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WO2016118118A1 - Modulation de l'expression de l'isoforme kcnh2 par des oligonucléotides à titre d'approche thérapeutique pour le syndrome du qt long - Google Patents

Modulation de l'expression de l'isoforme kcnh2 par des oligonucléotides à titre d'approche thérapeutique pour le syndrome du qt long Download PDF

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WO2016118118A1
WO2016118118A1 PCT/US2015/012060 US2015012060W WO2016118118A1 WO 2016118118 A1 WO2016118118 A1 WO 2016118118A1 US 2015012060 W US2015012060 W US 2015012060W WO 2016118118 A1 WO2016118118 A1 WO 2016118118A1
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Prior art keywords
oligonucleotide
seq
kcnh2
sequence
kvll
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Zhengfeng ZHOU
Qiuming GONG
Matthew STUMP
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Oregon Health and Science University
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Oregon Health and Science University
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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
    • C12N15/1138—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 receptors or cell surface proteins
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3233—Morpholino-type ring
    • 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
    • C12N2320/34—Allele or polymorphism specific uses

Definitions

  • the field is pharmaceutical compositions, more specifically; the field is pharmaceutical compositions comprising oligonucleotides.
  • KCNH2 or human ether-a-go-go-related gene 1 encodes the Kvll.l channel that conducts the rapidly activating delayed rectifier K + current (I Kr) in the heart (Warm ke JW and Ganetzky B, Proc Natl Acad Sci U S A 91, 3438-3442 (1994); Sanguinetti MC et al, Cell 81, 299-307 (1995); Trudeau MC et al, Science 269, 92-95 (1995); and Zhou Z et al, Biophys J 74, 230-241 (1998); all of which are incorporated by reference herein).
  • I Kr delayed rectifier K + current
  • Kvll.l channels are essential for cardiac action potential repolarization and mutations in KCNH2 cause long OT syndrome type 2 (L T2) (Curran M E et al, Cell 80, 795-803 (1995); incorporated by reference herein).
  • L T2 long OT syndrome type 2
  • Alternative intronic polyadenylation has been shown to direct the expression of two Kvll.l C-terminal isoforms, the functional Kvll.la isoform and the non-functional Kvll.la- USO isoform (Gong et al, J Biol Chem 285, 32233-32241 (2010); incorporated by reference herein).
  • Kvll.la is produced by splicing from exon 9 to exon 10 and use of a distal poly(A) site in exon 15, whereas Kvll.la-USO is generated by the activation of a proximal poly(A) site within intron 9.
  • the last 359 amino acids of Kvll.la are absent in Kvll.la-USO and the truncated isoform fails to form functional channels when expressed in mammalian cells (Kupersmidt S et al, J Biol Chem 273, 27231-27235 (1998); incorporated by reference herein).
  • a pharmaceutical composition that acts directly on the poly(A) intron 9 signal of KCNH2 is necessary.
  • One example of such a pharmaceutical composition is an oligonucleotide in an antisense configuration relative to downstream elements of the poly(A) intron 9 signal that facilitate formation of the poly(A) tail.
  • oligonucleotides that include a first sequence of SEO ID NO: 1 (5'- CAAAAC-3') and a second sequence of SEO ID NO: 2 (5'-AACACA-3').
  • the first sequence is the antisense of a site termed DSE-1 herein.
  • the second sequence is the antisense of a site termed DSE-2 herein.
  • the oligonucleotide includes both SEO ID NO: 1 and SEO ID NO: 2.
  • the oligonucleotide is at least 19 nucleotides in length.
  • oligonucleotides including the addition of a locked nucleotide, a G-clamp nucleotide, a nucleotide base analog, a 3'- terminal cap moiety, or a phosphate backbone modification.
  • a locked nucleotide e.g., a nucleotide base analog
  • a 3'- terminal cap moiety e.g., a phosphate backbone modification
  • Figure 1A is a diagram of the KCNH2 minigene luciferase reporter construct described herein and includes the sequence of downstream elements of the KCN H2 intron 9 poly(A) signal. Downstream element deletions and mutations are indicated.
