disease, stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, venous thrombosis, or any combination thereof.
  • the cardiovascular disease is cardiomyopathy.
  • the ASO, conjugate, salt, or pharmaceutical composition is administered intracardially, orally, parenterally, intrathecally, intra-cerebroventricularly, pulmorarily, topically, intraventricularly, or any combination thereof.
  • FIG. 1 provides exemplary ASOs targeting the target region (i.e., exon 14) of a
  • FIG. 1 show (i) the SEQ ID number designated for the sequence only of the ASO, (ii) the target start and end positions on the CAMK2D pre-mRNA sequence, (iii) the ASO sequence (i.e., sequence motif) without any particular design or chemical structure, (iv) the ASO number, (v) the sequence motif with a particular design, (vi) the ASO design number ("DES No.”), and (vii) compound no.
  • FIG. 2 provides a table summarizing the potency of different exemplary ASOs in inhibiting and/or reducing the expression of CAMK2D-E14 mRNA in human cardiomyocytes.
  • the potency of the ASOs are shown as the proportion (; i.e ., number) of exon 14 containing transcripts ( CAMK2D-E14 mRNA) remaining in cells treated with the ASO indicated, as compared to the level (i.e., number) of exon 14 containing transcripts ( CAMK2D-E14 mRNA) in non-treated cells (normalized to 100%).
  • FIG. 3 shows the potency of different ASOs in increasing CAMK2DA14 mRNA expression in human cardiomyocytes.
  • the potency of the ASOs are shown as the proportion ( i.e ., number) of CAMK2DA14 mRNA in cells treated with the ASO, as compared to the level ⁇ i.e., number) of CAMK2DA14 mRNA present in non-treated ⁇ i.e., control) cells (normalized to 100%).
  • Data shown for the ASOs is the average of the following 17 ASOs: compounds 12 1, 13 1, 20_1, 26_1, 26_2, 27_1, 28 1, 29_1, 31 1, 32 1, 33_2, 34 1, 35 1, 36 1, 38 1, 39 1, and 39_2.
  • Control refers to human cardiomyocytes treated with PBS only, p-value was calculated using student’s T-test.
  • the present disclosure is directed to an ASO comprising a contiguous nucleotide sequence of at least 10 nucleotides that has at least about 90% complementarity to a nucleic acid sequence within SEQ ID NO: 1, wherein the ASO has a length of 10 to 40 nucleotides and is capable of modulating the splicing of a mammalian CAMK2D pre-mRNA transcript.
  • the ASO is capable of reducing the expression of a CAMK2D mRNA transcript comprising exon 14 ( CAMK2D-E14 ), or the protein encoded thereof, in a target cell ⁇ e.g., expressing the CAMK2D pre-mRNA transcript).
  • a or “an” entity refers to one or more of that entity; for example, "a nucleotide sequence,” is understood to represent one or more nucleotide sequences.
  • the terms “a” (or “an”), “one or more,” and “at least one” can he used interchangeably herein.
  • the term "about” is used herein to mean approximately, roughly, around, or in the regions of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). For example, if it is stated that an ASO reduces expression of a CAMK2D transcript (e.g, CAMK2D-E14 mRNA) in a cell following administration of the ASO "by at least about 20%, " it is implied that the CAMK2D transcript levels are reduced by a range of 18% to 22%.
  • a CAMK2D transcript e.g, CAMK2D-E14 mRNA
  • oligonucleotide as used herein is defined as it is generally understood by the skilled person as a molecule comprising two or more covalently linked nucleosides. Such covalently bound nucleosides can also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification and isolation. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides.
  • the oligonucleotides of the disclosure are man-made, and are chemically synthesized, and are typically purified or isolated.
  • the oligonucleotides of the disclosure can comprise one or more modified nucleosides, such as 2’ sugar modified nucleosides.
  • the oligonucleotides of the disclosure can comprise one or more modified intemucleoside linkages, such as one or more phosphorothioate internucleoside linkages.
  • antisense oligonucleotide or "ASO,” as used herein, is defined as an oligonucleotide capable of modulating expression of a target gene by hybridizing to a target nucleic acid, in particular to a contiguous sequence on a target nucleic acid.
  • ASOs are not essentially double stranded and are therefore not siRNAs or shRNAs. In some aspects, the ASOs of the present disclosure can be single stranded.
  • single stranded oligonucleotides of the present disclosure can form hairpins or intermolecular duplex structures (duplex between two molecules of the same oligonucleotide), as long as the degree of intra or inter self-complementarity is less than approximately 50% across of the full length of the oligonucleotide.
  • the single stranded ASOs of the disclosure do not contain RNA nucleosides.
  • ASOs of the disclosure comprise one or more modified nucleosides or nucleotides, such as T sugar modified nucleosides.
  • the non-modified nucleosides of an ASO disclosed herein are DNA nucleosides.
  • sequence motif represents the sequence of nucleobases, independent of the nucleoside sugar chemistry and/or design.
  • nucleobases A, T, C and G can be modified, for example, capital C can be 5-methyl cytosine beta-D-oxy LNA nucleoside, and in RNA sequences, T can be U.
  • all the nucleosides of an antisense oligonucleotide constitute the contiguous nucleotide sequence.
  • the contiguous nucleotide sequence is the sequence of nucleotides in the ASOs of the disclosure which are complementary to, and in some instances fully complementary to, the target nucleic acid or target sequence.
  • an ASO comprises the contiguous nucleotide sequence, and can optionally comprise further nucleotide(s), for example a nucleotide linker region which can be used to attach a functional group (e.g. a conjugate group) to the contiguous nucleotide sequence.
  • the nucleotide linker region can be complementary to the target nucleic acid.
  • the nucleotide linker region is not complementary to the target nucleic acid. It is understood that the contiguous nucleotide sequence of an ASO cannot be longer than the ASO as such, and that the ASO cannot be shorter than the contiguous nucleotide sequence.
  • nucleic acids or “nucleotides” is intended to encompass plural nucleic acids.
  • the term “nucleic acids” or “nucleotides” refers to a target sequence, e.g., pre-mRNAs, mRNAs, or DNAs in vivo or in vitro.
  • the nucleic acids or nucleotides can be naturally occurring sequences within a cell.
  • nucleic acids or nucleotides refer to a sequence in the ASOs of the disclosure.
  • the nucleic acids or nucleotides are not naturally occurring, i.e., chemically synthesized, enzymatically produced, recombinantly produced, or any combination thereof.
  • the nucleic acids or nucleotides in the ASOs are produced synthetically or recombinantly, but are not a naturally occurring sequence or a fragment thereof.
  • the nucleic acids or nucleotides in the ASOs are not naturally occurring because they contain at least one nucleotide analog that is not naturally occurring in nature.
  • nucleic acid refers to a single nucleic acid segment, e.g., a DNA, an RNA, or an analog thereof, present in a polynucleotide.
  • Nucleic acid or “nucleotide” includes naturally occurring nucleic acids or non -naturally occurring nucleic acids.
  • nucleotide or “unit” and “monomer” are used interchangeably. It will be recognized that when referring to a sequence of nucleotides or monomers, what is referred to is the sequence of bases, such as A, T, G, C or U, and analogs thereof
  • nucleotide refers to a glycoside comprising a sugar moiety, a base moiety and a covalently linked group (linkage group), such as a phosphate or phosphorothioate intemucleotide linkage group, and covers both naturally occurring nucleotides, such as DNA or RNA, and non-naturally occurring nucleotides comprising modified sugar and/or base moieties, which are also referred to as "nucleotide analogs" herein.
  • a single nucleotide (unit) can also be referred to as a monomer or nucleic acid unit.
  • nucleotide analogs refers to nucleotides having modified sugar moieties.
  • nucleotides having modified sugar moieties e.g., LNA
  • nucleotide analogs refers to nucleotides having modified nucleobase moieties.
  • nucleotides having modified nucleobase moieties include, but are not limited to, 5 -methyl-cytosine, isocytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, pseudoisocytosine, 5-bromouracil, 5-propynyl -uracil, thiazolo-uracil, 2-thio-uracil, 2- thiothymine, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2,6-diaminopurine, and 2- chloro-6-aminopurine.
  • the 5' terminal nuc!eotide of an oligonucleotide ⁇ e.g., ASOs disclosed herein does not comprise a 5' interaudeotide linkage group, although it can comprise a 5' terminal group.
  • nucleoside is used to refer to a glycoside comprising a sugar moiety and a base moiety, and can therefore be used when referring to the nucleotide units, which are covalently linked by the internucieotide linkages between the nucleotides of the ASO.
  • nucleotide is often used to refer to a nucleic acid monomer or unit.
  • nucleotide can refer to the base alone, i.e., a nucieobase sequence comprising cytosine (DNA and RNA), guanine (DNA and RNA), adenine (DNA and RNA), thymine (DNA) and uracil (RNA), in which the presence of the sugar backbone and internucieotide linkages are implicit.
  • nucleotide can refer to a "nucleoside.”
  • nucleoside can be used, even when specifying the presence or nature of the linkages between the nucleosides.
  • nucleotide length or the “length” of an ASO, or contiguous nucleotide sequence thereof, as used herein means the total number of the nucleotides (monomers) in a given sequence.
  • sequence of cctgagtgagaacaa SEQ ID NO: 12
  • nucleotide length of the sequence is 15.
  • nucleotide length or “length” is therefore used herein interchangeably with “nucleotide number.”
  • Nucleotides and nucleosides are the building blocks of oligonucleotides and polynucleotides, and for the purposes of the present disclosure include both naturally occurring and non-naturally occurring nucleotides and nucleosides.
  • nucleotides such as DNA and RNA nucleotides comprise a ribose sugar moiety, a nucieobase moiety and one or more phosphate groups (which is absent in nucleosides).
  • Nucleosides and nucleotides can also interchangeably be referred to as "units" or "monomers”.
  • modified nucleoside refers to nucleosides modified as compared to the equivalent DNA or RNA nucleoside by the introduction of one or more modifications of the sugar moiety or the (nucleo)base moiety.
  • one or more of the modified nucleosides of the ASOs of the disclosure comprise a modified sugar moiety.
  • modified nucleoside can also be used herein interchangeably with the term “nucleoside analogue,” modified “units,” or modified “monomers.” Nucleosides with an unmodified DNA or RNA sugar moiety are termed DNA or RNA nucleosides herein.
  • nucleosides with modifications in the base region of the DNA or RNA nucleoside are still termed DNA or RNA if they allow Watson Crick base pairing.
  • modified nucleosides which can be used in the ASOs of the disclosure include LNA, 2’-0- MOE and morpholino nucleoside analogues. Examples of other modified nucleosides are provided elsewhere in the present disclosure.
  • a "high affinity modified nucleoside,” as used herein, is a modified nucleotide which, when incorporated into the oligonucleotide, enhances the affinity of the oligonucleotide for its complementary target, for example, as measured by the melting temperature (T m ).
  • a high affinity modified nucleoside of the present disclosure can result in an increase in melting temperature between +0.5 to +12°C, in some instances between +1.5 to +10°C and in others between +3 to +8°C per modified nucleoside.
  • Numerous high affinity modified nucleosides are known in the art and include, for example, many T substituted nucleosides as well as locked nucleic acids (LNA) (see e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Elhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213).
  • modified internucleoside linkage is defined as generally understood by the skilled person as linkages other than phosphodiester (PO) linkages, that covalently couple two nucleosides together.
