CN117957321A - Products and compositions - Google Patents

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CN117957321A
CN117957321A CN202280055021.6A CN202280055021A CN117957321A CN 117957321 A CN117957321 A CN 117957321A CN 202280055021 A CN202280055021 A CN 202280055021A CN 117957321 A CN117957321 A CN 117957321A
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oligomeric compound
region
nucleoside
sugar
nucleosides
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D.萨马斯基
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Sirnaomics Inc
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Sirnaomics Inc
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Priority claimed from PCT/US2022/034965 external-priority patent/WO2022272108A2/en
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Abstract

The present invention provides a nucleic acid product and composition and uses thereof. In particular, nucleic acid products are provided that modulate, interfere with, or inhibit the expression of the APOC3 gene. The product may be an oligomeric compound comprising at least a first region comprising a linked nucleoside, the region having at least a first nucleobase sequence complementary to at least a portion of an RNA transcribed from the APOC3 gene, wherein the first nucleobase sequence is selected from the group consisting of: SEQ ID NOs 1 to 39.

Description

Products and compositions
Cross-reference to related patent applications
The present application claims the benefits and priorities of two U.S. provisional patent applications, 63/214,608 filed on month 6 of 2021 and 24 and 63/318,287 filed on month 3 of 2022, which are incorporated herein by reference in their entireties.
Technical Field
The present invention provides nucleic acid products and compositions capable of modulating, particularly interfering with or inhibiting the expression of the apolipoprotein C3 (APOC 3) gene and uses thereof. Particular embodiments provide methods, compounds, and compositions for reducing APOC3 mRNA and protein expression in an animal. Such methods, compounds, and compositions are useful for treating, preventing, or ameliorating APOC 3-related diseases such as dyslipidemia, hypertriglyceridemia, hypercholesterolemia, and atherosclerotic cardiovascular disease (ASCVD).
Background
Triglycerides are esters of glycerol with three fatty acids. It can store fat and energy and is transported through the blood. The excessive levels of triglycerides in the blood have long been recognized as a direct or indirect causative factor for a range of diseases. Recent evidence suggests that in the acute cardiovascular disease ASCVD and diseases included under or associated with this term, it is partly associated with elevated cholesterol levels, in particular LDL cholesterol. A more comprehensive list of diseases associated with elevated triglyceride levels is also presented in the embodiments disclosed below.
Apolipoprotein C3 is secreted by the liver and small intestine. It is present in triglyceride-rich lipoproteins, including Very Low Density Lipoproteins (VLDL) and chylomicrons. APOC3 is involved in the negative regulation of lipid breakdown, particularly triglyceride breakdown, and clearance of VLDL, LDL and HDL lipoproteins. The molecular functions of APOC3 include inhibition of lipoprotein lipase and liver lipase.
Disease of the human body
Abnormal levels of circulating triglycerides, also known as hypertriglyceridemia, are a recognized disease in itself, as such abnormal levels, particularly if of a longer duration, may lead to diseases in the cardiovascular system and/or inflammation.
Treatment of
Existing methods of treatment include the use of statin drugs, such as rosuvastatin and simvastatin, and fibrates, such as fenofibrate. However, statin drugs may have side effects and some patients are intolerant to statin drugs.
Thus, there remains a need to seek to treat APOC 3-related diseases. Here we aim to provide compounds, methods and pharmaceutical compositions for the treatment of such diseases.
Double-stranded RNA (dsRNA) capable of complementarily binding to expressed mRNA has been shown to block gene expression (Fire et al, 1998, nature, 1998, month 2, 19; 391 (6669): 806-11 and Elbashir et al, 2001, nature, 5, 24; 41 (6836): 494-8), the mechanism of which is known as RNA interference (RNAi). Short dsrnas directly lead to gene-specific post-transcriptional silencing in many organisms, including vertebrates, have become useful tools for studying gene function. RNAi is mediated by the RNA-induced silencing complex (RISC), a multicomponent nuclease of a specific sequence that disrupts messenger RNA homologous to the silencing trigger loaded into the RISC complex. Interfering RNAs (irnas), such as sirnas, antisense RNAs, and micrornas, are oligonucleotides capable of preventing protein formation by degradation of mRNA molecules, i.e., inhibiting gene translation of a protein by gene silencing. Gene silencing agents are becoming increasingly important in medical therapeutic applications. Watts and Corey, in J.Pat.2012 (Vol 226, p 365-379), indicate that there are some algorithms available for designing nucleic acid silencing triggers, but all have serious limitations. Since the algorithm does not take into account the tertiary structure of the target mRNA or the involvement of RNA binding proteins, various experimental methods may be required to determine effective siRNA. Thus, it was found that a potent nucleic acid silencing trigger with minimal off-target effects is a complex process. For drug development of these highly charged molecules, these molecules must be synthesized in an economical manner, distributed to the targeted tissues, into cells, and function within an acceptable toxicity range. Accordingly, it is an object to provide compounds, methods and pharmaceutical compositions described herein for the treatment of thromboembolic disorders, including oligomers that modulate, and in particular inhibit, gene expression by RNAi.
Disclosure of Invention
The present invention provides nucleic acid products that modulate, particularly interfere with or inhibit, the expression of the apolipoprotein C3 (APOC 3) gene, and related therapeutic uses. Specific oligomeric compounds and sequences are described herein. This summary provides a simplified form that is further described in the detailed description that follows. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to determine the scope of the claimed subject matter.
Drawings
FIG. 1a shows a dose curve of candidate APOC3 precursor in primary human hepatocytes;
FIG. 1b shows a dose curve of APOC3 lead in a humanized mouse study in primary human hepatocytes;
FIG. 2 shows a time axis including the time point of dosing the mice and the time point of sample collection;
FIG. 3 shows the residual levels of APOC3 mRNA and plasma APOC3 protein in the liver of animals treated with the APOC3 targeting mxRNA construct, as compared to control animals;
FIG. 4 shows the levels of triglycerides and total cholesterol in the serum of animals treated with APOC3 targeted mxRNA construct compared to control (PBS);
FIG. 5a shows the average percentage of liver tissue remaining APOC3 mRNA in plasma of animals receiving treatment with APOC3 targeted mxRNA construct (10 mg/kg) as compared to control animals using an immunoELISA method;
FIG. 5b shows the determination of APOC3 protein levels in plasma of animals treated with APOC3 targeting mxRNA construct (10 mg/kg) using ELISA compared to control animals;
FIG. 6a shows the average percent of Triglycerides (TG) in serum of animals treated with the APOC3 targeted mxRNA construct at weeks 2 and 6, compared to animals in the control group;
FIG. 6b shows Total Cholesterol (TC) levels at week 2 and week 6 in serum of animals treated with the APOC3 targeted mxRNA construct, as compared to control animals;
FIG. 7 is a schematic diagram of a continuous study of Compound A28 (14-4) mF (also known as STP 125G) in humanized liver mice;
FIG. 8a shows the change in APOC3 mRNA over time observed in a continuous study between control and treatment groups;
FIG. 8b shows the APOC3 protein knockout as a function of time observed in duration studies between control and treatment groups;
FIG. 9a shows serum triglyceride levels versus time for control and treatment groups;
FIG. 9b shows serum total cholesterol levels as a function of time for control and treatment groups; and
Figure 10 shows a mouse humanized liver for duration study.
Detailed Description
The following are non-limiting aspects:
Aspect 1. An oligomeric compound capable of inhibiting APOC3 expression, wherein the compound comprises at least a first region of linked nucleosides, the region having a first nucleobase sequence complementary to at least a portion of an RNA transcribed from an APOC3 gene, wherein the first nucleobase sequence is selected from the group consisting of: the sequences of SEQ ID NOs1 to 391, preferably wherein the part has a length of at least 18 nucleosides.
Particularly preferred embodiments relate to mxRNA: see the embodiments and discussion below for details.
Furthermore, the antisense and sense regions disclosed herein can serve as building blocks for compounds directed against multiple targets. The general structure of such compounds ds is described in WO 2020/065602.
Furthermore, as described below, the disclosed embodiments also relate to double stranded RNAs (dsRNA). Unlike mxRNA, which has a hairpin structure linking the sense and antisense RNA strands, dsRNA lacks hairpin loops, and thus, consists of two strands.
Aspect 2. A composition comprising the oligomeric compound according to aspect 1 and a physiologically acceptable excipient.
Aspect 3. A pharmaceutical composition comprising the oligomeric compound according to aspect 1.
Aspect 4. The oligomeric compound according to aspect 1 for use in human or veterinary medicine or therapy.
Aspect 5. The oligomeric compound according to aspect 1, for use in a method of treating a disease or disorder.
Aspect 6. A method of treating a disease or disorder comprising administering to a subject in need of treatment the oligomeric compound of aspect 1.
Aspect 7. Use of the oligomeric compound according to aspect 1 as a tool for gene function analysis.