  • Figure 2A is a diagram of the structure of the short KCN H2 gene construct described herein and includes the sequence of downstream elements of the KCN H2 intron 9 poly(A) signal. DSE-1 and DSE-2 mutations are indicated.
  • Figure 2C is an image of an immunoblot analysis of Kvll.l protein expressed in cells expressing WT and DSE mutant short KCNH2 genes.
  • Figure 2D is a set of two plots showing representative currents recorded from cells stably expressing WT and Mutl + 2 short KCNH2 genes.
  • Figure 3A is a diagram of the tandem poly(A) signal construct and the sequence of a morpholino oligonucleotide that includes SEO ID NO: 1.
  • Figure 3B is an image of a gel resulting from RNAse protection assay analysis of relative usage of intron 9 poly(A) signal and synthetic poly(A) signal following the treatment with invert or antisense MO.
  • Figure 3C is a histogram quantifying results of RNAse protection assays as described in
  • Figure 5A is a set of two plots showing representative currents recorded from Flp-ln HEK293 cells stably expressing short KCNH2 gene following treatment with 10 ⁇ negative control or antisense morpholino oligonucleotide comprising SEQ. ID NO: 1 for 48 h.
  • Figure 5B is an l-V plot of tail current density measured at -50 mV following test voltages from -70 to +50 mV for negative control ( ⁇ ) and antisense morpholino oligonucleotide comprising SEQ ID NO: 1 ( ⁇ ).
  • Figure 6C is a set of two plots showing representative currents recorded from Flp-ln HEK293 cells stably expressing short KCNH2 gene containing the canonical poly(A) signal following treatment with 10 ⁇ negative control or antisense morpholino oligonucleotide comprising SEQ ID NO: 1 for 48 h.
  • SEQ ID NO: 1 is a sequence that blocks the DSE-1 site of intron 9 of human KCNH2 5'-CAAAAC-3'
  • SEQ ID NO: 3 is the sequence of an oligonucleotide that blocks both DSE-1 and DSE-2 5'-AACACAXXXXXXXCAAAAC-3'
  • SEQ ID NO: 4 is the sequence of an oligonucleotide that blocks both DSE-1 and DSE-2 5'-AACACAGTAGTGAATCAAAAC-3'
  • SEQ ID NO: 6 is the sequence of an inverted morpholino oligonucleotide that acts as a negative control. 5'-CCAAAACTAAGTGATGACACCAAGAC-3'. It is also called Invert MO or Inv MO herein.
  • Binding or stable binding An association between two substances or molecules, such as the association between an antisense nucleic acid to a sense nucleic acid. Binding can be detected by any procedure known to one skilled in the art, such as by physical or functional properties. For example, binding can be detected functionally by determining whether binding has an observable effect upon a biosynthetic process such as expression of a gene, DNA replication, transcription, translation, protein activity, and the like.
  • Contacting Placement in direct physical association, including contacting of a solid with a solid, a liquid with a liquid, a liquid with a solid, or either a liquid or a solid with a cell or tissue, whether in vitro or in vivo. Contacting can occur in vitro with isolated cells or tissue or in vivo by administering to a subject.
  • Effective amount An amount of agent, such as an antisense oligonucleotide that is sufficient to generate a desired response, such as reducing or eliminating a sign or symptom of a condition or disease, such as long-QT syndrome.
  • an "effective amount" is one that treats (including prophylaxis) one or more symptoms and/or underlying causes of any of a disorder or disease, for example long-QT syndrome.
  • An effective amount can be a therapeutically effective amount, including an amount that prevents one or more signs or symptoms of a particular disease or condition from developing, such as one or more signs or symptoms associated with long-QT syndrome.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of metastases, an improvement in the overall health or well-being of the subject, or by other clinical or physiological parameters associated with a particular disease.
  • a "prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • a “therapeutic” treatment is a treatment administered after the development of significant signs or symptoms of the disease.
  • a mutation is any difference in a nucleic acid or polypeptide sequence from a normal, consensus or "wild type" sequence.
  • a mutant is any protein or nucleic acid sequence comprising a mutation.