  • the oligonucleotides of the disclosure can therefore comprise one or more modified intemucleoside linkages, such as one or more phosphorothioate internucleoside linkage.
  • phosphorothioate such as at least about 60%, such as at least about 70%, such as at least about 75%, such as at least about 80%, or such as at least about 90% of the intemucleoside linkages of the ASO, or contiguous nucleotide sequence thereof, are phosphorothioate.
  • all of the intemucleoside linkages of the ASO, or contiguous nucleotide sequence thereof are phosphorothioate.
  • all the intemucleoside linkages of the contiguous nucleotide sequence of the oligonucleotide are phosphorothioate, or all the intemucleoside linkages of the oligonucleotide are phosphorothioate linkages.
  • nucleobase includes the purine (e.g. adenine and guanine) and pyrimidine (e.g. uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
  • pyrimidine e.g. uracil, thymine and cytosine
  • nucleobase also encompasses modified nucleobases which can differ from naturally occurring nucleobases, but which are functional during nucleic acid hybridization.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are, for example, described in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
  • the nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C or U, wherein each letter can optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties of the ASOs disclosed herein are selected from A, T, G, C, and 5-methyl cytosine.
  • 5-methyl cytosine LNA nucleosides can be used for LNA gapmers.
  • modified oligonucleotide describes an oligonucleotide
  • chimeric oligonucleotide is a term that has been used in the literature to describe oligonucleotides comprising modified nucleosides (e.g, sugar modified nucleosides) and DNA nucleosides.
  • the ASO of the disclosure is a chimeric oligonucleotide.
  • alkyl signifies a straight- chain or branched-chain alkyl group with 1 to 8 carbon atoms, particularly a straight or branched- chain alkyl group with 1 to 6 carbon atoms and more particularly a straight or branched-chain alkyl group with 1 to 4 carbon atoms.
  • Examples of straight-chain and branched-chain C i-Cx alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, the isomeric pentyls, the isomeric hexyls, the isomeric heptyls and the isomeric octyls, particularly methyl, ethyl, propyl, butyl and pentyl.
  • Particular examples of alkyl are methyl.
  • Further examples of alkyl are mono, di or trifluoro methyl, ethyl or propyl, such as cyclopropyl (cPr), or mono, di or tri fluoro cycloproyl.
  • alkoxy signifies a group of the formula alkyl-O- in which the term “alkyl” has the previously given significance, such as methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec.butoxy and tert.butoxy. Particular "alkoxy” are methoxy.
  • bicyclic sugar refers to a modified sugar moiety comprising a 4 to 7 membered ring comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure.
  • the bridge connects the C2' and C4' of the ribose sugar ring of a nucleoside (i.e., 2'-4' bridge), as observed in LNA nucleosides.
  • exons or “exonic regions” refer to nucleic acid molecules containing a sequence of nucleotides that is transcribed into RNA and is represented in a mature form of RNA, such as mRNA (messenger RNA), after splicing and other RNA processing.
  • An mRNA contains one or more exons operatively linked.
  • exons can encode polypeptides or a portion of a polypeptide.
  • exons can contain non- translated sequences, for example, translational regulatory sequences.
  • introns or “intronic regions” refer to nucleic acid molecules containing a sequence of nucleotides that is transcribed into RNA and is then typically removed from the RNA by splicing to create a mature form of an RNA, for example, an mRNA.
  • nucleotide sequences of introns are not incorporated into mature RNAs, nor are intron sequences or portions thereof translated and incorporated into a polypeptide.
  • Splice signal sequences such as splice donors and acceptors, are used by the splicing machinery of a cell to remove introns from RNA.
  • an intron in one splice variant can be an exon (i.e present in the spliced transcript) in another variant.
  • spliced mRNA encoding an intron fusion protein can include an exon(s) and introns.
  • splicing refers to a process of RNA maturation in which introns in the RNA are removed and exons are operatively linked to create a messenger RNA (mRNA).
  • mRNA messenger RNA
  • alternate splicing refers to the process of producing multiple mRNAs from a gene.
  • alternate splicing can include operatively linking less than all the exons of a gene, and/or operatively linking one or more alternate exons that are not present in all transcripts derived from a gene.
  • splice modulation refers to a process that can be used to correct cryptic splicing, modulate alternative splicing, restore the open reading frame, and induce protein knockdown.
  • a splice modulation can be used to modulate alternative splicing to generate a CAMK2DA14 mRNA and thereby enhance expression of CAMK2DA14 protein.
  • Splice modulation can be assayed by RNA sequencing (RNA-Seq), which allows for a quantitative assessment of the different splice products of a pre- mRNA.
  • the ASOs modulate the splicing of the CAMK2D pre- mRNA so as to reduce the level of mature CAMK2D mRNA which comprises an exon 14 (CAMK2D-E14 mRNA), and to increase the expression of the level of mature CAMK2D mRNA which lacks exon 14 ( CAMK2DA14 mRNA).
  • a "coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids.
  • a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it can be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions (“UTRs”), and the like, are not part of a coding region.
  • a coding region typically determined by a start codon at the 5' terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3' terminus, encoding the carboxyl terminus of the resulting polypeptide.
  • non-coding region means a nucleotide sequence that is not a coding region.
  • non-coding regions include, but are not limited to, promoters, ribosome binding sites, transcriptional terminators, introns, untranslated regions ("UTRs"), noncoding exons and the like. Some of the exons can be wholly or part of the 5' untranslated region (5' UTR) or the 3' untranslated region (3' UTR) of each transcript.
  • the untranslated regions are important for efficient translation of the transcript and for controlling the rate of translation and half-life of the transcript.
  • region when used in the context of a nucleotide sequence refers to a section of that sequence.
  • region within a nucleotide sequence or region within the complement of a nucleotide sequence refers to a sequence shorter than the nucleotide sequence, but longer than at least 10 nucleotides located within the particular nucleotide sequence or the complement of the nucleotides sequence, respectively.
  • sequence or “subsequence” can also refer to a region of a nucleotide sequence.
  • downstream when referring to a nucleotide sequence, means that a nucleic acid or a nucleotide sequence is located 3' to a reference nucleotide sequence.
  • downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.
  • upstream refers to a nucleotide sequence that is located 5' to a reference nucleotide sequence.
  • regulatory region refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, UTRs, and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • target sequence refers to a sequence of nucleotides present in the target nucleic acid which comprises the nucleobase sequence which is complementary to the ASOs of the disclosure (e.g ., exon 14 of a CAMK2D pre-mRNA transcript).
  • the target sequence consists of a region on the target nucleic acid with a nucleobase sequence that is complementary to the contiguous nucleotide sequence of the ASO of the disclosure. This region of the target nucleic acid can interchangeably be referred to as the target nucleotide sequence, target sequence, or target region.
  • the target sequence is longer than the complementary sequence of a single ASO, and can, for example, represent a preferred region of the target nucleic acid which can be targeted by several oligonucleotides of the disclosure.
  • the term "target cell” refers to a cell which expresses the target nucleic acid.
  • the target cell comprises a mammalian cell, such as a rodent cell, such as a mouse cell or a rat cell, or a primate cell such as a monkey cell or a human cell.
  • the target cell is a transgenic animal cell which is expressing the human CAMK2D target nucleic acid.
  • the target cell is a myeloid cell or a myeloid lineage cell.
  • the target cell is a heart cell, such as a cardiomyocyte.
  • the target cell is a cardiomyocyte derived from iPSC (e.g., iCell Cardiomyocytes2, available from Cellular Dynamics).
  • the target cell of the present disclosure expresses the CAMK2D pre- mRNA, which is processed in the cell to the mature CAMK2D mRNA, resulting in the expression of the both CAMK2D-E14 and/or CAMK2DA14 protein.
  • the ASOs of the disclosure modulate the splicing of the CAMK2D pre-mRNA to increase the proportion of CAMK2D mRNA which lacks CAMK2D exon 14 (or a portion of exon 14).
  • thereby the expression of CAMK2DA14 transcript variants can be increased, as compared to CAMK2D-E14 transcript variants.
  • nucleobase complementarity refers to the ability of a nucleobase (e.g, those present in an ASO disclosed herein) to pair with another nucleobase (e.g, those present in the target sequence, e.g, CAMK2D pre-mRNA transcript).
  • guanine (G) pairs with cytosine (C)
  • adenine (A) pairs with thymine (T) or uracil (U).
  • ASOs of the present disclosure can comprise nucleosides with modified nucleobases, for example 5-methyl cytosine is often used in place of cytosine, and as such, the term complementarity encompasses base-paring between non-modified and modified nucleobases (see for example Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1).
  • complement indicates a sequence that is complementary to a reference sequence. It is well known that complementarity is the base principle (Watson-Crick base pairing) of DNA replication and transcription as it is a property shared between two DNA or RNA sequences, such that when they are aligned antiparallel to each other, the nucleotide bases at each position in the sequences will be complementary, much like looking in the mirror and seeing the reverse of things. Therefore, for example, the complement of a sequence of 5"'ATGC"3' can be written as 3"'TACG"5' or 5"'GCAT"3'.
  • reverse complement refers to 100% match or complementarity (i.e., fully complementary) to a contiguous nucleic acid sequence within a CAMK2D transcript.
  • the term "complementary" refers to 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% match or complementarity to a contiguous nucleic acid sequence within a CAMK2D transcript.
  • % complementary refers to the proportion of nucleotides (in percent) of a contiguous nucleotide sequence in a nucleic acid molecule (e.g. ASO) which across the contiguous nucleotide sequence, are complementary to a reference sequence (e.g., a target sequence or sequence motif).
  • ASO nucleic acid molecule
  • the percentage of complementarity is thus calculated by counting the number of aligned nucleobases that are complementary (from Watson Crick base pairs) between the two sequences (when aligned with the target sequence 5’ -3’ and the oligonucleotide sequence from 3’ -5’), dividing that number by the total number of nucleotides in the oligonucleotide and multiplying by 100.
  • a nucleobase/nucleotide which does not align (form a base pair) is termed a "mismatch.” Insertions and deletions are not allowed in the calculation of % complementarity of a contiguous nucleotide sequence.
  • a contiguous nucleotide sequence in a nucleic acid molecule e.g. oligonucleotide
  • a reference sequence e.g. a sequence motif
  • 80.11, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.
  • naturally occurring variant thereof refers to variants of the CAMK2D polypeptide sequence or CAMK2D nucleic acid sequence (e.g., transcript) which exist naturally within the defined taxonomic group, such as mammalian, such as mouse, monkey, and human.
  • CAMK2D nucleic acid sequence e.g., transcript
  • the term also can encompass any allelic variant of the CAMK2D-Q ncoding genomic DNA which is found at Chromosomal position 4q26 (i.e., residues 113,451,032 to 113,761,927 of GenBank Accession No.
  • RNA such as mRNA derived therefrom.
  • “Naturally occurring variants” can also include variants derived from alternative splicing of the CAMK2D mRNA.
  • the term also includes naturally occurring forms of the protein, which can therefore be processed, e.g, by co- or post-translational modifications, such as signal peptide cleavage, proteolytic cleavage, glycosylation, etc.
  • the naturally occurring variants have at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to a mammalian CAMK2D target nucleic acid, such as that set forth in SEQ ID NO: 1. In some aspects, the naturally occurring variants have at least 99% homology to the human CAMK2D target nucleic acid of SEQ ID NO: 1.