Aspect 8. Use of the oligomer according to aspect 1 in the manufacture of a medicament for the treatment of a disease or disorder.
Further embodiments are described below, by way of example only. These examples represent ways of putting the disclosed compositions and methods into practice, but those skilled in the art will recognize that they are not the only way to achieve this.
It is to be appreciated that the benefits and advantages described herein may relate to one embodiment or multiple embodiments. Embodiments of the invention are not limited to embodiments that solve any or all of the problems, nor to embodiments that have any or all of the benefits and advantages.
The features of the different aspects and embodiments described herein may be combined as appropriate, as will be apparent to those skilled in the art, and may be combined with any other aspect.
Definition of the definition
The following definitions relate to the entire embodiment disclosed. In many cases, these definitions provide a non-exhaustive list of possible embodiments, in addition to the respective definitions, which correspond to the preferred embodiments.
Unless specifically defined otherwise, the terms, procedures and techniques of analytical chemistry, organic synthetic chemistry, pharmaceutical chemistry and pharmaceutical chemistry described herein are all terms and techniques known and commonly used in the art. Standard techniques can be used for chemical synthesis and chemical analysis. For example, certain such techniques and procedures can be found in "modification of carbohydrates in antisense studies" (Carbohydrate Modifications IN ANTISENSE RESEARCH), sangvi, and the Cook editions, american society of chemistry, washington, D.C., 1994; remington pharmaceutical science (Remington's Pharmaceutical Sciences), mcSton, pa., 21 st edition, 2005; and "antisense drug technology, principles, strategies and applications", by Stanley t.rooke, CRC Press, bocaroton, florida; "molecular cloning, laboratory Manual", 2 nd edition, cold spring harbor laboratory Press, 1989, by Sambrook et al. All patents, applications, published applications and other publications mentioned in this disclosure, as well as other data, are incorporated herein by reference in their entirety, where permitted.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, "excipient" refers to any compound or mixture of compounds suitable for delivery of an oligomeric compound added to the compositions provided herein.
As used herein, "nucleoside" refers to a compound comprising a nucleobase and a sugar group. Nucleosides include, but are not limited to, natural nucleosides (such as those in DNA and RNA) and modified nucleosides. Nucleosides can be linked to a phosphate molecule, and nucleosides linked to phosphate are also referred to as "nucleosides".
As used herein, "chemical modification" or "chemical modification" refers to a chemical difference in a compound as compared to a naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including glycosyl modifications and nucleobase modifications) and modifications of linkages between nucleosides. In the case of oligonucleotides, chemical modifications do not include differences that exist only in nucleobase sequences.
As used herein, "furanosyl" refers to a five-membered ring structure consisting of four carbon atoms and one oxygen atom.
As used herein, "native glycosyl" refers to a ribofuranosyl in native RNA or a deoxyribofuranosyl in native DNA. The "natural glycosyl" is also referred to as "unmodified glycosyl". In particular, the "natural glycosyl" or "unmodified glycosyl" has a-H (DNA glycosyl) or-OH (RNA glycosyl) at the 2 '-position of the glycosyl, especially a-H (DNA glycosyl) at the 2' -position of the glycosyl.
As used herein, "glycosyl" refers to a naturally occurring glycosyl or a modified glycosyl of a nucleoside. As used herein, "modified glycosyl" refers to a substituted glycosyl or a glycosyl.
As used herein, "substituted glycosyl" refers to a substituted furanglycosyl. Substituted glycosyl groups include, but are not limited to, in the 2' -position, 3' -position, 5' -position and +.
Or a furanosyl group having a substituent at the 4' -position. In some embodiments, the substituted glycosyl is a bicyclic glycosyl molecule.
As used herein, "2 '-substituted glycosyl" refers to a furanglycosyl containing substituents other than H or OH in the 2' -position. Unless otherwise indicated, a 2' -substituted glycosyl is not a bicyclic glycosyl (i.e., the 2' -substituent of a 2' -substituted glycosyl does not form a bridge with another atom of the furanosyl ring).
As used herein, "MOE" refers to-OCH 2CH2OCH3.
As used herein, "2'-F nucleoside" refers to a nucleoside consisting of a sugar that contains fluorine in the 2' position. Unless otherwise indicated, the fluorine in the 2' -F nucleoside is located at the ribose position (substituting OH of natural ribose). The double strand of the homogeneously modified 2' -fluorinated (ribose) oligonucleotide hybridized to the RNA strand is not a RNaseH substrate, whereas the ara analogue retains RNaseH activity.
As used herein, the term "sugar substituent" refers to a structure that does not contain furanosyl groups, and is capable of replacing the naturally occurring glycosyl groups in a nucleoside, thereby allowing the nucleoside subunits thus produced to be linked together and/or to other nucleosides to form an oligomeric compound that is capable of hybridizing to a complementary oligomeric compound. . Such structures include rings containing a different number of atoms than furanose (e.g., rings of 4, 6, or 7 members); replacement of furanose oxygen with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or the change in the number of atoms and the replacement of oxygen occur simultaneously. Such structures may also include substitutions corresponding to the substituents of the substituted saccharide groups (e.g., 6-member carbocyclylcyclosaccharide compounds containing additional substituents). Sugar substitutes also include more complex sugar substitutes (e.g., acyclic systems of peptide nucleic acids). Sugar substitutes include, but are not limited to, morpholino, cyclohexenyl, and cyclohexenyl alcohols.
As used herein, a "bicyclic sugar molecule" refers to a modified sugar molecule comprising one 4 to 7 membered ring (including but not limited to furanosyl) comprising two atoms connecting the 4 to 7 membered ring to form a bridge of a second ring, thereby forming a bicyclic structure. In some embodiments, the 4-to 7-position ring is a sugar ring. In certain embodiments, the 4-to 7-membered ring is furanosyl. In certain such embodiments, the bridge connects the 2 '-carbon and the 4' -carbon of the furanosyl group.
As used herein, "nucleotide" refers to a nucleoside further comprising a phosphate linker. As used herein, "linked nucleosides" may or may not be linked by phosphate, thus including but not limited to "linked nucleotides". As used herein, "linked nucleosides" refers to nucleosides that are linked in a continuous sequence (i.e., no other nucleosides are present between the linked nucleosides).
As used herein, "nucleobase" refers to a radical that can be linked to a glycosyl to produce a nucleoside that can be incorporated into an oligonucleotide, wherein the radical can be bonded, more specifically hydrogen bonded, to a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or modified.
As used herein, the term "unmodified nucleobase" or "naturally occurring nucleobase" refers to a heterocyclic nucleobase that naturally occurs in RNA or DNA: purine bases adenine (A) and guanine (G), and pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U).
As used herein, "modified nucleobase" refers to any non-naturally occurring nucleobase.
As used herein, "modified nucleoside" refers to a nucleoside that comprises at least one chemical modification as compared to a naturally occurring RNA or DNA nucleoside. The modified nucleoside can include a modified glycosyl and/or a modified nucleobase.
As used herein, "bicyclic nucleoside" or "BNA" refers to a nucleoside comprising a bicyclic sugar molecule.
As used herein, "locked nucleic acid nucleoside" or "LNA" refers to a nucleoside consisting of a bicyclic sugar molecule comprising a 4'-CH 2 -O-2' bridge.
As used herein, "2 '-substituted nucleoside" refers to a nucleoside that contains a substituent other than H or OH at the 2' -position of the glycosyl group. Unless otherwise indicated, a 2' -substituted nucleoside is not a bicyclic nucleoside.
As used herein, "deoxynucleoside" refers to a nucleoside comprising a 2' -H furanosyl group, such as the nucleoside in naturally occurring Deoxynucleosides (DNA). In some embodiments, the 2' -deoxynucleoside can include a modified nucleobase or include an RNA nucleobase (e.g., uracil).
As used herein, "oligonucleotide" refers to a compound that consists of multiple linked nucleosides. In some embodiments, the oligonucleotide comprises one or more unmodified Ribonucleosides (RNA) and/or unmodified Deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
As used herein, "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage. Preferred modified internucleoside linkages have greater stability than naturally occurring phosphodiesters. "stability" refers primarily to stability to hydrolysis, including enzyme-catalyzed hydrolysis, enzymes (including exonucleases and endonucleases).
Preferred positions for such modified internucleoside linkages include the terminal end of a single-stranded oligomeric compound and hairpin loops. For example, the linkage between a nucleoside that links the first nucleoside to the second nucleoside and links the second nucleoside to the third nucleoside, counted from the 5 'end, and/or the linkage between a nucleoside that links the first nucleoside to the second nucleoside and links the second nucleoside to the third nucleoside, counted from the 3' end, is modified. In addition, the linkage of the terminal nucleoside linked to the 3' -end to a ligand (e.g., galNAc) may also be modified.