  • a cell or an organism with a mutation may also be referred to as a mutant.
  • Sequence identity/similarity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are.
  • NCBI Basic Local Alignment Search Tool (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • a larger or smaller scale of synthesis is encompassed by the invention, including any method of synthesis now known or yet to be disclosed.
  • Suitable reagents for synthesis of the oligonucleotides disclosed herein are known to those of skill in the art.
  • a single stranded oligonucleotide can comprise a modified nucleotide.
  • modified nucleotides include, but are not limited to, nucleotides having a 2'-0-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-0-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, or 2'-C-allyl group.
  • Modified nucleotides having a conformation such as those described in, for example in Sanger, Principles of Nucleic Acid Structure, Springer- Verlag Ed. (1984), are also suitable for use in oligonucleotides.
  • LNA nucleotides include, without limitation: locked nucleic acid (LNA) nucleotides, G-clamp nucleotides, or nucleotide base analogs.
  • LNA nucleotides include but need not be limited to 2'-0, 4'-C-methylene-(D- ribofuranosyl)nucleotides), 2'-0-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy-2'-chloro (2CI) nucleotides, and 2'- azido nucleotides.
  • MOE 2-methoxyethyl
  • Non-limiting examples of phosphate backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, peptide nucleic acid, and alkylsilyl substitutions (see, e.g., Hunziker et al, Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al, Antisense Research , ACS, 24-39 (1994); Bennett CF and Swayze EE, Ann Rev Pharmacol Toxicol 50, 259-293 (2010); incorporated by reference herein).
  • modified nucleotides and types of chemical modifications that can be introduced into the modified oligonucleotides of the present invention are described, e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos. 20040192626 and 20050282188.
  • the conjugate can be attached at the 5'-and/or the 3'-end of an oligonucleotide via a covalent attachment such as a nucleic acid or non-nucleic acid linker.
  • the conjugate can also be attached to the oligonucleotide through a carbamate group or other linking group (see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and
  • a conjugate may be added to the oligonucleotide for any of a number of purposes.
  • the conjugate may be a molecular entity that facilitates the delivery of the oligonucleotide into a cell or the conjugate a molecule that comprises a drug or label.
  • oligonucleotides include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.),
  • steroids such as cholesterol
  • glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.),
  • phospholipids phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Publication Nos. 20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).
  • Other examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Publication Nos. 20050119470 and 20050107325.
  • conjugate molecules include the 2'-0-alkyl amine, 2'-0-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337. Additional examples of conjugate molecules include a hydrophobic group, a membrane active compound, a cell penetrating compound, a cell targeting signal, an interaction modifier, or a steric stabilizer as described in U.S. Patent Publication No. 20040167090. Further examples include the conjugate molecules described in U.S. Patent Publication No. 20050239739.
  • the type of conjugate used and the extent of conjugation to the oligonucleotide can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the oligonucleotide while retaining activity.
  • one skilled in the art in light of this disclosure can screen oligonucleotides having various conjugates attached thereto to identify
  • oligonucleotide conjugates having improved properties using any of a variety of well-known in vitro cell culture or in vivo animal models, such as for long-QJ syndrome caused by mutations in the KCNH2 gene.
  • a morpholino oligonucleotide is a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by phosphodiester bonds which is typical of nucleotides and nucleosides.
  • the morpholino oligonucleotide instead contains a ring nitrogen with coupling through the ring nitrogen.
  • Morpholino oligonucleotides are structures from 12 to 25 nucleotides, including a targeting base sequence that is complementary to a target region of a selected preprocessed mRNA such as preprocessed intron 9 of KCNH2.
  • the morpholino antisense oligonucleotide inhibits formation of a poly(A) signal at a site within intron 9 and thereby results in the proper processing of the mRNA to form KCNH2 protein.
  • the antisense compound employed in the present invention is one that does not activate RNase H.
  • RNase-H active oligomers of which phosphorothioate oligonucleotides are the most prominent example, operate primarily by a mechanism in which the target mRNA is cleaved.
  • RNase-incompetent oligomers are believed to act by a steric blocking mechanism.