  • nucleotide sequences when referencing two separate nucleic acid or nucleotide sequences can be used to clarify regions of the sequences that correspond or are similar to each other based on homology and/or functionality, although the nucleotides of the specific sequences can be numbered differently. For example, different isoforms of a gene transcript can have similar or conserved portions of nucleotide sequences whose numbering can differ in the respective isoforms based on alternative splicing and/or other modifications.
  • nucleic acid or nucleotide sequence e.g ., a gene transcript and whether to begin numbering the sequence from the translation start codon or to include the 5'UTR.
  • nucleic acid or nucleotide sequence of different variants of a gene or gene transcript can vary. As used herein, however, the regions of the variants that share nucleic acid or nucleotide sequence homology and/or functionality are deemed to "correspond" to one another.
  • a nucleotide sequence of a CAMK2D transcript corresponding to nucleotides X to Y of SEQ ID NO: 1 refers to an CAMK2D transcript sequence (e.g., CAMK2D pre-mRNA or mRNA) that has an identical sequence or a similar sequence to nucleotides X to Y of SEQ ID NO: 1, wherein X is the start site and Y is the end site (as shown in FIG. 1).
  • a person of ordinary skill in the art can identify the corresponding X and Y residues in the CAMK2D transcript sequence by aligning the CAMK2D transcript sequence with SEQ ID NO: 1.
  • hybridizing or “hybridizes,” as used herein, is to be understood as two nucleic acid strands (e.g. an ASO and a target nucleic acid) forming hydrogen bonds between base pairs on opposite strands thereby forming a duplex.
  • the affinity of the binding between two nucleic acid strands is the strength of the hybridization. It is often described in terms of the melting temperature (T m ) defined as the temperature at which half of the oligonucleotides are duplexed with the target nucleic acid. At physiological conditions T m is not strictly proportional to the affinity (Mergny and Lacroix, 2003, Oligonucleotides 13:515-537).
  • ⁇ G° is the energy associated with a reaction where aqueous concentrations are 1M, the pH is 7, and the temperature is 37°C.
  • the hybridization of oligonucleotides to a target nucleic acid is a spontaneous reaction and for spontaneous reactions ⁇ G° is less than zero.
  • DO° can be measured experimentally, for example, by use of the isothermal titration calorimetry (ITC) method as described in Hansen et al., 1965, ( hem. Comm. 36-38 and Holdgate et al., 2005, Drug Discov Today.
  • ITC isothermal titration calorimetry
  • the skilled person will know that commercial equipment is available for ⁇ G° measurements.
  • ⁇ G° can also be estimated numerically by using the nearest neighbor model as described by SantaLucia, 1998, Proc Nad Acad Sci USA. 95: 1460-1465 using appropriately derived thermodynamic parameters described by Sugimoto et al., 1995, Biochemistry 34:11211-11216 and McTigue et al., 2004, Biochemistry 43:5388-5405.
  • oligonucleotides of the present disclosure hybridize to a target nucleic acid with estimated ⁇ G° values below -10 kcal for oligonucleotides that are 10-30 nucleotides in length. In some aspects the degree or strength of hybridization is measured by the standard state Gibbs free energy ⁇ G°.
  • the oligonucleotides can hybridize to a target nucleic acid with estimated ⁇ G° values below the range of -10 kcal, such as below -15 kcal, such as below - 20 kcal and such as below -25 kcal for oligonucleotides that are 8-30 nucleotides in length.
  • the oligonucleotides hybridize to a target nucleic acid with an estimated ⁇ G° value of -10 to -60 kcal, such as -12 to -40, such as from -15 to -30 kcal or- 16 to -27 kcal such as -18 to -25 kcal.
  • transcript can refer to a primary transcript that is synthesized by transcription of DNA and becomes a messenger RNA (mRNA) after processing, i.e., a precursor messenger RNA (pre-mRNA), and the processed mRNA itself.
  • mRNA messenger RNA
  • pre-mRNA precursor messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • pre-mRNA precursor messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • mRNA messenger RNA
  • pre-mRNA precursor messenger RNA
  • miRNA miRNA
  • RNA messenger RNA
  • expression produces a "gene product.”
  • a gene product can be either a nucleic acid, e.g ., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript.
  • Gene products described herein further include nucleic acids with post transcriptional modifications, e.g. , polyadenylation or splicing, or polypeptides with post translational modifications, e.g, methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.
  • DES Number refers to a unique number given to a nucleotide sequence having a specific pattern of nucleosides (e.g, DNA) and nucleoside analogs (e.g, LNA).
  • ASO ASO
  • DES-0012 refers to an ASO sequence of CCTGAGTGAGAACAA (SEQ ID NO: 12) with an ASO design of LDDDLDLDDDLLDLL (i.e., CctgAgTgagAAcAA), wherein the L (i.e., upper case letter) indicates a nucleoside analog (e.g, beta-D-oxy-LNA) and the D (i.e., lower case letter) indicates a nucleoside (e.g, DNA).
  • L i.e., upper case letter
  • the D i.e., lower case letter
  • ASO Number refers to a unique number given to a nucleotide sequence having the detailed chemical structure of the components, e.g, nucleosides (e.g, DNA), nucleoside analogs (e.g, LNA, e.g, beta-D-oxy-LNA), nucleobase (e.g, A, T, G, C, U, or MC), and backbone structure (e.g, phosphorothioate or phosphorodiester).
  • nucleosides e.g, DNA
  • nucleoside analogs e.g, LNA, e.g, beta-D-oxy-LNA
  • nucleobase e.g, A, T, G, C, U, or MC
  • backbone structure e.g, phosphorothioate or phosphorodiester
  • a reference to a SEQ ID number includes a particular nucleic acid sequence but does not include any design or full chemical structure. Furtherfore, the ASOs disclosed in the figures (e.g, FIG. 1) herein show a representative design but are not limited to the specific design shown unless otherwise indicated.
  • ICso is the median inhibitory concentration of a therapeutic molecule.
  • ECso is the median effective concentration of a therapeutic molecule relative to a vehicle or control (e.g, saline).
  • ICso is the concentration of a therapeutic molecule that reduces a biological response, e.g, transcription of mRNA or protein expression, by 50% of the biological response that is achieved by the therapeutic molecule.
  • ECso is the concentration of a therapeutic molecule that produces 50% of the biological response, e.g, transcription of mRNA or protein expression.
  • ICso or ECso can be calculated by any number of means known in the art.
  • subject or “individual” or “animal” or “patient” or “mammal,” is meant any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired.
  • Mammalian subjects include humans, domestic animals, farm animals, sports animals, and zoo animals including, e.g., humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, bears, and so on.
  • the subject is a human.
  • composition refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition would be administered.
  • Such composition can be sterile.
  • an “effective amount” of a composition disclosed herein refers to an amount sufficient to carry out a specifically stated purpose.
  • An “effective amount” can be determined empirically and in a routine manner, in relation to the stated purpose.
  • Terms such as “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent and/or slow the development of a targeted pathologic condition or disorder.
  • those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented.
  • a subject is successfully "treated” for a disease or condition disclosed elsewhere herein according to the methods provided herein if the patient shows, e.g, total, partial, or transient alleviation or elimination of symptoms associated with the disease or disorder.
  • the present disclosure employs antisense oligonucleotides (ASOs) for use in modulating the function of nucleic acid molecules encoding mammalian CAMK2D, such as the CAMK2D nucleic acid, e.g., CAMK2D transcript, including CAMK2D pre-mRNA, and CAMK2D mRNA, or naturally occurring variants of such nucleic acid molecules encoding mammalian CAMK2D.
  • ASO in the context of the present disclosure, refers to a molecule formed by covalent linkage of two or more nucleotides (i.e., an oligonucleotide).
  • the ASOs of the present disclosure modulate the splicing of a mammalian CAMK2D pre-mRNA transcript, such as that described herein. In some aspects, modulating the splicing of a mammalian CAMK2D pre-mRNA transcript can regulate the expression and/or activity of certain CAMK2D variants.
  • the ASOs disclosed herein are capable of reducing or inhibiting the expression (e.g, number) of a CAMK2D transcript (e.g, mRNA) comprising exon 14 (" CAMK2D-E14 ") in a cell.
  • a CAMK2D transcript e.g, mRNA
  • the ASOs disclosed herein are capable of selectively reducing the expression of CAMK2D-E14 in a cell without reducing the expression of CAMK2DA14 in the cell by more than about 5% or by more than about 10%.
  • the ASOs disclosed herein are capable of selectively reducing the expression of CAMK2D-E14 in a cell without reducing the expression of CAMK2DA14 in the cell by more than about 5%.
  • the ASOs disclosed herein are capable of selectively reducing the expression of CAMK2D-E14 in a cell without reducing the expression of CAMK2DA14 in the cell by more than about 10%. In certain aspects, the ASOs are capable of selectively reducing the expression of CAMK2D-E14 in the cell without significantly reducing the expression of CAMK2DA14 in the cell. As described herein, in certain aspects, the ASOs are capable of increasing the expression of CAMK2DA14 in the cell.
  • reducing or “inhibiting” the expression of, e.g., CAMK2D-E14 transcript, as used herein, is to be understood as an overall term for an ASO’s ability to inhibit or reduce the amount or the activity of CAMK2D-E14 protein in a target cell (e.g, by reducing or inhibiting the expression of CAMK2D-E14 mRNA and thereby reducing the expression of a CAMK2D-E14 protein). Inhibition of activity can be determined by measuring the level (e.g, number) of CAMK2D-E14 mRNA, or by measuring the level (e.g, number) or activity of CAMK2D-E14 protein in a cell.
  • Inhibition of expression can therefore be determined in vitro or in vivo. It will be understood that splice modulation can result in an inhibition of expression (e.g, number) of CAMK2D-E14 transcript (e.g, mRNA), or the protein encoded thereof, in the cell. In certain aspects, the expression (e.g, number) of CAMK2D-E14 transcript (e.g, mRNA) is reduced by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASOs. As used herein, the term “corresponding cell that is not exposed to the ASOs” can refer to the same cell but prior to the treatment with an ASO disclosed herein.
  • treating a cell with an ASO of the present disclosure reduces (e.g, by at least about 10% or by at least about 20%) the expression of CAMK2D-E14 transcript (e.g, mRNA) in the cell compared to the expression of CAMK2D-E14 transcript (e.g., mRNA) in the same cell prior to the ASO treatment.
  • the term “corresponding cell that is not exposed to the ASOs” can refer to the same cell type (but not necessarily the same cell, e.g, both human cardiomyocytes derived from pluripotent stem cells) that has not been treated with an ASO described herein.
  • treating a cell with an ASO of the present disclosure reduces (e.g, by at least about 10% or by at least about 20%) the expression of CAMK2D-E14 transcript (e.g, mRNA) in the cell compared to the expression of CAMK2D-E14 transcript (e.g., mRNA) in a reference cell prior to the ASO treatment, wherein the reference cell in the cell treated with the ASO are of the same type.