As mentioned above, preferred positions are in the hairpin loop of the single stranded oligomeric compound. In particular, all but one, or most of the connections in the hair clip ring may be modified. As used herein, "ligation in a hairpin loop" refers to ligation between nucleosides that are not involved in base pairing. For example, in a hairpin loop consisting of five nucleosides, there are four linkages between nucleosides that are not involved in base pairing. Preferably, the term "ligation in a hairpin loop" also extends to a ligation of a stem to a loop, i.e. a ligation of one base-paired nucleoside to one non-base-paired nucleoside. Generally, there are two such positions in the hairpins and mxRNA described herein.
Most preferably, the modified internucleoside linkage is located at both ends and in the hairpin loop. As used herein, "attached" or "linking group" refers to an atomic group that connects two or more other groups together.
As used herein, "internucleoside linkage" refers to covalent linkage between adjacent nucleosides in an oligonucleotide.
As used herein, "natural nucleoside linkage" refers to a3 'to 5' phosphodiester linkage. As used herein, "modified internucleoside linkage" refers to any internucleoside linkage other than a natural internucleoside linkage. In particular, the term "modified internucleoside linkage" as referred to herein may include a modified phosphorus linking group, such as phosphorothioate or phosphorodithioate internucleoside linkage.
As used herein, "terminal nucleoside linkage" refers to the linkage between the last two nucleosides of an oligonucleotide or defined region thereof.
As used herein, "phosphorus-linking group" refers to a linking group comprising one phosphorus atom, and may include phosphorus linking groups present in natural RNA or DNA, such as phosphodiester linking groups, or modified phosphorus linking groups not typically present in natural RNA or DNA, such as phosphorothioate or phosphorodithioate linking groups. Thus, phosphorus linking groups may include, but are not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, methylphosphonate, phosphoramidate, phosphorothioate, phosphotriester, phosphorothioate alkyl phosphotriester, and borophosphoester.
As used herein, "internucleoside phosphorus-linking group" refers to a phosphorus-linking group that directly links two nucleosides.
As used herein, "oligomeric compound" refers to a polymeric structure consisting of two or more substructures. In some embodiments, the oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide. In some embodiments, the oligomeric compound further comprises one or more conjugated groups and/or terminal groups and/or ligands. In some embodiments, the oligomeric compound consists of oligonucleotides. In some embodiments, the oligomeric compound comprises a backbone of one or more linked monosaccharide molecules, wherein each linked monosaccharide molecule is directly or indirectly linked to a heterocyclic base group. In some embodiments, the oligomeric compounds may also include monomeric sugar groups that are not linked to heterocyclic base groups, thereby providing racemization sites. Oligomeric compounds may be defined in terms of nucleotide sequences only, i.e., the sequence of A, G, C, U (or T) is specified. In this case, the structure of the sugar-phosphorus skeleton is not particularly limited, and may or may not include a modified sugar and/or a modified phosphate. On the other hand, the definition of oligomeric compounds may be more comprehensive, i.e. not only to specify the nuclear nucleotide sequence, but also to specify the backbone structure, in particular the modified state of the sugar (unmodified, 2'-OMe modified, 2' -F modified, etc.) and/or the modified state of the phosphate.
As used herein, a "terminal group" refers to one or more atoms attached to the 3 'or 5' end of an oligonucleotide. In some embodiments, the terminal groups comprise one or more terminal group nucleosides.
As used herein, "conjugate" or "conjugated group" refers to an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. In some embodiments, a conjugated group links the ligand to the modified oligonucleotide or oligomeric compound. In general, conjugated groups can alter one or more properties of the attached compound, including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge and/or scavenging properties. As used herein, "conjugated linker" or "linker" in the context of a conjugated group refers to a portion of the conjugated group, including any atom or group of atoms, that covalently links an oligonucleotide to another portion of the conjugated group. In some embodiments, the point of attachment on the oligomeric compound is the 3' -oxygen atom of the 3' -hydroxyl group of the 3' -terminal nucleoside of the oligonucleotide. In some embodiments, the point of attachment to the oligomer is the 5' -oxygen atom of the 5' hydroxyl group of the 5' terminal nucleoside of the oligonucleotide. In certain embodiments, the bond forming the linkage with the oligomeric compound is a cleavable bond. In some such embodiments, such cleavable linkages constitute all or part of the cleavable molecule.
In some embodiments, the conjugated group includes a cleavable molecule (e.g., a cleavable bond or a cleavable nucleoside) and a ligand moiety, which may include one or more ligands, such as carbohydrate cluster moieties, such as N-acetylgalactose (also referred to as "GalNAc") cluster moieties. In certain embodiments, the carbohydrate cluster moiety is identified by the numbering and identification of the ligand. For example, in some embodiments, the carbohydrate cluster moiety comprises 2 GalNAc groups. For example, in some embodiments, it is particularly preferred that the carbohydrate cluster moiety comprises 3 GalNAc groups. In some embodiments, the carbohydrate cluster moiety comprises 4 GalNAc groups. These ligand moieties are linked to the oligomeric compound by a cleavable molecule (e.g., a cleavable bond or a cleavable nucleoside). The ligands may be arranged in a linear or branched configuration, such as a binary configuration or a ternary configuration. A preferred carbohydrate cluster (also referred to as a "toothbrush") has the formula:
in the above formulae, one, two or three phosphodiester linkages may also be replaced by phosphorothioate linkages.
As used herein, "cleavable molecule" refers to a bond or group that is capable of being cleaved under physiological conditions. In some embodiments, the cleavable molecule is cleaved within a cell or subcellular compartment (e.g., endosome or lysosome). In some embodiments, the cleavable molecule is cleaved by an endogenous enzyme such as a nuclease. In some embodiments, the cleavable molecule comprises a atomic group having one, two, three, four, or more cleavable bonds. In some embodiments, the cleavable molecule is a phosphodiester linkage.
As used herein, "cleavable bond" refers to any chemical bond that can be cleaved.
As used herein, a "carbohydrate cluster" refers to a compound having one or more carbohydrate residues attached to a linking group.
As used herein, "modified carbohydrate" refers to any carbohydrate having one or more chemical modifications relative to a naturally occurring carbohydrate.
As used herein, "carbohydrate derivative" refers to any compound synthesized starting from a carbohydrate or intermediate.
As used herein, "carbohydrate" refers to a natural carbohydrate, modified carbohydrate, or carbohydrate derivative. Carbohydrates are biological macromolecules comprising carbon atoms (C), hydrogen atoms (H) and oxygen atoms (O). The carbohydrate may include a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide, such as one or more galactose molecules, one or more lactose molecules, one or more N-acetylgalactosamine molecules, and/or one or more mannose molecules. A particularly preferred carbohydrate is N-acetyl-galactosamine.
As used herein, "strand" refers to an oligomer comprising linked nucleosides.
As used herein, "single-stranded" or "single-stranded" refers to an oligomer comprising linked nucleosides that are linked in a contiguous sequence without cleavage. Such a single strand may include a region of sufficient self-complementarity to be able to form a hairpin structure capable of stabilizing its own duplex.
As used herein, "hairpin" refers to a single-stranded oligomer compound that includes double strands formed by base pairing between self-complementary and oppositely directed sequences in the strand.
As used herein, "hairpin loop" refers to the unpaired loop of linked nucleosides in a hairpin that results from hybridization of complementary sequences. The resulting structure looks like a ring or U-shape.
In particular short hairpin ribonucleic acids, also known as shRNA, comprise a double-stranded region and a loop connecting the double-stranded region. The ends of the double stranded region without a loop may be blunt ended or may be overhanging with (one) 3 'and/or (one) 5'. The blunt tip configuration is preferably selected.
As used herein, "directionality" refers to the chemical orientation of an oligonucleotide based on the numbering of the carbon atoms of the sugar groups, i.e., the 5 'carbon of the sugar groups defines the 5' end, and the 3 'carbon of the sugar groups defines the 3' end. In double-stranded or duplex oligonucleotides, the 5 'to 3' directions of the strands are reversed for base pairing.
As used herein, "double strand" or abbreviated "dup" refers to an oligonucleotide or two or more complementary strand regions of an oligonucleotide that hybridize together by non-covalent, sequence-specific interactions. Most often, hybridization in the duplex occurs between the nucleobases adenine (A) and thymine (T), and/or (A) adenine and uracil (U), and/or guanine (G) and cytosine (C). Double strands may be part of a single-stranded structure, in which self-complementarity results in hybridization, or may be the result of hybridization between strands in a double-stranded structure.
As used herein, "double-stranded" or "double-stranded" refers to a pair of oligomeric compounds that hybridize to each other. In some embodiments, the double-stranded oligomer compound comprises a first and a second oligomer compound.
As used herein, "expression" refers to the process by which a gene ultimately produces a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenylation, addition of 5' -cap), and translation.