  • Such compounds include morpholino oligonucleotides, PNA's (peptide nucleic acids), methylphosphonates, and 2'-0-alkyl or -allyl modified oligonucleotides, all of which are known in the art.
  • the morpholino oligonucleotides are composed of morpholino subunits of the form shown in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337.
  • the synthesis, structures, and binding characteristics of morpholino oligonucleotides are detailed in these patents so that one of skill in the art in light of this disclosure can create the morpholino oligonucleotides with the sequences disclosed herein.
  • I n a morpholino oligonucleotide
  • the morpholino groups are linked together by uncharged phosphorus-containing linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit
  • the base attached to the morpholino group is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine.
  • Such morpholino oligonucleotides have shown high binding affinity for RNA targets, and the uncharged backbone favors uptake into cells and reduces non-specific binding interactions, relative to charged analogs such as phosphorothioates. They have been shown to provide significantly improved activity and selectivity in inhibiting translation of targeted sequences in comparison to phosphorothioate oligonucleotides. See, for example, Summerton et al., Antisense & Nucleic Acid Drug Dev. 7, 63-70, (1997).
  • the morpholino oligonucleotides have very high nuclease resistance and good water solubility, making them good candidates for in vivo use.
  • the solubility of the morpholino oligonucleotides, and the ability of the compound to resist precipitation on storage in solution, can be further enhanced by derivatizing the oligomer with a solubilizing moiety, such as a hydrophilic oligomer, or a charged moiety, such as a charged amino acid or organic acid.
  • a solubilizing moiety such as a hydrophilic oligomer, or a charged moiety, such as a charged amino acid or organic acid.
  • the moiety may be any biocompatible hydrophilic or charged moiety that ca n be coupled to the antisense compound and that does not interfere with compound binding to the target sequence.
  • the moiety can be chemically attached to the antisense compound, e.g., at its 5' end, by well-known derivatization methods.
  • One preferred moiety is a defined length oligo ethylene glycol moiety, such as triethyleneglycol, coupled covalently to the 5' end of the antisense compound through a carbonate linkage, via a piperazine linking group forming a carbamate linkage with triethyleneglycol, where the second piperazine nitrogen is coupled to the 5'-end phosphorodiamidate linkage of the antisense.
  • the compound may be designed to include one a small number of charged backbone linkages, such as a phosphodiester linkage.
  • the compound is designed to hybridize to the target sequence under physiological conditions with a T m substantially greater than 37° C, e.g., at least 50° C. and preferably 60° C- 80° C.
  • the compound may not be necessarily 100% complementary to the target sequence, so long as it is effective to stably and specifically bind to the target sequence such that formation of a poly(A) at intron 9 is inhibited.
  • the appropriate length of the oligomer to allow stable, effective binding combined with good specificity is about 8 to 40 nucleotide base units, and preferably about 12-25 base units. Mismatches, if present, are less destabilizing toward the end regions of the hybrid duplex than in the middle. Oligomer bases that allow degenerate base pairing with target bases are also contemplated, assuming base-pair specificity with the target and inhibition of poly(A) formation at intron 9 is maintained.
  • morpholino oligonucleotides have bases that are analogs of those of nucleic acids
  • a description of a morpholino oligonucleotide as having a particular sequence (or a homolog thereof) is a recitation of the structure of the morpholino oligonucleotide.
  • Chemical modification of the morpholino oligonucleotide can involve attaching a conjugate to the morpholino oligonucleotide.
  • the conjugate can be attached at the 5'- and/or the 3'-end of the sense and/or the antisense strand of the oligonucleotide via a covalent attachment such as a nucleic acid or non-nucleic acid linker.
  • the conjugate can also be attached to the morpholino oligonucleotide through a carbamate group or other linking group (see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and 20050158727).
  • a conjugate ca n be added to the morpholino oligonucleotide for any of a number of purposes.
  • the conjugate may be a molecular entity that facilitates the delivery of the morpholino
  • oligonucleotide into a cell or the conjugate a molecule that comprises a drug or label.