  • Nonlimiting examples of ASOs disclosed herein that can reduce the expression of CAMK2D-E14 transcript include the following compounds: 29_1, 33_2, 35 1, 35_2, 31 1, 34 1, 39_2, 32 1, 26_2, 30 1, 36 1, 28 1, 26_1, 39 1, 12 1, 20_1, 13 1, 38 1, 27_1, 18 2, 20_2, 37_2, 14 1, 17 1, 19 1, 33 1, 15 1, 25 1, 37 1, 22_2, 16 1, 18 1, 24_1, 40_2, 21 1, 23 1, and 22_1 ( see FIG. 2).
  • reducing the expression of CAMK2D-E14 transcript (e.g, mRNA) in a cell results in the cell expressing reduced levels of CAMK2D-E14 protein.
  • the level of CAMK2D-E14 protein in a cell treated with the ASOs disclosed herein is reduced by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASOs.
  • reducing the expression of CAMK2D-E14 transcript (e.g, mRNA) in a cell can decrease the total CAMK2D protein expression (i.e., including all variants) in the cell.
  • the total CAMK2D protein expression in a cell treated with the ASOs of the present disclosure is reduced by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASOs.
  • the ASOs disclosed herein can change the ratio of alternative
  • CAMK2D splice variants expressed in a cell For instance, in certain aspects, reducing the expression of CAMK2D-E14 transcript in a cell can result in increased or enhanced expression of other CAMK2D transcript variants in the cell, such as those that lack exon 14 (CAMK2DA14). Accordingly, in some aspects, the ASOs disclosed herein can enhance or increase the expression of CAMK2DA14 transcript (e.g, mRNA) in a cell. In certain aspects, expression of the CAMK2DA14 transcript is enhanced or increased by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASOs.
  • CAMK2DA14 transcript e.g, mRNA
  • the level of CAMK2DA14 protein in a cell treated with the ASOs disclosed herein is increased by at least about 10% or at least about 20% compared to a corresponding cell that is not exposed to the ASOs.
  • ASOs disclosed herein that can increase the expression of CAMK2DA14 transcript include the following compounds: 40_1, 39 1, 20_2, 21 1, 15 1, 35 1, 14 1, 18 1, 19 1, 36 1, 13 1, 38 1, 33_2, 29_1, 20_1, 26_1, 24_1, 25 1, 18 2, 12 1, 26_2, 37 1, 32 1, 17 1, 28 1, 33 1, 34 1, 22_2, 24_2, 37_2, 40_2, and 39_2.
  • ASOs that can enhance or increase the expression of CAMK2DA14 activity can be identified by measuring the increased expression (i.e., number) of CAMK2DA14 mRNA or CAMK2DA14 protein as compared to control cells (e.g., corresponding cells that were not exposed to the ASOs), or by measuring an increased ratio of expression ( i.e ., number) of CAMK2DA14 as compared to CAMK2D-E14 at the mRNA or protein level.
  • this can be determined by comparing the proportion (i.e., number) of CAMK2D-E14 mRNA remaining in cells treated with an ASO of the present disclosure, as compared to the level (i.e., number) of CAMK2D-E14 mRNA in untreated cells (normalized to 100%).
  • a change of ratio (CAMK2DA 14/CAMK2D-E14) of > 1 upon ASO treatment is indicative of an effective CAMK2DA14 enhancer - i.e., a compound which is capable of reducing the level of CAMK2D-E14 as compared to CAMK2DA14 transcripts in the cell.
  • the CAMK2DA14 enhancer is capable of eliciting a response of > about 1.5, > about 2, > about 2.5, > about 3, > about 3.5, > about 4, > about 4.5, > about 5, > about 5.5, > about 6, > about 6.5, > about 7, > about 7.5, > about 8, > about 8.5, > about 9, > about 9.5, > about 10, > about 11, > about 12, > about 13, > about 14, > about 15, > about 16, > about 17, > about 18, > about 19, > about 20, > about 21, > about 22, > about 23, > about 24, > about 25, > about 26, > about 27, > about 28, > about 29, > about 30, > about 31, > about 32, > about 33, > about 34, or > about 35.
  • the ASOs disclosed herein are capable of both i) increasing the amount of CAMK2DA14 mRNA or CAMK2DA14 protein in the target cell and ii) decreasing the amount of CAMK2D-E14 mRNA and CAMK2D-E14 protein in a target cell.
  • the ratio of the expression of CAMK2DA14 transcript (e.g, mRNA) to the expression of CAMK2D-E14 transcript (e.g, mRNA) is increased compared to a corresponding ratio of a cell that is not exposed to the ASOs of the present disclosure.
  • the ratio of the expression of CAMK2DA14 transcript (e.g, mRNA) to the expression of CAMK2D-E14 transcript (e.g, mRNA) is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 50-fold.
  • the ratio of the expression of CAMK2DA14 transcript (e.g, mRNA) to the expression of CAMK2D-E14 transcript (e.g, mRNA) is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, or at least about 50-fold, as measured using the assay described in Example 2.
  • the ratio of the expression of CAMK2DA14 transcript (e.g., mRNA) to the expression of CAMK2D-E14 transcript (e.g, mRNA) increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20- fold, at least about 25-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, or at least about 50-fold, as measured in cardiomyocytes (e.g, human cardiomyocytes derived from pluripotent stem cells, e.g, iCell Cardiomyocytes2 from Cellular Dynamics).
  • cardiomyocytes e.g, human cardiomyocytes derived from pluripotent stem cells, e.g, iCell Cardiomyocytes2 from Cellular Dynamics.
  • the ratio of the expression of CAMK2DA14 protein to the expression of CAMK2D-E14 protein is increased in a cell treated with the ASOs of the present disclosure. In certain aspects, the ratio of the expression of CAMK2DA14 protein to the expression of CAMK2D-E14 protein is increased by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 25-fold.
  • CAMK2DA14 can be measured by comparing mRNA levels, or levels of the corresponding protein products.
  • ratio refers to a comparison of the number of different molecules (e.g, protein or mRNA) in a sample (e.g, cell).
  • ratio of different transcript products e.g., CAMK2DA14 vs. CAMK2D-E14 ) refers to a comparison of the number of CAMK2DA14 mRNA molecules to the number of CAMK2D-E14 mRNA molecules in a sample.
  • ratio of different transcript products e.g., CAMK2DA14 vs.
  • CAMK2D-E14 can refer to a comparison of the number of CAMK2DA14 protein molecules to the number of CAMK2D-E14 protein molecules in a sample.
  • Anti-CAMK2D antibodies which can be used for assaying the protein levels of CAMK2D-E14 and CAMK2DA14 include monoclonal or polyclonal antibodies raised against CAMK2D (e.g., antibody for detection of both CAMK2D-E14 and CAMK2DA14 is available from Abeam (AB-181052) and antibody for detection of CAMK2D-E14 was generated by immunizing rabbits with the 14 amino acid peptide EPQTTVIHNPDGNK (i.e., exon 14; SEQ ID NO: 11) as described in Zhang et al., (2019)).
  • the CAMK2DA14 protein shown in SEQ ID NO: 5 has a mass that is about 56.4 kDa.
  • the CAMK2D-E14 protein (SEQ ID NO: 3) has a mass of approximately 57.9 kDa.
  • the mass of the E14 protein (i.e., SEQ ID NO: 11) is about 1.55 kDa.
  • the amount of each CAMK2D protein present in a cell can be measured using conventional assays, such as Protein Simple or conventional western blot analysis.
  • the amount of each CAMK2D mRNA molecules present in a cell can be measured by assays available in the art, such as quantitative-PCR.
  • ASOs of the present disclosure are or comprise gapmers, mixmers or totalmers. In certain aspects, the ASOs disclosed herein are mixmers. In some aspects, the ASOs disclosed herein are totalmers. In some aspects, the ASOs disclosed herein are not gapmers. In some aspects, the ASO is a morpholino oligonucleotide. In some aspects, the ASO is a 2’-0-M0E oligonucleotide, i.e. comprises one or more 2’-0-M0E nucleosides. In some aspects, the ASO is an LNA oligonucleotide, i.e. comprises one or more LNA nucleosides.
  • the ASO or contiguous nucleotide sequence thereof is 10 - 25 or 10 - 20 nucleotides in length.
  • the ASO consists or comprises of an oligonucleotide provided herein, such as a compound selected from Compound NOs #12_1 - #40_2, as shown in FIG. 1.
  • the ASO comprises one or more high affinity modified nucleosides.
  • the ASO comprises one or more sugar modified nucleosides, such as T sugar modified nucleosides.
  • the ASOs of the disclosure comprise one or more T sugar modified nucleoside independently selected from 2’-0-alkyl-RNA, 2’-0-methyl-RNA, T - alkoxy-RNA, 2’-0-methoxyethyl-RNA, 2’-amino-DNA, 2’-fluoro-DNA, arabino nucleic acid (ANA), 2’-fluoro-ANA, LNA nucleosides, or combinations thereof.
  • one or more of the modified nucleoside(s) can be a locked nucleic acid (LNA).
  • the ASO disclosed herein comprises at least one modified internucleoside linkage.
  • At least 75% of the internucleoside linkages of an ASO, or contiguous nucleotide sequence thereof, disclosed herein are phosphorothioate internucleoside linkages. In some aspects, all of the intemucleotide linkages are phosphorothioate linkages.
  • ASOs, or contiguous nucleotide sequence thereof are complementary to the CAMK2D target nucleic acid, such as SEQ ID NO: 1, or target sequence (such as a sequence selected from the group consisting of SEQ ID NO: 70, 71, 6, 7, 8, 9, 10, or target site region thereof (such as a sequence selected from the group consisting of SEQ ID NOs: 41 - 69), as measured across the length of the oligonucleotide, optionally with the exception of a mismatch, and optionally excluding nucleotide based linker regions which can link the oligonucleotide to an optional functional group such as a conjugate, or other non-complementary terminal nucleotides.
  • target nucleic acid such as SEQ ID NO: 1
  • target sequence such as a sequence selected from the group consisting of SEQ ID NO: 70, 71, 6, 7, 8, 9, 10, or target site region thereof (such as a sequence selected from the group consisting of SEQ ID NOs: 41 - 69
  • ASOs, or contiguous nucleotide sequence thereof, described herein comprises a sequence motif as shown in a sequence selected from SEQ ID NOs: 12 - 40, or at least 10 contiguous nucleotides thereof.
  • the ASO, or contiguous nucleotide sequence thereof comprises a sequence motif as shown in a sequence selected from SEQ ID NOs: 12 - 40, or at least 12 contiguous nucleotides thereof.
  • the ASO, or contiguous nucleotide sequence thereof comprises a sequence motif as shown in a sequence selected from SEQ ID NOs: 12 - 40, or at least 14 contiguous nucleotides thereof.
  • the ASO, or contiguous nucleotide sequence thereof comprises a sequence motif as shown in a sequence selected from SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18,
  • the ASO of the disclosure is selected from the following groups of ASOs, which are identified herein as Compound Nos. 12 1, 13 1, 14 1, 15 1, 16 1, 17 1, 18 1, 18 2, 19 1, 20_1, 20_2, 21 1, 22_1, 22_2, 23 1, 24_1, 24_2, 25 1, 26_1, 26_2, 27_1,
  • the ASOs of the disclosure are in solid powdered form, such as in the form of a lyophilized powder.
  • Splice modulating oligonucleotides typically operate via an occupation-based mechanism rather than via a degradation mechanism (such as RNaseH mediated inhibition). However, in some aspects, splice modulation can result in the alternative transcript being degraded, e.g, via non-sense mediated decay.