As used herein, "transcription" or "transcription" refers to the first of several steps of DNA-based gene expression, in which a target sequence of DNA is replicated into RNA (especially mRNA) by an RNA polymerase. During transcription, the DNA sequence is read by RNA polymerase, producing complementary, antiparallel RNA sequences, called the primary transcript.
As used herein, "target sequence" refers to a sequence to which an oligomeric compound hybridizes to produce the desired APOC3 expression activity. The oligonucleotide is sufficiently complementary to its sequence of interest to hybridize under physiological conditions.
As used herein, "nucleobase complementarity" or "complementarity" when referring to a nucleobase refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (a) is complementary to thymine (T). For example, in RNA adenine (A) is complementary to uracil (U). In DNA and RNA, guanine (G) is complementary to cytosine (C). In some embodiments, complementary nucleobases refer to nucleobases in an oligomer compound that are capable of base pairing with nucleobases of their target sequences. For example, if a nucleobase at a position in an oligomeric compound is capable of hydrogen bonding with a nucleobase at a position in a target sequence, the hydrogen bonding position between the oligomeric compound and the target sequence is considered to be the complementary position of the nucleobase pair. Nucleobases comprising certain modifications may retain the ability to pair with the corresponding nucleobase and thus still have nucleobase complementarity.
As used herein, "non-complementarity" of nucleobases refers to the fact that hydrogen bonds do not form between a pair of nucleobases.
As used herein, "complementarity" of an oligomeric compound (e.g., linked nucleoside, oligonucleotide) refers to the ability of such oligomeric compound or region thereof to hybridize to a region of interest or the oligomeric compound itself by nucleobase complementarity.
The complementary oligomeric compounds do not necessarily have nucleobase complementarity at each nucleoside. Conversely, some mismatches may be tolerated. In some embodiments, complementary oligomeric compounds or regions are complementary over 70% nucleobases (70% complementary). In some embodiments, the complementarity of the complementary oligomeric compound or region is >80%. In certain embodiments, the complementarity of the complementary oligomer compounds or regions is >90%. In some embodiments, the complementarity of the complementary oligomer compounds or regions is at least 95%. In some embodiments, the complementarity of the complementary oligomer compounds or regions is 100%.
As used herein, "self-complementarity" refers to a compound that an oligomeric compound can fold back upon itself, forming a double strand as a result of nucleobase hybridization of an internal complementary strand region. Depending on the tightness and/or length of the chain region, the compound may form a hairpin loop, junction, bulge or inner loop.
As used herein, "mismatch" refers to the inability of an oligomer compound to base pair with a corresponding position of a target sequence or with the corresponding position of the oligomer compound itself when the oligomer compound is aligned with the target sequence and/or with the self-complementary region of the oligomer compound.
As used herein, "hybridization" refers to pairing of complementary oligomeric compounds (e.g., oligomeric compounds and their target sequences). Although not limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleobases, which may be Watson-Crick, hoogsteen or reverse Hoogsteen hydrogen bonding. As used herein, "specific hybridization" refers to an oligomeric compound that hybridizes with one nucleic acid site with a greater affinity than another nucleic acid site.
As used herein, "fully complementary" refers to each nucleobase of an oligomeric compound or region thereof being capable of pairing with a nucleobase of a complementary nucleic acid target sequence or a self-complementary region of an oligomeric compound. Thus, a perfectly complementary oligomeric compound or region thereof does not contain mismatched or unhybridized nucleobases relative to its target sequence or the self-complementary region of the oligomeric compound.
As used herein, "percent complementary" refers to the percentage of nucleobases in an oligomeric compound that are partially complementary to the same length of the target nucleic acid. The method for calculating the complementarity percentage comprises the following steps: the number of nucleobases in the oligomeric compound that are complementary to nucleobases at the corresponding positions in the target nucleic acid is divided by the total length of the oligomeric compound.
As used herein, "percent identity" refers to the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as the nucleobases in the corresponding position in a second nucleic acid divided by the total number of nucleobases in the first nucleic acid.
As used herein, "modulating" refers to a process in which the number or mass of a molecule, function, or activity is changed compared to the molecule, function, or activity prior to modulation. For example, modulation includes changes in gene expression, which may be increased (stimulation or induction) or decreased (inhibition or reduction).
As used herein, "type of modification" refers to a nucleoside or "type" of nucleoside, meaning chemical modification of a nucleoside, including modified and unmodified nucleosides. Thus, the first and second substrates are bonded together,
Unless otherwise indicated, "nucleoside having a first type of modification" may be an unmodified nucleoside.
As used herein, "different modifications" refers to different chemical modifications or chemical substituents, including no modifications. Thus, for example, MOE nucleosides and unmodified natural RNA nucleosides are "differently modified" even though the natural nucleosides are unmodified. Likewise, DNA and RNA oligonucleotides are also "differently modified," although both are naturally occurring unmodified nucleosides. Identical nucleosides consisting of different nucleobases do not belong to different modified nucleosides. For example, a nucleoside consisting of a 2'-OMe modified glycosyl and an unmodified adenine nucleobase and a nucleoside consisting of a 2' -OMe modified glycosyl and an unmodified thymine nucleobase are not modified differently.
As used herein, "the same type of modification" refers to modifications that are identical to each other, including no modifications. Thus, for example, two unmodified RNA nucleosides have "the same type of modification", even though the two RNA nucleosides are not modified. Nucleosides having the same type of modification may comprise different nucleobases.
As used herein, "region" or "region," or "portion" refers to a plurality of linked nucleosides having a function or feature defined herein, particularly with reference to the claims and definitions provided herein. Typically, these regions or portions comprise at least 10, at least 11, at least 12 or at least 13 linked nucleosides. For example, such a region may comprise 13 to 20 linked nucleosides, such as 13 to 16 or 18 to 20 linked nucleosides. Typically, the first region defined herein consists essentially of 18 to 20 nucleosides, while the second region defined herein consists essentially of 13 to 16 linked nucleosides.
As used herein, "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for use in an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile physiological saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
As used herein, "substituent" and "substituent group" refer to an atom or group that replaces the parent compound atom or group. For example, a substituent of a modified nucleoside refers to any atom or group that is different from the atom or group in the natural nucleoside (e.g., a modified 2 '-substituent refers to any atom or group other than H or OH at the 2' -position of the nucleoside). The substituents may be protected or unprotected. In certain embodiments, the compounds described herein have substituents at one or more positions of the parent compound. The substituents may be further substituted with other substituent groups and may be attached to the parent compound directly or through a linking group such as oxygen or alkyl or hydrocarbyl.
These substituents may exist as modifications to the glycosyl group, particularly substituents present at the 2' -position of the glycosyl group. Groups that may be used as substituents include, but are not limited to, one or more substituents of halo, hydroxy, alkyl, alkenyl, alkynyl, acyl, carboxyl, alkoxy, alkoxyalkylene, and amino, unless otherwise indicated. Certain substituents described herein may be attached directly to the sugar ring (e.g., directly to the halogen and fluorine on the sugar ring), or indirectly to an oxygen-linking atom of the sugar ring, which is itself directly attached to the sugar group (e.g., an alkoxyalkylene and methoxyethylene group attached to the oxygen atom, through the oxygen atom to the 2 '-position of the sugar group, thereby providing the MOE substituent at the 2' -position of the sugar group of the oligonucleotide as described generally).
As used herein, "alkyl" refers to a saturated straight or branched monovalent C1-6 hydrocarbon radical, wherein methyl is the most preferred alkyl substituent at the 2' -position of the sugar molecule. The alkyl group is typically attached to the oxygen linking atom at the 2' position of the sugar, and thus, in general, provides an-alkyl substituent, such as an-OCH 3 substituent, on the sugar molecule of the oligomeric compounds described herein. As will be well understood by those skilled in the art.
As used herein, "olefin" refers to a saturated straight or branched divalent hydrocarbon radical of the formula-C nH2n -wherein n is 1 to 6. Methylene or vinyl are preferred alkylene groups.
As used herein, "alkenyl" refers to a straight or branched unsaturated monovalent C2-6 hydrocarbon group, with vinyl or propenyl being the most preferred alkenyl group at the 2' -position of the sugar molecule. As is well known in the art, the degree of unsaturation of an alkenyl group means that at least one carbon-carbon double bond is present. Alkenyl groups are typically attached to the oxygen linking atom at the 2' -position of the saccharide, thus, in general, providing a-Oalkenyl substituent, such as the-OCH 2CH＝CH2 substituent, on the saccharide molecule of the oligomeric compounds described herein. As will be well understood by those skilled in the art.
As used herein, "alkynyl" refers to a straight or branched chain unsaturated C 2-6 hydrocarbon group, with ethynyl being the most preferred alkynyl substituent at the 2' -position of the glycosyl group. As is well known in the art, the degree of unsaturation of an alkynyl group means that at least one carbon-carbon triple bond is present. Alkynyl groups are typically attached to the oxygen linking atom at the 2' -position of the saccharide, and thus, in general, provide an-alkynyl substituent on the saccharide molecule of the oligomeric compounds described herein. As will be well understood by those skilled in the art.