  • conjugate molecules suitable for attachment to a morpholino are examples of conjugate molecules suitable for attachment to a morpholino
  • oligonucleotide include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Publication Nos.
  • steroids such as cholesterol
  • glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof),
  • conjugate molecules include the 2'-0-alkyl amine, 2'-0-alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337. Additional examples of conjugate molecules include a hydrophobic group, a membrane active compound, a cell penetrating compound, a cell targeting signal, an interaction modifier, or a steric stabilizer as described in U.S. Patent Publication No. 20040167090. Further examples include the conjugate molecules described in U.S. Patent Publication No. 20050239739.
  • oligonucleotide can be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the morpholino oligonucleotide while retaining activity.
  • one skilled in the art can screen oligonucleotides having various conjugates attached thereto to identify oligonucleotide conjugates having improved properties using any of a variety of well-known in vitro cell culture or in vivo animal models.
  • a pharmaceutical composition may be any chemical compound or composition capable of inducing a desired therapeutic or prophylactic effect when properly administered to a subject.
  • a pharmaceutical composition can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent.
  • a therapeutic agent is one that alone or together with one or more additional compounds induces the desired response (such as inducing a therapeutic effect when administered to a subject).
  • a pharmaceutical agent is an agent that significantly reduces one or more symptoms associated with long-OT syndrome.
  • One example is a pharmaceutical composition comprising an oligonucleotide comprising a sequence of SEQ. ID NO: 1 and/or a sequence of SEQ. ID NO: 2 that is sufficient to block formation of a poly(A) signal in intron 9 of KCNH2.
  • a pharmaceutically acceptable carrier can be any material or molecular entity that facilitates the administration or other delivery of the morpholino oligonucleotides described herein.
  • a vehicle can be any material or molecular entity that facilitates the administration or other delivery of the morpholino oligonucleotides described herein.
  • the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral
  • formulations usually comprise injectable fluids that include pharmaceutically and
  • the carrier is one that allows trafficking of the morpholino oligonucleotide to the heart or one that allows the morpholino oligonucleotide to be taken up by the heart.
  • An effective amount or concentration of a compound may be any amount of a composition that alone, or together with one or more additional therapeutic agents is sufficient to achieve a desired effect in a subject, or in a cell being treated with the agent.
  • the effective amount of the agent will be dependent on several factors, including, but not limited to, the subject or cells being treated and the manner of administration of the therapeutic composition.
  • an effective amount or concentration is one that is sufficient to prevent advancement, delay progression, or to cause regression of a disease, or which is capable of reducing symptoms caused by any disease, including long-OT syndrome.
  • a desired effect is to reduce or inhibit one or more symptoms associated with long-OT syndrome. The one or more symptoms do not have to be completely eliminated for the composition to be effective.
  • a composition can decrease the sign or symptom by a desired amount, for example by at least 20%, at least 50%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100%, as compared to the sign or symptom in the absence of the composition.
  • a therapeutically effective amount of a pharmaceutical composition can be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • an effective amount of such agent can vary from about 100 ⁇ g -10 mg per kg body weight if administered intravenously.
  • the actual dosages will vary according to factors such as the particular status of the subject (for example, the subject's age, size, fitness, extent of symptoms, susceptibility factors, and the like) time and route of administration, other drugs or treatments being administered concurrently, as well as the specific pharmacology of treatments for long-QT syndrome for eliciting the desired activity or biological response in the subject. Dosage regimens can be adjusted to provide an optimum prophylactic or therapeutic response.
  • An effective amount is also one in which any toxic or detrimental side effects of the compound and/or other biologically active agent is outweighed in clinical terms by
  • a non-limiting range for a therapeutically effective amount of treatments for long-QT syndrome within the methods and formulations of the disclosure is about 0.0001 ⁇ g/kg body weight to about 10 mg/kg body weight per dose, such as about 0.0001 ⁇ g/kg body weight to about 0.001 ⁇ g/kg body weight per dose, about 0.001 ⁇ g/kg body weight to about 0.01 ⁇ g/kg body weight per dose, about 0.01 ⁇ g/kg body weight to about 0.1 ⁇ g/kg body weight per dose, about 0.1 ⁇ g/kg body weight to about 10 ⁇ g/kg body weight per dose, about 1 ⁇ g/kg body weight to about 100 ⁇ g/kg body weight per dose, about 100 ⁇ g/kg body weight to about 500 ⁇ g/kg body weight per dose, about 500 ⁇ g/kg body weight per dose to about 1000 ⁇ g/kg body weight per dose, or about 1.0 mg/kg body weight to about 10 mg/kg body weight per dose.