  • the ASOs of the present disclosure are capable of modulating the splicing of a mammalian CAMK2D pre-mRNA transcript and thereby, regulate the expression and/or activity of certain CAMK2D variants.
  • the ASOs disclosed herein are capable of targeting exon 14 of a CAMK2D pre-mRNA transcript and thereby, reducing the expression of a CAMK2D transcript (e.g, mRNA) comprising exon 14 (" CAMK2D-E14 ").
  • the ASOs of the present disclosure can affect the indirect inhibition of CAMK2D-E14 protein through the reduction in CAMK2D-E14 transcript (e.g, mRNA) levels in a cell.
  • CAMK2D can refer to CAMK2D from one or more species (e.g, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).
  • CAMK2D-E14 refers to CAMK2D transcripts (e.g., mRNA) and proteins, which comprises E14 exon, or comprise the amino acid sequence encoded by exon 14 of CAMK2D. Accordingly, the CAMK2D-E14 protein is encoded by a CAMK2D mRNA which comprises exon 14. Depending on the system, typically between about 10 - 30% of CAMK2D transcripts in tissues or cells, e.g. , heart tissue or cells, can comprise exon 14.
  • CAMK2DA14 refers to a CAMK2D transcript which lacks exon 14 (a CAMK2DA14 variant), or a CAMK2D protein which lacks the amino acids encoded by CAMK2D exon 14.
  • a CAMK2DA14 protein lacks all or essentially all of the amino acids encoded by CAMK2D exon 14.
  • exon 14 of SEQ ID NO: 1 ⁇ i.e., SEQ ID NO: 71
  • target is used to refer to the gene that the ASOs of the present disclosure specifically hybridizes/binds to (i.e., calcium/calmodulin dependent protein kinase II delta, referred to herein as "CAMK2D").
  • CAMK2D calcium/calmodulin dependent protein kinase II delta
  • CAMK2D is also known as CaM kinase II subunit delta and CamK-II subunit delta. Synonyms of CAMK2D are known and include CaMKIId or CAMKD.
  • the sequence for the human CAMK2D gene can be found under publicly available GenBank Accession Number NC_000004.12.
  • the sequence for the human CAMK2D pre-mRNA transcript (SEQ ID NO: 1) corresponds to residues 113,451,032 - 113,761,927 of NC_000004.12 (Chromosome 4) reverse strand.
  • An exemplary CAMK2D mRNA sequence (GenBank Accession No.
  • CAMK2DA14 NM_001221.3; referred to herein as “CAMK2DA14”
  • SEQ ID NO: 4 SEQ ID NO: 4
  • the sequence for the corresponding exemplary human CAMK2D protein can be found under publicly available Accession Numbers: Q13557-1 (canonical sequence, SEQ ID NO: 5; referred to herein as “CAMK2DA14"), A8MVS8, Q52PK4, Q59G21, Q8N553, Q9UGH6, and Q9UQE9.
  • CAMK2D-E14 a CAMK2D protein variant which comprises the amino acid sequence encoded by CAMK2D exon 14 is referred to herein as "CAMK2D-E14.”
  • the sequence of an exemplary CAMK2D-E14 protein (GenBank Accession No. NP OO 1308495.1) is provided in SEQ ID NO: 3, which differs from the canonical sequence (SEQ ID NO: 5) in that the amino acid E at position 329 of SEQ ID NO: 5 ⁇ i.e., the canonical sequence) is replaced with the following sequence:
  • CAMK2D-E14 protein comprises the amino acid sequence set forth in SEQ ID NO: 11 and one or more amino acids at both the 5’- and 3’ -ends of the amino acid sequence set forth in SEQ ID NO: 11.
  • An exemplary CAMK2D-E14 mRNA sequence (GenBank Accession No. NM_00 1321566.2) is provided in SEQ ID NO: 2, except that the nucleotide "t" in SEQ ID NO: 2 is shown as "u" in the mRNA.
  • Table 1 (below) provides exemplary exon and intron regions of a human CAMK2D pre-mRNA transcript (SEQ ID NO: 1).
  • An exemplary nucleic acid sequence for exon 14 i.e SEQ ID NO: 71
  • the amino acid sequence encoded by the exemplary nucleic acid for exon 14 is set forth in SEQ ID NO: 11.
  • the target nucleic acid of the ASOs disclosed herein is a nucleic acid which encodes a mammalian CAMK2D, e.g., human CAMK2D, and, in particular, CAMK2D-E14 protein.
  • the nucleic acid comprises a RNA, a mRNA, a pre-mRNA, a mature mRNA, or a cDNA sequence.
  • the target can therefore be referred to as a CAMK2D target nucleic acid.
  • a target nucleic acid is a CAMK2D pre-mRNA, as illustrated by SEQ ID NO: 1, or naturally occurring variant thereof.
  • the present disclosure identifies a region of the human CAMK2D pre-mRNA
  • SEQ ID NO: 1 SEQ ID NO: 1
  • ASOs disclosed herein e.g. , ASOs disclosed herein, e.g. , in order to modulate the splicing of exon 14 of the CAMK2D pre-mRNA.
  • the CAMK2D splicing can be modulated to reduce the proportion of CAMK2D transcripts which comprise exon 14 sequence.
  • the ASO of the present disclosure comprises a contiguous nucleotide sequence (e.g, at least 10 nucleotides in length, e.g., 10 to 40 nucleotides in length) that is complementary (e.g, fully complementary) to a region within a CAMK2D transcript ("target region"), e.g, a region corresponding to SEQ ID NO: 1.
  • target region e.g, a region corresponding to SEQ ID NO: 1.
  • the ASO comprises a contiguous nucleotide sequence that hybridizes to a region within a CAMK2D transcript (e.g, SEQ ID NO: 1) and thereby, modulate the splicing of the mammalian CAMK2D pre-mRNA transcript.
  • the ASOs described herein target exon 14 of a mammalian
  • the target region of an ASO disclosed herein corresponds to nucleotides 258,951 - 258,992 of SEQ ID NO: 1 (i.e., SEQ ID NO: 71).
  • the target region corresponds to nucleotides 258,637 - 259,107 of SEQ ID NO: 1 (i.e., SEQ ID NO: 70).
  • the target region corresponds to nucleotides 258,793-259,057 of SEQ ID NO: 1 (i.e., SEQ ID NO: 10).
  • the target region corresponds to nucleotides 258,887 - 259,057 of SEQ ID NO: 1 (i.e., SEQ ID NO: 6). In some aspects, the target region corresponds to nucleotides 258,937 - 259,007 of SEQ ID NO: 1 (i.e., SEQ ID NO: 9). In certain aspects, the target region corresponds to nucleotides 258,937 - 258,965 of SEQ ID NO: 1 (i.e., SEQ ID NO: 7). In some aspects, the target region corresponds to nucleotides 258,979 - 259,007 of SEQ ID NO: 1 (i.e., SEQ ID NO: 8). Table 2 (below) provides the sequences for the above described target regions. Table 2. Sequences for Exemplary Target Regions
  • an ASO described herein comprises a contiguous nucleotide sequence that is complementary ( e.g ., fully complementary) to a region within SEQ ID NO: 70.
  • an ASO described herein comprises a contiguous nucleotide sequence that is complementary (e.g., fully complementary) to a region within SEQ ID NO: 10.
  • an ASO described herein comprises a contiguous nucleotide sequence that is complementary (e.g, fully complementary) to a region within SEQ ID NO: 6.
  • an ASO described herein comprises a contiguous nucleotide sequence that is complementary (e.g, fully complementary) to a region within SEQ ID NO: 9.
  • an ASO described herein comprises a contiguous nucleotide sequence that is complementary (e.g, fully complementary) to a region within SEQ ID NO: 7. In some aspects, an ASO described herein comprises a contiguous nucleotide sequence that is complementary (e.g, fully complementary) to a region within SEQ ID NO: 8. [0133] Non-limiting examples of target regions of the ASOs disclosed herein are set forth in SEQ ID NOs: 41 to 69.
  • the ASO of the present disclosure is capable of targeting a
  • CAMK2D transcript from one or more species (e.g, humans, non-human primates, dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, and bears).
  • the ASO disclosed herein is capable of targeting both human and rodent (e.g, mice or rats) CAMK2D transcript.
  • the ASO is capable of down-regulating (e.g, reducing or removing) expression of the CAMK2D-E14 mRNA or protein both in humans and in rodents (e.g, mice or rats).
  • Non-human CAMK2D transcript sequences are known in the art and described in, e.g, International Publication No. WO 2019/165067 Al.
  • the ASOs of the disclosure comprise a contiguous nucleotide sequence which corresponds to the complement of a region (i.e., target regions described herein) of CAMK2D transcript, e.g, a nucleotide sequence corresponding to SEQ ID NO: 1, and thereby, modulate the splicing of a mammalian CAMK2D transcript.
  • the present disclosure provides an ASO of 10 to 40 nucleotides in length, such as 10 to 30 nucleotides, 10 to 25 nucleotides, 10 to 20 nucleotides, or 10 to 15 nucleotides in length, which comprises a contiguous nucleotide sequence with at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity to a region within the complement of a CAMK2D transcript (e.g, SEQ ID NO: 1) or a variant thereof.
  • a CAMK2D transcript e.g, SEQ ID NO: 1
  • the ASOs disclosed herein can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to the equivalent region of a nucleic acid which encodes a mammalian CAMK2D protein (e.g, SEQ ID NO: 1).
  • the ASO can comprise a contiguous nucleotide sequence which is fully complementary (perfectly complementary) to a nucleic acid sequence, or a region within the sequence, corresponding to nucleotides X-Y of SEQ ID NO: 1, wherein X and Y are the start site and the end site, respectively, as shown in FIG. 1.
  • the ASOs of the present disclosure has at least about 80% sequence identity to a sequence selected from SEQ ID NOs: 12 to 40 (i.e., the sequences in FIG. 1), such as 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% sequence identity, at least about 97% sequence identity, at least about 98% sequence identity, at least about 99% sequence identity, such as about 100% sequence identity.
  • the ASOs, or contiguous nucleotide sequence thereof, disclosed herein are fully complementary (i.e., 100% complementary) to a region of the target nucleic acid (i.e., the target region).
  • the ASOs, or contiguous nucleotide sequence thereof comprise one or two mismatches between the oligonucleotide and the target nucleic acid.
  • the ASOs have a design and/or chemical structure described elsewhere herein ( e.g ., FIG. 1).
  • the ASOs, or contiguous nucleotide sequence thereof are selected from, or comprises, one of the sequences set forth in SEQ ID NOs: 12 to 40 or a region of at least 10 contiguous nucleotides thereof, wherein the ASO (or contiguous nucleotide portion thereof) can optionally comprise one, two, three, or four mismatches when compared to the corresponding CAMK2D transcript.
  • the ASOs described herein can tolerate 1, 2, 3, or 4 (or more) mismatches, when hybridizing to the target region (e.g., exon 14) and still sufficiently bind to the target to show the desired effect, e.g, modulate the splicing of a CAMK2D pre-mRNA transcript.
  • Mismatches can, for example, be compensated by increased length of the ASO nucleotide sequence and/or an increased number of nucleotide analogs, which are disclosed elsewhere herein.
  • an ASO comprises no more than 3 mismatches when hybridizing to the target region. In some aspects, the contiguous nucleotide sequence comprises no more than 2 mismatches when hybridizing to the target region. In some aspects, the contiguous nucleotide sequence comprises no more than 1 mismatch when hybridizing to the target region.