As used herein, "carboxyl" is a group having the general formula-CO 2 H.
As used herein, "acyl" refers to a group formed by removing a hydroxyl group from a carboxyl group as defined herein, having the general formula-C (O) -X, wherein X is typically a C 1-6 alkyl group.
As used herein, "alkoxy" refers to a group formed between an alkyl group (e.g., a C1-6 alkyl group) and an oxygen atom, wherein the oxygen atom is used to attach the alkoxy group to the parent molecule (e.g., the 2' -position of a sugar molecule) or another group (e.g., an alkylene group as defined herein). Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy and tert-butoxy. Herein, alkoxy groups may optionally include further substituents.
As used herein, alkoxyalkyl refers to an alkoxy group as defined herein that is attached to an alkylene group as defined herein, wherein the oxygen atom of the alkoxy group is attached to the alkylene group and the alkylene group is attached to the parent molecule. The alkylene group is typically attached to the oxygen linking atom at the 2' -position of the saccharide, thus providing a-Oalkylene alkoxy substituent, such as a-OCH 2CH2OCH3 substituent, on the saccharide molecule of the oligomeric compounds described herein as a whole. It will be well understood by those skilled in the art that the MOE substituents, which are generally referred to as defined herein, are also known in the art.
As used herein, "amino" includes primary, secondary and tertiary amino groups.
As used herein, "halo" and "halogen" refer to atoms selected from fluorine, chlorine, bromine and iodine.
As used herein, the term "mxRNA" is to be understood as, inter alia, the term defined in WO2020/044186A2, which is incorporated herein by reference in its entirety. It is also understood that the oligomeric compounds described herein may have one or more non-hybridizing nucleosides (overhangs) and/or one or more internal non-hybridizing nucleosides (mismatches) at one or both strands, provided that there is sufficient complementarity under physiologically relevant conditions to maintain hybridization. In addition, at least one end of the oligomeric compounds described herein may be blunt-ended.
The term "comprising" as used herein means including the determined method steps or elements, but these steps or elements do not include an exclusive list, so that other steps or elements may be present.
Furthermore, if the term "includes" is used in the detailed description or claims, that term is intended to be inclusive in a manner similar to the term "comprising" as "comprising" is interpreted when employed as a transitional word in a claim.
The following exemplary embodiments (items/clauses) are now provided:
1. An oligomeric compound capable of inhibiting APOC3 expression, wherein the compound comprises a first region comprising at least a linked nucleoside, the region having a first nucleobase sequence that is complementary to at least a portion of RNA transcribed from an APOC3 gene, wherein the first nucleobase sequence is selected from the group consisting of: the sequences of tables 1a and 2a (SEQ ID NOs:1 to 391), preferably wherein the moiety has a length of at least 18 nucleosides.
The first region is also referred to as an antisense region and the second region is also referred to as a sense region. As described in the preferred embodiments below, the two regions may be located on the same strand, preferably in an adjacent manner, thereby forming a hairpin molecule, also referred to as mxRNA. Alternatively, the two regions may be located on different strands, which results in double stranded RNA (dsRNA), preferably wherein each strand preferably comprises a corresponding region.
Furthermore, the region may be used as a building block for muRNA (see aspect 1 above). In other words, the first and second regions may be used as muRNA first and third regions, respectively, according to the definition of muRNA below:
The nucleic acid construct (muRNA) comprises at least:
(a) A first nucleic acid portion: at least partially complementary to a first portion of RNA transcribed from the APOC3 gene;
(b) A second nucleic acid portion: a second portion at least partially complementary to RNA transcribed from another gene;
(c) Third nucleic acid portion: at least partially complementary to the first nucleic acid portion of (a) to form a first nucleic acid duplex region; and
(D) Fourth nucleic acid portion: at least partially complementary to said second nucleic acid portion of (b) so as to form a second nucleic acid duplex region therewith.
Preferred embodiments and further aspects related to muRNA are disclosed in WO 2020/065602.
2. The oligomeric compound according to claim 1, further comprising a second region comprising at least a linking nucleoside, the region having a second nucleobase sequence of at least a partially complementary pair to the first nucleobase sequence, the second nucleobase sequence selected from the group consisting of: the sequences of tables 1c and 2c (SEQ ID NOs:401 to 791), preferably the portion has a length of at least 11 nucleosides, or the portion preferably has a length of at least 8, 9, 10 or 11 nucleosides, more preferably at least 10 nucleosides.
3. The oligomeric compound according to item 1 or item 2, the first nucleobase sequence is selected from the following sequences or a portion ：SEQ ID NOs：175,293,262,297,277,366,337,254,274,286,137,149,280,343,225,221,185,373,121,281,331,367,296,28,345,328,339,278,271,212,223,369,276,332,300,341,334,138,193,340,31,167,275,191,336,90,346,219,283,213,23,24,285,347,370,206,282,342,272,303,220,209,29,89,291,117,372,218,368,148,217,128,338,171,94,324 and 299 thereof.
4. The oligomeric compound according to claim 3, wherein the second nucleobase sequence is selected from the following sequences or a portion thereof ：SEQ ID NOs：575,693,662,697,677,766,737,654,674,686,537,549,680,743,625,621,585,773,521,681,731,767,696,428,745,728,739,678,671,612,623,769,676,732,700,741,734,538,593,740,431,567,675,591,736,490,746,619,683,613,423,424,685,747,770,606,682,742,672,703,620,609,429,489,691,517,772,618,768,548,617,528,738,571,494,724 and 699.
5. The oligomeric compound according to any of items 1 to 4, wherein the first nucleobase sequence is selected from the following sequences or a portion ：SEQ ID NOs：277,337,28,343,369,366,274,367,336,332,293,373,280,221,334,286,149,193,328,175,262,254,185,328,271,137,225,167,297 and 191 thereof.
6. The oligomeric compound according to claim 5, wherein the second nucleobase sequence is selected from the group consisting of seq id nos. ：SEQ ID Nos：677,737,428,743,769,766,674,767,736,732,693,773,680,621,734,686,549,593,728,575,662,654,585,728,671,537,625,567,697 and 591.
7. The oligomeric compound according to any of items 1 to 6, the first nucleobase sequence being selected from the following sequences or a part thereof: SEQ ID NOs: 28. 277, 336, 337, 366, 367 and 369, preferably SEQ ID NO:28 or 277, more preferably SEQ ID NO:28. these examples define antisense nucleotide sequences that provide excellent performance. To demonstrate this, please refer to the examples.
8. The oligomeric compound according to claim 7, wherein the second nucleobase sequence is selected from the group consisting of: SEQ ID NOs: 428. 677, 736, 737, 766, 767 and 769, preferably SEQ ID NO:428 or 677, more preferably SEQ ID NO:428.
9. The oligomeric compound according to any of claims 1 to 8, the first region of linked nucleosides consisting essentially of 18 to 35, preferably 18 to 20, more preferably 18 or 19, more preferably 19 linked nucleosides.
10. The oligomeric compound according to any of claims 2 to 9, the second region of linked nucleosides consisting essentially of 11 to 35, preferably 11 to 20, more preferably 13 to 16, yet more preferably 14 or 15, most preferably 14 linked nucleosides; or the second region of linked nucleosides consists essentially of 10 to 35, preferably 10 to 20, more preferably 10 to 16, and more preferably 10 to 15 linked nucleosides.
11. The oligomeric compound according to any of claims 2 to 10, comprising at least one complementary double stranded region comprising at least a portion of a first nucleoside region directly or indirectly linked to at least a portion of a second nucleoside region. Preferably, the double-stranded region has a length of 11 to 19, more preferably 14 or 15 base pairs, most preferably 14 base pairs, wherein the double-stranded region optionally comprises a mismatch; or the compound comprises at least one complementary double-stranded region comprising at least a portion of the first nucleoside region linked directly or indirectly to at least a portion of the second nucleoside region, wherein preferably the double-stranded region is 10to 19 base pairs in length, more preferably 12 to 19 base pairs, more preferably 12 to 15 base pairs, wherein preferably there is a mismatch in the double-stranded region.
12. The oligomeric compound according to claim 11, each of the first and second nucleoside regions having 5 'to 3' directionality therein, thereby defining their 5 'and 3' regions, respectively.
13. The oligomeric compound according to claim 12, wherein the 5 'region of the first nucleoside region is directly or indirectly linked to the 3' region of the second nucleoside region, e.g. by complementary base pairing, and/or wherein the 3 'region of the first nucleoside region is directly or indirectly linked to the 5' region of the second nucleoside region, wherein preferably the 5 'terminal nucleoside of the first nucleoside region is base paired with the 3' terminal nucleoside of the second nucleoside region; or the 5 'region of the first nucleoside region is directly or indirectly linked to the 3' region of the second nucleoside region, such as by complementary base pairing, wherein preferably the 5 'terminal nucleoside of the first nucleoside region base pairs with the 3' terminal nucleoside of the second nucleoside region.