  • the effective amount can be selected based on the mode of delivery, for example, trans-epidermal, rectal, oral, pulmonary, intranasal delivery, intravenous or subcutaneous delivery.
  • Determination of effective amount is typically based on animal model studies followed up by human clinical trials and is guided by administration protocols that significantly reduce the occurrence or severity of targeted disease symptoms or conditions in the subject.
  • suitable models in this regard include, for example, murine, rat, porcine, feline, non-human primate, and other accepted animal model subjects known in the art.
  • effective dosages can be determined using in vitro models (for example, mouse models of long-QT syndrome). Using such models, only ordinary calculations and adjustments are required to determine an appropriate concentration and dose to administer a therapeutically effective amount of the treatments for long-QJ syndrome (for example, amounts that are effective to alleviate one or more symptoms of long-QJ syndrome).
  • compositions for treatment of long-QT syndrome can be for either prophylactic or therapeutic purposes.
  • the pharmaceutical composition is provided in advance of any clinical symptom of long-QT syndrome.
  • Prophylactic administration serves to prevent or ameliorate any subsequent disease process.
  • the compounds are provided in response to symptoms of the disease.
  • compositions described herein can be administered to the subject in a single bolus delivery, via continuous delivery (for example, continuous transdermal, mucosal or intravenous delivery) over an extended time period, or in a repeated administration protocol (for example, by an hourly, daily or weekly, repeated administration protocol) or as repeated doses within a prolonged prophylaxis or treatment regimen that will yield clinically significant results to alleviate one or more symptoms or detectable conditions associated with long-QT syndrome in the subject.
  • continuous delivery for example, continuous transdermal, mucosal or intravenous delivery
  • a repeated administration protocol for example, by an hourly, daily or weekly, repeated administration protocol
  • a subject can be any multi-cellular vertebrate organism, a category that includes human and non-human mammals, such as mice.
  • a subject is a male.
  • a subject is a female.
  • Further types of subjects to which the pharmaceutical composition may be properly administered include subjects known to have long-QT syndrome (through, for example, a molecular diagnostic test or clinical diagnosis,) subjects having a predisposition to long-QT syndrome or subjects displaying one or more symptoms of long-QT syndrome.
  • Treating a subject encompasses any therapeutic intervention that can ameliorate a sign or symptom of a disease or pathological condition after it has begun to develop, whether or not the subject has developed symptoms of the disease.
  • Ameliorating, with reference to a disease, pathological condition or symptom refers to any observable beneficial effect of the treatment.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the number of relapses of the disease, an improvement in the memory and/or cognitive function of the subject, a qualitative improvement in symptoms observed by a clinician or reported by a patient, or by other parameters well known in the art that are specific to long-QT syndrome.
  • a symptom may be any subjective evidence of disease or of a subject's condition, for example, such evidence as perceived by the subject; a noticeable change in a subject's condition indicative of some bodily or mental state.
  • a sign may be any abnormality indicative of disease, discoverable on examination or assessment of a subject.
  • a sign is generally an objective indication of disease.
  • Example 1 Inhibition of lntronic Polyadenylation with Antisense Morpholino
  • a minigene luciferase reporter construct was generated by subcloning the Renilla luciferase gene downstream of a splicing competent KCNH2 minigene comprising KCNH2 genomic DNA from exon 8 to exon 11.
  • the construction of the KCNH2 minigene has been previously described in Gong Q. et al, J Mol Cell Cardiol 44, 502-509 (2008) which is incorporated by reference herein.
  • the N-terminus of the minigene was Myc tagged.