  • the ASOs of the present disclosure can comprise a contiguous nucleotide sequence of a total of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous nucleotides in length. It should be understood that when a range is given for the length of an ASO, or contiguous nucleotide sequence thereof, the range includes the lower and upper lengths provided in the range, for example from (or between) 10-40, includes both 10 and 40.
  • the ASOs comprise a contiguous nucleotide sequence of a total of about 14-20, e.g, 14, 15, 16, 17, 18, 19, or 20 contiguous nucleotides in length. In certain aspects, the ASOs disclosed herein comprise a contiguous nucleotide sequence that is 15 nucleotides in length.
  • the ASOs disclosed herein comprise one or more non-naturally occurring nucleoside analogs.
  • Nucleoside analogs are variants of natural nucleosides, such as DNA or RNA nucleosides, by virtue of modifications in the sugar and/or base moieties. Analogs could in principle be merely “silent” or “equivalent” to the natural nucleosides in the context of the oligonucleotide, i.e., have no functional effect on the way the oligonucleotide works to inhibit target gene expression.
  • Such "equivalent" analogs can nevertheless be useful if, for example, they are easier or cheaper to manufacture, or are more stable to storage or manufacturing conditions, or represent a tag or label. In some aspects, however, the analogs will have a functional effect on the way in which the ASO works to inhibit expression; for example, by producing increased binding affinity to the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell.
  • nucleoside analogs are described by e.g. Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann; Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1.
  • the ASOs of the present disclosure can contain more than one, more than two, more than three, more than four, more than five, more than six, more than seven, more than eight, more than nine, more than 10, more than 11, more than 12, more than 13, more than 14, more than 15, more than 16, more than 18, more than 19, or more than 20 nucleoside analogs.
  • the nucleoside analogs in the ASOs are the same. In some aspects, the nucleoside analogs in the ASOs are different.
  • the nucleotide analogs in the ASOs can be any one of or combination of the following nucleoside analogs.
  • nucleobase includes the purine (e.g, adenine and guanine) and pyrimidine (e.g, uracil, thymine and cytosine) moiety present in nucleosides and nucleotides which form hydrogen bonds in nucleic acid hybridization.
  • nucleobase also encompasses modified nucleobases which can differ from naturally occurring nucleobases, but are functional during nucleic acid hybridization.
  • the nucleobase moiety is modified by modifying or replacing the nucleobase.
  • nucleobase refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants. Such variants are for example described in Hirao et al ., (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.
  • the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl- cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo- uracil, 2-thio-uracil, 2'thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6- diaminopurine, and 2-chloro-6-aminopurine.
  • a nucleobase selected from isocytosine, pseudoisocytosine, 5-methyl- cytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil
  • the nucleobase moieties can be indicated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, wherein each letter can optionally include modified nucleobases of equivalent function.
  • the nucleobase moieties are selected from A, T, G, C, and 5-methyl-cytosine.
  • 5-methyl-cytosine LNA nucleosides can be used.
  • the ASOs of the disclosure can comprise one or more nucleosides which have a modified sugar moiety, i.e. a modification of the sugar moiety when compared to the ribose sugar moiety found in DNA and RNA.
  • nucleosides with modification of the ribose sugar moiety have been made, primarily with the aim of improving certain properties of oligonucleotides, such as affinity and/or nuclease resistance.
  • Such modifications include those where the ribose ring structure is modified, e.g. by replacement with a hexose ring (HNA), or a bicyclic ring, which typically have a biradical bridge between the C2 and C4 carbons on the ribose ring (LNA), or an unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons (e.g. UNA).
  • HNA hexose ring
  • LNA ribose ring
  • UPA unlinked ribose ring which typically lacks a bond between the C2 and C3 carbons
  • Other sugar modified nucleosides include, for example, bicyclohexose nucleic acids (see, e.g, WO2011/017521) or tricyclic nucleic acids (see, e.g, WO2013/154798). Modified nucleosides also include nucleosides where the sugar moiety is replaced with
  • Sugar modifications also include modifications made via altering the substituent groups on the ribose ring to groups other than hydrogen, or the T -OH group naturally found in DNA and RNA nucleosides. Substituents can, for example, be introduced at the 2’, 3’, 4’ or 5’ positions.
  • Nucleosides with modified sugar moieties also include 2' modified nucleosides, such as 2' substituted nucleosides. Indeed, much focus has been spent on developing 2' substituted nucleosides, and numerous 2' substituted nucleosides have been found to have beneficial properties when incorporated into oligonucleotides, such as enhanced nucleoside resistance and enhanced affinity.
  • a 2' sugar modified nucleoside is a nucleoside which has a substituent other than
  • a 2' sugar modified nucleoside is a nucleoside which has a substituent other than
  • the 2’ modified sugar can provide enhanced binding affinity and/or increased nuclease resistance to the oligonucleotide.
  • 2’ substituted modified nucleosides are 2’-0-alkyl-RNA, 2’ - O-methyl-RNA, 2’-alkoxy-RNA, 2’-0-methoxyethyl-RNA (MOE), 2’-amino-DNA, 2’-Fluoro- RNA, and 2’-F-ANA nucleoside.
  • T substituted sugar modified nucleosides does not include T bridged nucleosides like LNA.
  • LNA Locked Nucleic Acid Nucleosides
  • LNA nucleosides are 2'-sugar modified nucleosides which comprise a linker group (referred to as a biradical or a bridge) between C2' and C4' of the ribose sugar ring of a nucleoside (i.e., 2'-4' bridge), which restricts or locks the conformation of the ribose ring.
  • These nucleosides are also termed bridged nucleic acid or bicyclic nucleic acid (BNA) in the literature.
  • BNA bicyclic nucleic acid
  • the locking of the conformation of the ribose is associated with an enhanced affinity of hybridization (duplex stabilization) when the LNA is incorporated into an oligonucleotide for a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide/complement duplex.
  • Non limiting, exemplary LNA nucleosides are disclosed in WO 99/014226, WO
  • LNA nucleosides are beta-D-oxy-LNA, 6’-methyl-beta-D-oxy
  • LNA such as (S)-6’-methyl-beta-D-oxy-LNA (ScET) and ENA.
  • the LNA nucleosides in the oligonucleotides are beta-D-oxy-LNA nucleosides.
  • the ASO of the disclosure comprises or consists of morpholino nucleosides (i.e. is a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO)).
  • morpholino nucleosides i.e. is a Morpholino oligomer and as a phosphorodiamidate Morpholino oligomer (PMO)
  • Splice modulating morpholino oligonucleotides have been approved for clinical use - see for example eteplirsen, a 30nt morpholino oligonucleotide targeting a frame shift mutation in DMD, used to treat Duchenne muscular dystrophy.
  • Morpholino oligonucleotides have nucleobases attached to six membered morpholine rings rather ribose, such as methylenemorpholine rings linked through phosphorodiamidate groups, for example as illustrated by the following illustration of 4 consecutive morpholino nucleotides:
  • morpholino ASOs of the disclosure can be, for example 20 - 40 morpholino nucleotides in length, such as morpholino 25 - 35 nucleotides in length.
  • Nuclease mediated degradation refers to an oligonucleotide capable of mediating degradation of a complementary nucleotide sequence when forming a duplex with such a sequence.
  • ASOs disclosed herein can function via nuclease mediated degradation of the target nucleic acid, where the oligonucleotides of the disclosure are capable of recruiting a nuclease, particularly and endonuclease, preferably endoribonuclease (RNase), such as RNase H.
  • RNase endoribonuclease
  • examples of oligonucleotide designs which operate via nuclease mediated mechanisms are oligonucleotides which typically comprise a region of at least 5 or 6 DNA nucleosides and are flanked on one side or both sides by affinity enhancing nucleosides, for example gapmers, headmers and tailmers.
  • the RNase H activity of an ASO refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule.
  • WOOl/23613 provides in vitro methods for determining RNaseH activity, which can be used to determine the ability to recruit RNaseH.
  • an oligonucleotide is deemed capable of recruiting RNase H if it, when provided with a complementary target nucleic acid sequence, has an initial rate, as measured in pmol/l/min, of at least 5%, at least 10%, or more than 20% of the of the initial rate determined when using a oligonucleotide having the same base sequence as the modified oligonucleotide being tested, but containing only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide, and using the methodology provided by Example 91 - 95 of WOOl/23613.
  • DNA oligonucleotides are known to effectively recruit RNaseH, as are gapmer oligonucleotides which comprise a region of DNA nucleosides (typically at least 5 or 6 contiguous DNA nucleosides), flanked 5’ and 3’ by regions comprising T sugar modified nucleosides, typically high affinity T sugar modified nucleosides, such as 2’-0-MOE and/or LNA.
  • T sugar modified nucleosides typically high affinity T sugar modified nucleosides, such as 2’-0-MOE and/or LNA.
  • the splice modulating ASOs of the disclosure are not gapmers. RNaseH recruitment can be avoided by limiting the number of contiguous DNA nucleotides in the oligonucleotide. Therefore, in some aspects, ASOs disclosed herein are or comprises mixmers or totalmers.
  • the ASO of the disclosure can comprise a nucleotide sequence which comprises both nucleosides and nucleoside analogs, and can be in the form of a gapmer, blockmer, mixmer, headmer, tailmer, or totalmer.
  • gapmer refers to an ASO which comprises a region of
  • RNase H recruiting oligonucleotides which is flanked 5' and 3' by one or more affinity enhancing modified nucleosides (flanks).
  • the terms "headmers” and “tailmers” are oligonucleotides capable of recruiting RNase H where one of the flanks is missing, i.e., only one of the ends of the oligonucleotide comprises affinity enhancing modified nucleosides.
  • the 3' flank is missing ⁇ i.e., the 5' flank comprise affinity enhancing modified nucleosides
  • the 5' flank is missing ⁇ i.e., the 3' flank comprises affinity enhancing modified nucleosides).
  • LNA gapmer is a gapmer oligonucleotide wherein at least one of the affinity enhancing modified nucleosides is an LNA nucleoside.
  • mixed wing gapmer refers to an LNA gapmer wherein the flank regions comprise at least one LNA nucleoside and at least one DNA nucleoside or non-LNA modified nucleoside, such as at least one 2' substituted modified nucleoside, such as, for example, 2'-0-alkyl-RNA, 2'-0-methyl- RNA, 2'-alkoxy-RNA, 2'-0-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-Fluoro-RNA, 2'- Fluro-DNA, arabino nucleic acid (ANA), and 2'-Fluoro-ANA nucleoside(s).
  • chimeric ASOs consist of an alternating composition of (i) DNA monomers or nucleoside analog monomers recognizable and cleavable by RNase, and (ii) non-RNase recruiting nucleoside analog monomers.
  • a “totalmer” is a single stranded ASO which only comprises non-naturally occurring nucleotides or nucleotide analogs.
  • ASOs which do not recruit RNAaseH are desired.
  • RNaseH activity requires a contiguous sequence of DNA nucleotides
  • RNaseH activity of ASO can be prevented by designing ASOs which do not comprise a region of more than 3 contiguous DNA nucleosides. This can be achieved by using ASOs or contiguous nucleoside regions thereof with a mixmer design, which comprise sugar modified nucleosides, such as T sugar modified nucleosides, and short regions of DNA nucleosides, such as 1, 2 or 3 DNA nucleosides.