14. The oligomeric compound according to claim 12 or 13, wherein the 3 'region of the first nucleoside region is directly or indirectly linked to the 5' region of the second nucleoside region, wherein preferably the first nucleoside region is directly covalently linked to the second nucleoside region, e.g. by a phosphate, phosphorothioate or phosphorodithioate.
15. The oligomeric compound of any one of items 1 to 14, further comprising one or more ligands.
16. The oligomeric compound according to claim 15, wherein one or more ligands are conjugated to the second nucleoside region and/or the first nucleoside region.
17. Oligomeric compound according to claim 12, item 16, wherein one or more ligands are conjugated in the 3' region, preferably in the 3' end of the second nucleoside region and/or the first nucleoside region and/or in the 5' end of the second nucleoside region.
18. The oligomeric compound according to any of claims 15 to 17, wherein the one or more ligands are any cell-directed molecule, such as a lipid, carbohydrate, aptamer, vitamin and/or peptide that binds to a cell membrane or a cell surface specific target.
19. The oligomeric compound of claim 18, the one or more ligands comprising one or more carbohydrates.
20. The oligomeric compound according to claim 19, wherein the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides or polysaccharides.
21. The oligomeric compound according to claim 20, wherein the one or more carbohydrates comprise or consist of one or more hexose molecules.
22. The oligomeric compound according to claim 21, wherein the one or more hexose molecules are one or more galactose molecules, one or more lactose molecules, one or more N-acetyl-galactosamine molecules and/or one or more mannose molecules.
23. The oligomeric compound according to claim 22, the one or more carbohydrates comprising one or more N-acetyl-galactosamine molecules.
24. The oligomeric compound according to claim 23, comprising two or three N-acetyl-galactosamine molecules, preferably three.
25. The oligomeric compound according to any of claims 15 to 24, the one or more ligands being attached to the oligomeric compound in a linear or branched configuration, preferably to the second nucleoside region thereof.
26. The oligomeric compound of claim 25, wherein the one or more ligands are attached to the oligomeric compound in a two-terminal configuration or a three-terminal configuration.
27. The oligomeric compound according to any of claims 1 to 26, wherein the compound consists of a first region of linked nucleosides and a second region of linked nucleosides.
Each of the regions may constitute a separate strand, thereby producing double-stranded RNA (dsRNA). Particularly preferred dsrnas are dsRNA having a first strand length of 19 nucleotides and a second region length of 14 or 15 nucleotides (preferably 14 nucleotides). When used to define the length of a region or strand, the terms "nucleoside" and "nucleotide" (sometimes abbreviated as "nt") are used interchangeably.
28. The oligomeric compound according to claim 12, wherein the oligomeric compound comprises a single strand consisting of a first nucleoside region and a second nucleoside region, wherein the single strand dimerizes, at least a portion of the first nucleoside region being directly or indirectly linked to at least a portion of the second nucleoside region, thereby forming an at least partially complementary double stranded region.
In other words, the oligomeric compound comprises a single strand consisting of a first nucleoside region and a second nucleoside region, wherein at least a portion of the first nucleoside region is directly or indirectly linked to at least a portion of the second nucleoside region, thereby forming an at least partially complementary double-stranded region.
29. The oligomeric compound according to claim 28, wherein the first nucleoside region has a greater number of linked nucleosides than the second nucleoside region, such that an additional number of linked nucleosides of the first nucleoside region form a hairpin loop linking the first and second nucleoside regions.
Such compounds are also referred to herein as hairpins or mxRNA.
30. The oligomeric compound according to claim 29, wherein a hairpin loop is present in the 3' region of the first nucleoside region.
31. The oligomeric compound of claim 29 or 30, wherein the hairpin loop comprises 4 or 5 linked nucleosides.
Preferably, the first region is 19 nucleosides in length, the second region is 14 nucleosides in length, and the hairpin loop is 5 nucleosides in length, wherein the 5 nucleosides in the hairpin loop are the 5 3' terminal nucleosides of the first region. This molecular structure of hairpin or mxRNA is also referred to herein as "14-5-14".
32. The oligomeric compound according to any of claims 28 to 31, wherein the single strand has a sequence selected from the group consisting of SEQ ID Nos:792 to 803, preferably selected from the group consisting of SEQ ID Nos: 792. 793, 796, 800 and 803, most preferably selected from the group consisting of SEQ ID Nos:796 and 803, in particular SEQ ID NO:803.
33. The oligomeric compound according to claim 32, wherein the single strand is selected from table 3b, in particular from constructs a28 (14-4) mF and a277 (12-5), wherein a28 (14-4) mF is particularly advantageous.
34. The oligomeric compound of any one of claims 1 to 33 comprising internucleoside linkages, wherein at least one internucleoside linkage is a modified internucleoside linkage.
Specific modified internucleoside linkages are the subject of the following examples. Certain modified internucleoside linkages are known in the art, for example, hu et al, signal transduction and targeting therapy (2020) 5:101.
35. The oligomeric compound of claim 34, wherein the modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
36. The oligomeric compound of claim 35 comprising 1 to 15 phosphorothioate or phosphorodithioate internucleoside linkages.
37. The oligomeric compound of claim 36 comprising 7, 8, 9, or 10 phosphorothioate or phosphorodithioate internucleoside linkages.
38. The oligomeric compound of any one of claims 35 to 37 comprising one or more phosphorothioate or phosphorodithioate internucleoside linkages in the 5' region of the first nucleoside region.
39. The oligomeric compound according to any of items 35 to 38, having one or more phosphorothioate or phosphorodithioate internucleoside linkages in the 5' region of the second nucleoside region.
40. The oligomeric compound of any of items 28 and 35 to 39 having phosphorothioate or phosphorodithioate internucleoside linkages between at least two, preferably at least three, more preferably at least four, more preferably at least five adjacent nucleosides of a hairpin loop, depending on the number of nucleosides present in the hairpin loop.
41. The oligomeric compound according to claim 40, wherein phosphorothioate or phosphorodithioate internucleoside linkages between each adjacent nucleoside are present in the hairpin loop.
42. The oligomeric compound according to any of items 1-41, wherein at least one nucleoside comprises a modified sugar.
Preferred modified sugars are the subject of the following examples. Certain modified sugars are known in the art and are described, for example, in Hu et al, signal transduction and Targeted therapy (2020) 5:101.
43. The oligomeric compound according to claim 42, wherein the modifying sugar is selected from the group consisting of a 2' modifying sugar, a Locked Nucleic Acid (LNA) sugar, an (S) -restricted ethyl bicyclic nucleic acid sugar, a tricyclic-DNA sugar, a morpholino, an Unlocked Nucleic Acid (UNA) sugar, and an ethylene Glycol Nucleic Acid (GNA) sugar.
44. The oligomeric compound according to item 43, wherein the 2' modified sugar is selected from the group consisting of a 2' -O-methyl modified sugar, a 2' -O-methoxyethyl modified sugar, a 2' -F modified sugar, a 2' -arabino-fluoro modified sugar, a 2' -O-benzyl modified sugar, and a 2' -O-methyl-4-pyridine modified sugar.
45. The oligomeric compound according to item 44, wherein at least one modified sugar is a 2' -O-methyl modified sugar.
46. The oligomeric compound according to claim 44 or 45, wherein at least one modified sugar is a 2' -F modified sugar.
47. The oligomeric compound according to claim 45 or 46, wherein the sugar is ribose.
48. The oligomeric compound according to any of items 12 and 45 to 48, comprising no 2 '-O-methyl modification of a sugar of a nucleoside at any of positions 2 and 14 downstream of a first nucleoside of the 5' region of the first nucleoside region.
49. The oligomeric compound according to any of items 45 to 48, wherein the sugar of the nucleoside of the second nucleoside region, in particular the sugar at any of positions 9 to 11 downstream of the first nucleoside of the 5 'region of the first nucleoside region, particularly sequences a277 (12-5) and a28 (14-4) mF, does not comprise A2' -O-methyl modification.
50. The oligomeric compound according to any of claims 45-49, wherein the 3 '-terminal position of the second nucleoside region is free of 2' -O-methyl modification.
51. The oligomeric compound according to item 49 or 50, wherein the sugar of the nucleoside at positions 2 and 14 downstream of the first nucleoside of the 5 'region of the first nucleoside region contains a 2' -F modification.
52. The oligomeric compound according to any of claims 49 to 51, wherein the sugar of the nucleoside of the second nucleoside region, in particular the sugar at any of positions 9 to 11 downstream of the first nucleoside of the 5 'region of the first nucleoside region, comprises a 2' -F modification.
53. The oligomeric compound according to item 51 or 52, comprising a 2'-F modification at the 3' -terminal position of the second nucleoside region.