  • the Myc tag was inserted in-frame with the KCNH2 and luciferase translation sequence. Expression of the minigene luciferase reporter is driven by a CMV promoter.
  • the vector also contains the firefly luciferase gene driven by the SV40 promoter, which was used as a control for transfection efficiency.
  • the deletion and mutations of U/GU-rich elements downstream of the KCNH2 intron 9 poly(A) signal were performed using the pAlter ® in vitro mutagenesis system (Promega, Madison, Wl).
  • HEK293 cells were transiently transfected with the minigene luciferase reporter construct using the Effectene ® method (Qjagen, Valencia, CA). After 24 hours, cells were harvested and assayed for both firefly and Renilla luciferase activity using the Dual-Luciferase assay kit (Promega). Data were analyzed by normalizing Renilla luciferase activity to firefly luciferase activity and presented as mean ⁇ SEM.
  • Stably transfected Flp-ln HEK293 cells were generated by the co- transfection of the KCNH2 gene constructs (0.1 ⁇ g) with the Flp recombinase expression vector pOG44 (0.9 ⁇ g) using the Effectene ® method and selected with 100 ⁇ g/ml hygromycin.
  • Morpholino oligonucleotides were synthesized by Gene Tools (Philomath, OR).
  • the KCNH2 antisense morpholino oligonucleotide was designed to target a 25 nt sequence within KCNH2 intron 9 containing U/GU-rich elements (underlined) essential for the activation of the intron 9 poly(A) signal, 5'-CAGAACACAGTAGTGAATCAAAACC-3'.
  • An inverted morpholino oligonucleotide with the same sequence but in a reverse orientation was used as a control 5'- CCAAAACTAAGTGATGACACAAGAC-3'.
  • the Endo-Porter ® delivery system (Gene Tools) was used to deliver antisense and control morpholino oligonucleotides into the cells.
  • RNA isolation and an RNase protection assay were performed as previously described in Gong et al, 2006 supra. Briefly, antisense RNA riboprobes were transcribed in vitro in the presence of biotin-14-CTP. Yeast RNA was used as a control for the complete digestion of the probes by RNase. The relative intensity of each band was quantified using ImageJ ® software and adjusted for the number of biotin-labeled cytidines in each protected fragment. The expression level of the hygromycin B resistance gene from the KCNH2 gene constructs was used to normalize the relative expression of Kvll.l isoforms.
  • Kvll.l current was activated by depolarizing steps between -70 and +50 mV from a holding potential of-80mVand Kvll.l tail current was recorded following repolarization to -50 mV.
  • the patch-clamp data are presented as mean ⁇ SEM and analyzed by Student's t-test. P ⁇ 0.05 is considered statistically significant.
  • Polyadenylation sites are primarily defined by a hexameric poly(A) signal AAUAAA or other close variants (Tian B and Manley JL, Trends Biochem Sci 38, 312-320 (2013); incorporated by reference herein).
  • a cis-acting, U/GU-rich downstream element DSE
  • DSE U/GU-rich downstream element
  • a reporter construct was designed to identify cis-acting elements required for intron 9 polyadenylation by subcloning the Renilla luciferase gene downstream of a splicing competent minigene composed of KCNH2 genomic DNA from exon 8 to exon 11.
  • the removal of intron 9 generated active luciferase and polyadenylation of intron 9 resulted in no luciferase activity ( Figure 1A).
  • Figure IB d-DS
  • DSE-1 was mutated from GUUUUG to CCACAA (Mutl), DSE-2 from UGUGUU to CAACCA (Mut2) and DSE-3 from UCUUU to CCAAC (Mut3) in the minigene luciferase reporter construct. Mutl and Mut2, but not Mut3, resulted in higher luciferase expression relative to unmutated ( Figure IB). When both DSE-1 and DSE-2 were mutated (Mutl + 2), luciferase expression was higher when compared to Mutl and Mut2 alone.
  • RNAse protection assay was performed using a probe specific to 249 nt of KCNH2 intron 9 (Gong, 2010 supra). Use of the probe results in termination of transcription after generation of a 158 nt fragment, indicating polyadenylation at the intron 9 poly(A) signal and termination of transcription after generation of a 249 nt fragment indicating polyadenylation at the downstream synthetic poly(A) signal.