  • Mixmers are exemplified herein by every second design, wherein the nucleosides alternate between 1 LNA and 1 DNA nucleoside, e.g., with 5’ and 3’ terminal LNA nucleosides, and every third design, such as where every third nucleoside is a LNA nucleoside.
  • each monomer is linked to the 3' adjacent monomer via a linkage group.
  • linkage groups Suitably, each monomer is linked to the 3' adjacent monomer via a linkage group.
  • linkage group or "intemucleoside linkage” are intended to mean a group capable of covalently coupling together two nucleosides. Specific and preferred examples include phosphate groups and phosphorothioate groups.
  • nucleosides of the ASO of the disclosure or contiguous nucleosides sequence thereof are coupled together via linkage groups.
  • each nucleoside is linked to the 3' adjacent nucleoside via a linkage group.
  • the intemucleoside linkage is modified from its normal phosphodiester to one that is more resistant to nuclease attack, such as phosphorothioate, which is cleavable by RNaseH, also allows that route of antisense inhibition in reducing the expression of the target gene.
  • nuclease attack such as phosphorothioate
  • at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of intemucleoside linkages are modified.
  • ASOs disclosed herein e.g ., gapmers, mixmers, and totalmers
  • the present disclosure further provides a conjugate comprising an ASO disclosed herein, and at least one conjugate moiety covalently attached to the ASO.
  • conjugate refers to an ASO which is covalently linked to a non-nucleotide moiety (e.g., conjugate moiety).
  • conjugate moiety cay be covalentaly linked to the ASO, optionally via a linker group.
  • Conjugation of the ASO of the disclosure to one or more non-nucleotide moieties can improve the pharmacology of the ASO, e.g., by affecting the activity, cellular distribution, cellular uptake, or stability of the ASO.
  • the non-nucleotide moieties modify or enhance the pharmacokinetic properties of the ASO by improving cellular distribution, bioavailability, metabolism, excretion, permeability, and/or cellular uptake of the ASO.
  • the non-nucleotide moieties can target the ASO to a specific organ, tissue, or cell type and thereby enhance the effectiveness of the ASO in that organ, tissue, or cell type.
  • the non-nucleotide moieties reduce the activity of the ASO in non-target cell types, tissues, or organs, e.g, off target activity or activity in non-target cell types, tissues, or organs.
  • Further suitable conjugate moieties are those capable of binding to the asialoglycoprotein receptor (ASGPr).
  • ASGPr asialoglycoprotein receptor
  • tri-valent N-acetylgalactosamine conjugate moieties are suitable for binding to the ASGPr.
  • the non-nucleotide moiety is selected from the group consisting of carbohydrates (e.g. GalNAc), cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g. bacterial toxins), vitamins, viral proteins (e.g., capsids) or combinations thereof.
  • Oligonucleotide conjugates and their synthesis has also been reported in comprehensive reviews by Manoharan in Antisense Drug Technology, Principles, Strategies, and Applications, S.T. Crooke, ed., Ch. 16, Marcel Dekker, Inc., 2001 and Manoharan, Antisense and Nucleic Acid Drug Development, 2002, 12, 103.
  • a linkage or linker is a connection between two atoms that links one chemical group or segment of interest to another chemical group or segment of interest via one or more covalent bonds.
  • Conjugate moieties can be attached to the oligonucleotide directly or through a linking moiety (e.g, linker or tether).
  • linkers serve to covalently connect a conjugate moiety to an oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid.
  • the conjugate or oligonucleotide conjugate of the disclosure can optionally comprise a linker, which is positioned between the oligonucleotide or contiguous nucleotide sequence complementary to the target nucleic acid and the conjugate moiety.
  • linkers can be biocleavable linkers comprising or consisting of a physiologically labile bond that is cleavable under conditions normally encountered or analogous to those encountered within a mammalian body.
  • Conditions under which physiologically labile linkers undergo chemical transformation include chemical conditions such as pH, temperature, oxidative or reductive conditions or agents, and salt concentration found in or analogous to those encountered in mammalian cells.
  • Mammalian intracellular conditions also include the presence of enzymatic activity normally present in a mammalian cell such as from proteolytic enzymes or hydrolytic enzymes or nucleases.
  • the biocleavable linker is susceptible to SI nuclease cleavage.
  • the nuclease susceptible linker comprises between 1 and 5 nucleosides, such as DNA nucleoside(s) comprising at least two consecutive phosphodiester linkages.
  • Phosphodiester containing biocleavable linkers are described in more detail in WO 2014/076195.
  • linkers are not biocleavable but primarily serve to covalently connect a conjugate moiety to an oligonucleotide.
  • the linkers can comprise a chain structure or an oligomer of repeating units such as ethylene glycol, amino acid units or amino alkyl groups
  • the linker is an amino alkyl, such as a C2 - C36 amino alkyl group, including, for example C6 to C12 amino alkyl groups.
  • the linker is a C6 amino alkyl group.
  • compositions can be used in pharmaceutical formulations and compositions.
  • such compositions comprise a pharmaceutically acceptable diluent, carrier, salt, or adjuvant.
  • the ASOs of the disclosure are in the form of a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable salt comprises a sodium salt, a potassium salt, or an ammonium salt.
  • a pharmaceutically acceptable diluent includes phosphate- buffered saline (PBS) and pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
  • the pharmaceutically acceptable diluent is sterile phosphate buffered saline.
  • the ASO is used in the pharmaceutically acceptable diluent at a concentration of 50 - 300 mM solution.
  • the ASO of the disclosure can be included in a unit formulation such as in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount without causing serious side effects in the treated patient.
  • serious side effects can be acceptable in terms of ensuring a positive outcome to the therapeutic treatment.
  • the formulated drug can comprise pharmaceutically acceptable binding agents and adjuvants.
  • Capsules, tablets, or pills can contain for example the following compounds: microcrystalline cellulose, gum or gelatin as binders; starch or lactose as excipients; stearates as lubricants; various sweetening or flavoring agents.
  • the dosage unit can contain a liquid carrier like fatty oils.
  • coatings of sugar or enteric agents can be part of the dosage unit.
  • the ASO formulations can also be emulsions of the active pharmaceutical ingredients and a lipid forming a micellular emulsion.
  • compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.
  • Administration can be (a) oral; (b) pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, (c) topical including epidermal, transdermal, ophthalmic and to mucous membranes including vaginal and rectal delivery; or (d) parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g, intrathecal, intra-cerebroventricular, or intraventricular, administration.
  • the ASO is administered intravenously, intraperitoneally, orally, topically, or as a bolus injection or administered directly in to the target organ. In some aspects, the ASO is administered intracardially or intraventricularly as a bolus injection. In some aspects, the ASO is administered subcutaneously. In some aspects, the ASO is administered orally. In some aspects, the ASO is administered intravenously. In some aspects, the ASO is administered subcutaneously.
  • compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, sprays, suppositories, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable.
  • topical formulations include those in which the ASO of the disclosure is in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants.
  • compositions and formulations for oral administration include but are not limited to powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets.
  • Compositions and formulations for parenteral, intrathecal, intra-cerebroventricular, or intraventricular administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.
  • compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Delivery of drug to the target tissue can be enhanced by carrier-mediated delivery including, but not limited to, cationic liposomes, cyclodextrins, porphyrin derivatives, branched chain dendrimers, polyethylenimine polymers, nanoparticles and microspheres (Dass CR. J Pharm Pharmacol 2002; 54(l):3-27).
  • compositions of the present disclosure can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the formulation can include a sterile diluent, buffers, regulators of tonicity and antibacterials.
  • the active ASOs can be prepared with carriers that protect against degradation or immediate elimination from the body, including implants or microcapsules with controlled release properties.
  • the carriers can be physiological saline or phosphate buffered saline.
  • kits that comprise an ASO of the disclosure described herein and that can be used to perform the methods described herein.
  • a kit comprises at least one ASO in one or more containers.
  • the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results.
  • the disclosed ASO can be readily incorporated into one of the established kit formats which are well known in the art.
  • the disclosure provides methods for manufacturing the ASOs of the disclosure comprising reacting nucleotide units and thereby forming covalently linked contiguous nucleotide units comprised in the oligonucleotide.
  • the method uses phophoramidite chemistry (see for example Caruthers et al, 1987, Methods in Enzymology vol. 154, pages 287-313).
  • the method further comprises reacting the contiguous nucleotide sequence with a conjugating moiety (ligand) to covalently attach the conjugate moiety to the oligonucleotide.
  • a method for manufacturing the composition of the disclosure comprising mixing the oligonucleotide or conjugated oligonucleotide of the disclosure with a pharmaceutically acceptable diluent, solvent, carrier, salt and/or adjuvant.
  • the present disclosure provides methods for modulating the splicing of a CAMK2D pre-mRNA in a cell which is expressing CAMK2D (e.g. a target cell).
  • the method comprises administering an ASO, a conjugate, or a pharmaceutical composition disclosed herein in an effective amount to the cell.
  • the administering can be done in vitro.
  • the administering can be done in vivo.
  • the administration of the ASO results in enhanced expression of CAMK2DA14 transcript (e.g., mRNA), or protein encoded thereof.
  • the administration of the ASO results in reduced expression of CAMK2D-E14 transcript (e.g, mRNA), or protein encoded thereof. In some aspects, the administration of the ASO results in reduced expression of CAMK2D-E14 transcript, or protein encoded thereof, and enhanced expression of CAMK2DA14 transcript, or protein encoded thereof.
  • CAMK2D-E14 transcript e.g, mRNA
  • the administration of the ASO results in reduced expression of CAMK2D-E14 transcript, or protein encoded thereof, and enhanced expression of CAMK2DA14 transcript, or protein encoded thereof.
  • the ASOs of the disclosure can be utilized as research reagents for, e.g, diagnostics, therapeutics, and prophylaxis.
  • CAMK2D protein in cells (e.g. in vitro cell cultures) and experimental animals thereby facilitating functional analysis of the target or an appraisal of its usefulness as a target for therapeutic intervention.
  • the target modulation is achieved by degrading or inhibiting the mRNA producing the protein, and thereby preventing protein formation or by degrading or inhibiting a modulator of the gene or mRNA producing the protein.
  • modulating the synthesis of CAMK2D protein comprises inhibiting or reducing the expression of CAMK2D-E14 protein and/or enhancing the expression of CAMK2DA14 protein.
  • the ASOs can be used to detect and quantitate CAMK2D expression (e.g, CAMK2D-E14 and/or CAMK2DA14 transcript expression) in cell and tissues by northern blotting, in-situ hybridisation or similar techniques.
  • detecting and quantitating CAMK2D-E14 and/or CAMK2DA14 transcript expression can be useful in determining whether a subject is suffering from or predisposed to a disease or disorder, such as those described herein (e.g, cardiovascular disease or disorder, e.g, cardiomyopathy).
  • the target nucleic acid can be a cDNA or a synthetic nucleic acid derived from DNA or RNA.
  • the ASOs can be administered to an animal or a human, suspected of having a disease or disorder, which can be treated by modulating the expression of CAMK2D (e.g., CAMK2D-E14 and/or CAMK2DA14), and thereby treating the disease or disorder.