54. The oligomeric compound according to claim 12, any of items 47 to 53, which is modified with one or more of the odd-numbered and/or even-numbered nucleosides starting from the 5' region of the first nucleoside region, wherein the modification of the even-numbered nucleoside is typically a second modification different from the modification of the odd-numbered nucleoside.
55. The oligomeric compound according to claim 54, wherein one or more of the odd numbered nucleosides starting from the 3' region of the second nucleoside region are modified differently than the modification of the odd numbered nucleosides of the first nucleoside region.
56. The oligomeric compound according to claim 54 or 55, wherein one or more even nucleosides starting from the 3' region of the second nucleoside region are modified differently than the even nucleosides of the first nucleoside region of item 55.
57. The oligomeric compound according to any of claims 54 to 56, wherein at least one or more modified even nucleosides of the first nucleoside region are adjacent to at least one or more differently modified odd nucleosides of the first nucleoside region.
58. The oligomeric compound according to any of claims 54 to 57, wherein at least one or more modified even nucleosides of the second nucleoside region are adjacent to at least one or more differently modified odd nucleosides of the second nucleoside region.
59. The oligomeric compound according to any of claims 54 to 58, wherein the sugar of one or more odd numbered nucleosides starting from the 5 'region of the first nucleoside region is a 2' -O-methyl modified sugar.
60. The oligomeric compound according to any of claims 54 to 59, wherein the sugar of one or more of the even nucleosides starting from the 5 'region of the first nucleoside region is a 2' -F modified sugar.
61. The oligomeric compound of any of claims 54 to 60, wherein the sugar of one or more odd numbered nucleosides starting from the 3 'region of the second nucleoside region is a 2' -F modified sugar.
62. The oligomeric compound according to any of claims 54 to 61, wherein one or more of the saccharides of the even numbered nucleosides starting from the 3 'region of the second nucleoside region are 2' -O-methyl modified saccharides.
63. The oligomeric compound according to any of claims 42-62, wherein the sugar of a plurality of adjacent nucleosides of the first nucleoside region is co-modified.
64. The oligomeric compound according to any of claims 42-63, wherein sugars of multiple adjacent nucleosides of the second nucleoside region are co-modified.
65. The oligomeric compound according to claim 31, any of items 54 to 64, wherein saccharides of multiple adjacent nucleosides of a hairpin loop are co-modified.
66. The oligomeric compound of any of claims 63-65, wherein the common modification is a 2' -F modified sugar.
67. The oligomeric compound according to any of items 63-65, the common modification of which is a 2' -O-methyl modified sugar.
68. The oligomeric compound according to claim 67, wherein a plurality of adjacent 2' -O-methyl modified sugars are present in at least eight adjacent nucleosides of the first and/or second nucleoside region.
69. The oligomeric compound according to claim 67, wherein a plurality of adjacent 2' -O-methyl modified sugars are present in three or four adjacent nucleosides of a hairpin loop.
70. The oligomeric compound according to item 29, item 42, wherein the hairpin loop comprises at least one nucleoside having a modified sugar.
71. The oligomeric compound according to claim 70, wherein at least one nucleoside is adjacent to a nucleoside having a different modified sugar.
72. The oligomeric compound according to item 71, wherein the modified sugar is a2 '-O-methyl modified sugar and the different modified sugar is a 2' -F modified sugar.
73. The oligomeric compound of any of claims 1 to 72 comprising one or more nucleosides with an unmodified sugar molecule.
74. The oligomeric compound according to claim 73, wherein an unmodified sugar is present in the 5' region of the second nucleoside region.
75. The oligomeric compound according to claim 29, item 73 or 74, wherein unmodified sugar is present in the hairpin loop.
76. The oligomeric compound according to any of claims 1 to 75, wherein one or more nucleosides of the first and/or second nucleoside region are inverted nucleosides and are linked to adjacent nucleosides by their 3 'carbon of a sugar and the 3' carbon of a sugar of an adjacent nucleoside and/or are linked to adjacent nucleosides by their 5 'carbon of a sugar and the 5' carbon of a sugar of an adjacent nucleoside.
77. The oligomeric compound according to any of claims 1 to 76, having a blunt end.
78. The oligomeric compound according to any of claims 1 to 76, wherein the first or second nucleoside region has overhang.
79. The oligomeric compound according to any of the preceding claims, wherein the first region of the linking nucleoside is selected from table 1b or table 2b, preferably from table 1b having a nucleobase sequence as defined in any of items 3, 5 or 7.
80. The oligomeric compound according to any of the preceding claims, the second region of the linking nucleoside of which is selected from table 1d or table 2d, preferably from table 1b having a nucleobase sequence as defined in any of items 4, 6 or 8.
81. A composition comprising the oligomeric compound of any one of claims 1 to 80 and a physiologically acceptable excipient.
82. A pharmaceutical composition comprising the oligomeric compound of any one of claims 1 to 80.
83. The pharmaceutical composition of claim 82, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant and/or preservative.
84. The pharmaceutical composition of item 82 or 83, wherein the oligomeric compound is the only pharmaceutically active agent thereof.
85. The pharmaceutical composition according to claim 84, which is to be used for the treatment of statin intolerant and/or statin contraindicated patients or individuals.
86. The pharmaceutical composition of claim 82 or 83, further comprising one or more pharmaceutically active agents.
87. The pharmaceutical composition of claim 86, further wherein the pharmaceutically active agent is directed against another oligomeric compound other than an APOC3 target, preferably PCSK9; vascepa; vupanorsen; statin drugs such as rosuvastatin and simvastatin; fibrates, such as fenofibrate; and/or low density lipoprotein cholesterol lowering compounds such as statins and ezetimibe.
88. The pharmaceutical composition of claim 86 or 87, wherein the oligomeric compound and the other pharmaceutically active agent can be administered simultaneously or in any order.
89. The oligomeric compound according to any of claims 1 to 80 for use in human or veterinary medicine or therapy.
90. The oligomeric compound according to any of claims 1 to 80 for use in the treatment, amelioration and/or prophylaxis of a disease or disorder.
91. The compound of claim 90, wherein the disease or disorder is a disease or disorder associated with APOC3, or a disease or disorder in which reduced expression levels of APOC3 are desired, preferably selected from dyslipidemia, including mixed dyslipidemia; hypercholesterolemia, including familial hypercholesterolemia; hypertriglyceridemia, preferably severe hypertriglyceridemia and/or hypertriglyceridemia with triglyceride levels in the blood exceeding 500 mg/dl; inflammation, including low grade inflammation; atherosclerosis; atherosclerotic cardiovascular disease (ASCVD) including Major Adverse Cardiovascular Events (MACE) such as myocardial infarction, stroke and peripheral arterial disease; and pancreatitis, including acute pancreatitis.
92. A method of treating a disease or disorder comprising administering to a subject in need of treatment an oligomeric compound according to any one of claims 1 to 80.
93. The method of claim 92, wherein the oligomeric compound is administered subcutaneously or intravenously.
93. Use of the oligomeric compound of any of items 1 to 80 as a tool for gene function analysis in research.
94. The oligomeric compound of any of claims 1 to 80, when used in the manufacture of a medicament for the treatment of a disease or disorder, the disease or disorder is preferably the same as described in item 91 above.
The effect achieved by the oligomeric compounds due to the use of the oligomeric compounds described herein, APOC3mRNA levels, in particular in liver tissue consisting of human hepatocytes in vitro or predominantly, can be significantly reduced, as shown in the examples disclosed herein. Furthermore, APCO3 protein levels in plasma can also be significantly reduced by using the oligomer constructs described herein, for example in mouse plasma where the liver is composed mainly of human hepatocytes. In particular, these effects can last for a longer time, for example for 6 weeks in mice whose liver is composed mainly of human hepatocytes.
Furthermore, by using the oligomeric compounds described herein, triglyceride levels in serum can be significantly reduced, especially in mice consisting essentially of human hepatocytes, as well as for longer periods of time, such as six weeks. An unexpected and surprising finding is that in addition to reducing the triglyceride content in serum, in particular in serum of the same mouse, the cholesterol content in serum can be significantly reduced at the same time over a longer period of time (e.g. six weeks).
It is also surprising that in certain embodiments, the above benefits can be achieved by using shRNA constructs in the form of oligomeric compounds described herein with a reduced number of fluorine substitutions, e.g., a total of 5 fluorine substitutions, at the respective 2' positions of their ribose units, as compared to conventional shRNA molecules having a series of alternating modifications of 2' -fluorine and 2' -O-methyl groups.
Furthermore, surprisingly, in certain embodiments, the above-described effects can be achieved by using the oligomer compounds as described herein, particularly in the form of shRNA constructs having a shorter length, e.g., 29 linked nucleotides, compared to conventional shRNA molecules. It is also surprising that the sense chain length of this construct is about 10 nucleosides, which also achieves the same effect.