  • Flp-ln HEK293 cells stably expressing the short KCNH2 gene were treated with the indicated concentrations of the antisense morpholino oligonucleotide of SEQ. ID NO: 5 for 48 hours or with 5 ⁇ for the indicated amount of time ( Figures 4B and 4C).
  • Treatment with the antisense morpholino oligonucleotide of SEQ ID NO: 5 resulted in significantly higher expression of Kvll.la protein and lower expression of Kvll.la- USO protein relative to controls.
  • the voltage dependence of Kvll.l channel activation was determined by fitting the normalized tail currents with a Boltzmann function (Figure 5C).
  • the half maximal activation voltages (Vi/ 2 ) in cells treated with the negative control was -4.2 ⁇ 2.9 mV.
  • the half maximal activation voltage in cells treated with antisense morpholino oligonucleotide of SEQ ID NO: 5 was -5.6 ⁇ 2.0 mV.
  • the slope factor (k) in cells treated with the negative control (SEQ ID NO: 6) was 8.7 ⁇ 0.2.
  • the slope factor in cells treated with antisense morpholino oligonucleotide of SEQ ID NO: 5 was 7.9 ⁇ 0.3.
  • HEK293 cells stably expressing the short KCNH2 gene construct containing a canonical poly(A) signal were treated with negative control (SEQ ID NO: 6) or antisense morpholino oligonucleotide of SEQ ID NO: 5.
  • RNAse protection assay shows that in the presence of the negative control, Kvll.la-USO was predominantly expressed with no detectable expression of Kvll.la mRNA. When treated with 10 ⁇ antisense oligonucleotide of SEQ ID NO: 5, the expression of the Kvll.la isoform was significantly higher.

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Abstract

Cette invention concerne des oligonucléotides manifestant une activité de prévention de l'adénylation poly(A) sur l'intron 9 du gène KCNH2, ainsi que des compositions pharmaceutiques contenant lesdits oligonucléotides, et des procédés d'utilisation de ceux-ci pour traiter le syndrome du QT long. Les oligonucléotides contiennent des séquences antisens correspondant aux sites appelés DSE 1 et DSE 2 dans l'intron 9.
PCT/US2015/012060 2015-01-20 2015-01-20 Modulation de l'expression de l'isoforme kcnh2 par des oligonucléotides à titre d'approche thérapeutique pour le syndrome du qt long Ceased WO2016118118A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050130190A1 (en) * 2003-08-22 2005-06-16 Charles Antzelevitch Mutations in ion channel proteins associated with sudden cardiac death
US20090220949A1 (en) * 2005-06-07 2009-09-03 Fondzione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione I.R.C.C.S. Mutations Associated with the Long QT Syndrome and Diagnostic Use Thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050130190A1 (en) * 2003-08-22 2005-06-16 Charles Antzelevitch Mutations in ion channel proteins associated with sudden cardiac death
US20090220949A1 (en) * 2005-06-07 2009-09-03 Fondzione Salvatore Maugeri Clinica Del Lavoro E Della Riabilitazione I.R.C.C.S. Mutations Associated with the Long QT Syndrome and Diagnostic Use Thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DONOVAN, ALEXANDER J. ET AL.: "Long QT2 mutation on the Kv11.1 ion channel inhibits current activity by ablating a protein kinase C a consensus site", MOLECULAR PHARMACOLOGY, vol. 82, no. 3, September 2012 (2012-09-01), pages 428 - 437 *
GONG, QIUMING ET AL.: "Identification of Kv11.1 isoform switch as a novel pathogenic mechanism of long-QT syndrome", CIRCULATION. CARDIOVASCULAR GENETICS, vol. 7, no. 4, August 2014 (2014-08-01), pages 482 - 490 *
GONG, QIUMING ET AL.: "Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides", JOURNAL OF MOLECULAR AND CELLULAR CARDIOLOGY, vol. 76, November 2014 (2014-11-01), pages 26 - 32 *

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