  • CAMK2D e.g., CAMK2D-E14 and/or CAMK2DA14
  • the present disclosure provides methods for treating or preventing a disease or disorder (e.g, cardiovascular disease or disorder, e.g, cardiomyopathy) in a subject in need thereof, comprising administering a therapeutically or prophylactically effective amount of the ASO, the conjugate, or the pharmaceutical composition of the disclosure to the subject.
  • the subject is suffering from or prone to a disease or disorder associated with increased expression of a CAMK2D transcript and/or a CAMK2D protein (e.g, CAMK2D-E14 transcript and/or CAMK2D-E14 protein).
  • a disease or disorder associated with increased expression of a CAMK2D transcript and/or a CAMK2D protein e.g, CAMK2D-E14 transcript and/or CAMK2D-E14 protein.
  • the ASO or conjugate of the disclosure is used in therapy.
  • the target cell described herein comprises a mammalian cell.
  • the target cell is a human cell.
  • the target cell comprises in vitro cell culture.
  • the target cell comprises in vivo cell forming part of a tissue in a mammal.
  • the target cell is a cardiomyocyte cell.
  • the target cell is present in, isolated from, or derived from heart tissue.
  • the disclosure further provides for an ASO, conjugate, or pharmaceutical composition of the disclosure for use in the treatment of a disease or disorder described herein.
  • a disease or disorder that can be treated with the present disclosure are associated with abnormal levels and/or activity of CAMK2D (e.g, CAMK2D-E14).
  • methods disclosed herein can be used for treatment or prophylaxis against diseases or disorders caused by abnormal levels and/or activity of CAMK2D (e.g, CAMK2D-E14).
  • a disease or disorder that can be treated with the present disclosure comprises a cardiovascular disease and cardiovascular related diseases.
  • Non-limiting examples of cardiovascular disease includes a cardiomyopathy, coronary artery disease, stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.
  • the cardiovascular disease is cardiomyopathy. Examples of other cardiovascular disease are provided elsewhere in the present disclosure.
  • the disclosure also provides for methods of inhibiting (e.g, by reducing) the expression of CAMK2D gene transcript and/or CAMK2D protein (e.g, CAMK2D-E14) in a cell or a tissue, the method comprising contacting the cell or tissue, in vitro or in vivo, with an effective amount of one or more ASOs, conjugates, or pharmaceutical compositions thereof, of the disclosure to affect degradation of expression of CAMK2D gene transcript thereby reducing CAMK2D protein.
  • CAMK2D gene transcript and/or CAMK2D protein e.g, CAMK2D-E14
  • the present disclosure also provides for an ASO, conjugate, or pharmaceutical composition of the disclosure for use as a medicament, e.g ., for the treatment of a disease or disorder as referred to herein, or for a method of the treatment of as a disorder as referred to herein.
  • the disclosure further provides for a method for inhibiting or reducing CAMK2D
  • CAMK2D-E14 CAMK2D-E14 protein in a cell which is expressing the CAMK2D protein, comprising administering an ASO or a conjugate according to the disclosure to the cell so as to affect the inhibition or reduction of CAMK2D (e.g, CAMK2D-E14) protein in the cell.
  • the disclosure also provides for a method for treating a disorder as referred to herein the method comprising administering an ASO or a conjugate according to the disclosure as herein described and/or a pharmaceutical composition according to the disclosure to a patient in need thereof.
  • compositions according to the disclosure can be used for the treatment of conditions associated with over expression of a CAMK2D (e.g, CAMK2D-E14) protein.
  • a CAMK2D e.g, CAMK2D-E14
  • the present disclosure is directed to a method of treating a mammal suffering from or susceptible to conditions associated with abnormal levels of CAMK2D (e.g, CAMK2D-E14), comprising administering to the mammal and therapeutically effective amount of an ASO targeted to CAMK2D transcript that comprises one or more LNA units.
  • CAMK2D e.g, CAMK2D-E14
  • the ASO, a conjugate, or a pharmaceutical composition according to the disclosure is typically administered in an effective amount.
  • the present disclosure is directed to the use of an ASO
  • cardiovascular diseases can include a coronary artery disease, stroke, heart failure, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, heart arrhythmia, congenital heart disease, valvular heart disease carditis, aortic aneurysms, peripheral artery disease, thromboembolic disease, and venous thrombosis.
  • the cardiovascular disease is a heart failure, which can include a left-sided heart failure, a right-sided heart failure, congestive heart failure, a heart failure with reduced ejection fraction (HFrEF), a heart failure with preserved ejection fraction (HFpEF), a heart failure with mid-range ejection fraction (HFmrEF), a hypertrophic cardiomyopathy (HCM), a hypertensive heart disease (HHD), or hypertensive hypertrophic cardiomyopathy.
  • HFrEF heart failure with reduced ejection fraction
  • HFpEF heart failure with preserved ejection fraction
  • HmrEF hypertrophic cardiomyopathy
  • HHD hypertensive heart disease
  • the disclosure is furthermore directed to a method for treating abnormal levels of CAMK2D protein (e.g., CAMK2D-E14), the method comprising administering an ASO, conjugate, or pharmaceutical composition of the disclosure to a subject in need thereof.
  • CAMK2D protein e.g., CAMK2D-E14
  • the disclosure also relates to an ASO, a composition or a conjugate as defined herein for use as a medicament.
  • the disclosure further relates to use of a compound, composition, or a conjugate as defined herein for the manufacture of a medicament for the treatment of abnormal levels of CAMK2D (e.g, CAMK2D-E14) protein or expression of mutant forms of CAMK2D protein (such as allelic variants, wherein the allelic variants are associated with one of the diseases referred to herein).
  • CAMK2D e.g, CAMK2D-E14
  • mutant forms of CAMK2D protein such as allelic variants, wherein the allelic variants are associated with one of the diseases referred to herein.
  • a subject who is in need of treatment is a patient suffering from or likely to suffer from the disease or disorder.
  • the ASO, conjugate, or pharmaceutical composition of the disclosure can be used in combination with one or more additional therapeutic agents.
  • the one or more additional therapeutic agents comprise a standard of care treatment for the diseases or disorders described herein.
  • the one or more additional therapeutic agents comprise a CAMK2D antagonist.
  • the CAMK2D antagonist comprises an anti-CAMK2D antibody or a fragment thereof.
  • ASOs described herein were designed to target a region within the CAMK2D pre- mRNA (SEQ ID NO: 1) to modulate the splicing of the CAMK2D pre-mRNA.
  • the ASOs were designed to target the exon 14 region of CAMK2D pre-mRNA (SEQ ID NO: 1).
  • the ASOs were constructed to target the regions denoted using the start and end sites of SEQ ID NO: 1, as shown in FIG. 1.
  • the exemplary sequences of the ASOs of the present disclosure are also provided in FIG. 1.
  • the ASOs were designed to be mixmers, as shown in FIG. 1.
  • the disclosed gapmers were constructed to contain locked nucleic acids - LNAs (upper case letters).
  • a gapmer can have beta-deoxy LNA at the 5' end and the 3' end and have a phosphorothioate backbone.
  • the LNA can also be substituted with any other nucleoside analogs and the backbone can be other types of backbones (e.g ., phosphodiester linkage, a phosphotriester linkage, a methylphosphonate linkage, a phosphoroamidate linkage, or any combinations thereof).
  • ASOs were synthesized using methods well known in the art. Exemplary methods of preparing such ASOs are described in Barciszewski et al ., Chapter 10 - “Locked Nucleic Acid Aptamers” in Nucleic Acid and Peptide Aptamers: Methods and Protocols , vol. 535, Gunter Mayer (ed.) (2009).
  • Example 2 Analysis of the Potency of the ASOs in Modulating CAMK2DA14 Expression
  • the ASOs of the present disclosure were tested for their ability to inhibit or reduce the expression of CAMK2D-E14 mRNA in a target cell. Briefly, human cardiomyocytes derived from pluripotent stem cells (iCell Cardiomyocytes2, Cellular Dynamic) were seeded in 96 well plates (40,000 cells / well) and incubated with 100 pL medium (cardiomyocytes maintenance medium, Cellular dynamic) alone (control) or with 100 pL medium (cardiomyocytes maintenance medium, Cellular dynamic) containing 50 pM (final concentration) of ASOs shown in FIG. 2. After 3 days of incubation, additional 100 pL of the maintenance medium were added to the wells for the remaining 4 days. In total, the cells were incubated in the maintenance medium alone or in the presence of ASOs for 7 days.
  • Droplet digital PCR was done using BioRad Automatic Droplet Generator (AutoDG) using Automated Droplet Generation Oil for Probes (BioRad) together with the 0X200 droplet digital reader.
  • AutoDG BioRad Automatic Droplet Generator
  • the ddPC Supermix for Probes (No dUTP) (BioRad 1863024) reactions were run according to the manufacturer’s instructions with an annealing temperature of 58°C.
  • the droplets were read in the 0X200 droplet digital reader, and the data were analyzed and quantified using the QuantaSoft Analysis Pro Software 1.0.596 (BioRad).
  • the thresholds for defining the different droplet groups in the triplex PCR reaction was set by free hand within the software according to the guidelines.
  • the following probe assay was used to measure CAMK2D-E14 mRNA transcript: CAMK2D exon 14 probe Assay (probe spanning from exon 13 to 14): Primer 1 ( Primer 2
  • the data is represented in FIG. 2 as the proportion (/.£., number) of exonl4 containing transcripts ( CAMK2D-E14 mRNA) remaining in cells treated with the ASO indicated, as compared to the level (i.e., number) of exon 14 containing transcripts ( CAMK2D-E14 mRNA) in non-treated cells (normalized to 100%).
  • Splice-switching oligos capable of skipping exon 14 of CAMK2D pre-mRNA should switch the transcript from CAMK2D-E14 transcripts into CAMK2DA14 transcripts (i.e., decrease CAMK2D-E14 while increasing CAMK2DA14 expression).
  • the ASOs shown in Example 2 as reducing the expression of CAMK2D-E14 were further assessed for their ability to also increase CAMK2DA14 expression. Briefly, human cardiomyocytes derived from pluripotent stem cells were treated with the different ASOs and the expression level of CAMK2DA14 was measured as described in Example 2.
  • CAMK2Ddeltal4 probe Assay (probe spanning from exon 13 to 15): Primer 1 HPTR1 probe assay: Hs.PT.58v.45621572 used both with a both 56-FAM and HEX fluorophore.
  • Primer 1 HPTR1 probe assay Hs.PT.58v.45621572 used both with a both 56-FAM and HEX fluorophore.
  • compounds 38 1, 39 1, and 39_2) are effective in selectively decreasing the level of CAMK2D- E14 in the cells, and the data is consistent with effective modulation of splicing of the CAM2K2D pre-mRNA in favour of mature mRNA transcripts which lack the CAMK2D exon 14.

Abstract

La présente divulgation concerne des oligonucléotides (oligomères) complémentaires du pré-ARNm CAMK2D et pouvant inhiber l'inclusion d'un exon 14 CAMK2D dans L'ARNm CAMK2D. En conséquence, dans certains aspects, les oligonucléotides de la présente divulgation sont capables de moduler l'épissage d'un pré-ARNm CAMK2D. Les oligonucléotides de la divulgation sont utiles dans le traitement des maladies cardiovasculaires telles que les cardiomyopathies.
PCT/US2021/016646 2020-02-05 2021-02-04 Oligonucléotides pour la modulation d'épissage de camk2d WO2021158810A1 (fr)

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