The above effect can be achieved by using a dose of about 10mg/kg body weight to 30mg/kg body weight, particularly for mice.
Structure of oligomeric compounds
The following table lists nucleobase sequences of antisense and sense strands of the oligomeric compounds described herein, as well as the definition of antisense and sense strands of modified oligomeric compounds (symbols include nucleobase sequences, sugar modifications, and, when applicable, modified phosphates).
The symbols used are common symbols in the art and have the following meanings:
A represents adenine;
u represents uracil;
c represents cytosine;
G represents guanine.
P represents a terminal phosphate group, which is preferred, but not indispensable;
m represents a methyl modification at the 2' position of the sugar in the base nucleoside;
f represents a fluorine modification at the 2' position of the sugar of the base nucleoside.
R represents an unmodified (2' -OH) ribonucleoside;
(ps) or # represents phosphorothioate internucleoside linkages;
i represents the reverse linkage between nucleosides, which may be 3'-3' or 5'-5';
vp represents vinyl phosphonate;
mvp represents methyl vinylphosphonate;
3xGalNAc represents trivalent GalNAc.
Sometimes, nucleosides are shown in brackets for ease of reading, in which case they do not represent structural elements or modifications.
The presence of the 5' -terminal phosphate ("P") is optional with respect to the moiety shown. Conversely, if the 5' -terminal phosphate is not shown, its presence is also optional. In general, there is no specific requirement for 5 '-terminal phosphate in compounds supplied to mammalian cells, because in the event of a deletion thereof, mammalian kinases will add 5' -terminal phosphate.
Furthermore, when a notation like "a277 (12-5) mF" is used, "a277" indicates a sequence suitable for RNAi of APOC3, wherein the first digit in parentheses, i.e., 12 in this example, indicates the number of base pairs in the double-stranded region in shRNA, and the second digit in parentheses, i.e., 5 in this example, indicates the number of hairpin loop nucleosides in shRNA. If not specified after the short dash in parentheses, this means that the hairpin loop consists of 5 nucleosides.
Tables 1a to 1d below show the base sequences and sugar-phosphate backbone modifications of the antisense and sense strands of 376 constructs selected according to the examples. From these 376 constructs, 30 preferred oligomeric compounds as disclosed above were selected. The numbers in table 1a are consistent with the numbers of the corresponding entries in the sequence listing. The numbers of table 1c are specified as follows: entry number in the sequence table = entry number in the table +400.
Table 1a: nucleobase sequence of the antisense strand of 376 example constructs:
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Table 1b: nucleobase sequence and sugar-phosphate backbone modification of the antisense strand of 376 example constructs:
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table 1c: nucleobase sequence of sense strand of 376 example constructs:
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Table 1d: nucleobase sequence and sugar phosphate backbone modification of the sense strand of 376 example constructs:
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Tables 2a to 2d show nucleobase sequences and sugar-phosphate backbone modifications of the antisense and sense strands of another 15 exemplary building peptides. For the corresponding entry in the sequence list, the following is specified: entry number +376=entry number in the sequence list in table 2 a; entry number +776=entry number in the sequence list in table 2 c.
Table 2a: nucleobase sequence of the antisense strand of 15 other example constructs:
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table 2b: nucleobase sequence and sugar-phosphate backbone modification of the antisense strand of 15 other example constructs:
# Modification of antisense strand
1 [mU][#][fA][#][mA][#][fC][mU][fC][mA][fG][mA][fG][mA][fA][mC][fU][#][mU][#][fG][#][mU][#][fC][#]rC
2 [mU][#][fU][#][mG][#][fU][mC][fC][mU][fU][mA][fA][mC][fG][mG][fU][#][mG][#][fC][#][mU][#][fC][#]rC
3 [mU][#][fA][#][mA][#][fU][mC][fC][mC][fA][mG][fA][mA][fC][mU][fC][#][mA][#][fG][#][mA][#][fG][#]rA
4 [mU][#][fC][#][mC][#][fU][mU][fG][mG][fC][mG][fG][mU][fC][mU][fU][#][mG][#][fG][#][mU][#][fG][#]rG
5 [mU][#][fC][#][mU][#][fG][mA][fA][mG][fC][mC][fA][mU][fC][mG][fG][#][mU][#][fC][#][mA][#][fC][#]rC
6 [mU][#][fC][#][mA][#][fG][mA][fG][mA][fA][mC][fU][mU][fG][mU][fC][#][mC][#][fU][#][mU][#][fA][#]rA
7 [mU][#][fA][#][mC][#][fU][mC][fA][mG][fA][mG][fA][mA][fC][mU][fU][#][mG][#][fU][#][mC][#][fC][#]rU
8 [mU][#][fG][#][mA][#][fA][mC][fU][mC][fA][mG][fA][mG][fA][mA][fC][#][mU][#][fU][#][mG][#][fU][#]rC
9 [mU][#][fA][#][mC][#][fU][mU][fG][mU][fC][mC][fU][mU][fA][mA][fC][#][mG][#][fG][#][mU][#][fG][#]rC
10 [mU][#][fC][#][mU][#][fC][mA][fG][mA][fG][mA][fA][mC][fU][mU][fG][#][mU][#][fC][#][mC][#][fU][#]rU
11 [mU][#][fU][#][mU][#][fG][mU][fC][mC][fU][mU][fA][mA][fC][mG][fG][#][mU][#][fG][#][mC][#][fU][#]rC
12 [mU][#][fU][#][mC][#][fC][mU][fU][mG][fG][mC][fG][mG][fU][mC][fU][#][mU][#][fG][#][mG][#][fU][#]rG
13 [mU][#][fG][#][mC][#][fU][mC][fC][mA][fG][mU][fA][mG][fU][mC][fU][#][mU][#][fU][#][mC][#][fA][#]rG
14 [mU][#][fC][#][mA][#][fU][mC][fC][mU][fC][mG][fG][mC][fC][mU][fC][#][mU][#][fG][#][mA][#][fA][#]rG
15 [mU][#][fU][#][mG][#][fG][mU][fG][mG][fC][mG][fU][mG][fC][mU][fU][#][mC][#][fA][#][mU][#][fG][#]rU
Table 2c:15 other examples construct nucleobase sequences of the sense strand of the peptide:
# Unmodified sense strand
1 AGUUCUCUGAGUUA
2 ACCGUUAAGGACAA
3 GAGUUCUGGGAUUA
4 AAGACCGCCAAGGA
5 CCGAUGGCUUCAGA
6 GACAAGUUCUCUGA
7 AAGUUCUCUGAGUA
8 GUUCUCUGAGUUCA
9 GUUAAGGACAAGUA
10 CAAGUUCUCUGAGA
11 CCGUUAAGGACAAA
12 AGACCGCCAAGGAA
13 AGACUACUGGAGCA
14 GAGGCCGAGGAUGA
15 AAGCACGCCACCAA
Table 2d: nucleobase sequence and sugar-phosphate backbone modification of the sense strand of 15 other example constructs:
tables 3a and 3b show nucleobase sequences and sugar-phosphate backbone modifications for another 12 example constructs.
Table 3a: nucleobase sequence of a strand of 12 other example constructs:
# Unmodified chain
A277(15) uuggauaggc agguggacuc accugccuau ccaa
A28(15) ucaacaagga guacccgggg guacuccuug uuga
A277(14) uuggauaggc agguggacua ccugccuauc caa
A28(14) ucaacaagga guacccgggg uacuccuugu uga
A277(12-5) uuggauaggc agguggacug ccuauccaa
A277(13-4) uuggauaggc agguggacuu gccuauccaa
A28(14-4) ucaacaagga guacccgggu acuccuuguu ga
A277(14)mF uuggauaggc agguggacua ccugccuauc caa
A28(14)mF ucaacaagga guacccgggg uacuccuugu uga
A277(12-5)mF uuggauaggc agguggacug ccuauccaa
A277(13-4)mF uuggauaggc agguggacuu gccuauccaa
A28(14-4)mF ucaacaagga guacccgggu acuccuuguu ga
Table 3b: nucleobase sequence and sugar-phosphate backbone modification of the strand of 12 other example constructs:
It should also be noted that the scope of the compositions and methods described herein encompasses sequences corresponding to those in the above-described tables, wherein the 5' nucleoside of the antisense (guide) strand (first region defined in the present item) may comprise any nucleobase that may be present in an RNA molecule, i.e., any one of adenine (a), uracil (U), guanine (G) or cytosine (C). In addition, the scope of the compositions and methods encompasses sequences corresponding to those in table 1a or table 1b, wherein the 3 'nucleoside of the sense (guest) strand (the second region defined in the present invention) may comprise any nucleoside that may be present in an RNA molecule, i.e., any one of adenine (a), uracil (U), guanine (G) or cytosine (C), but is preferably a nucleoside complementary to the 5' nucleoside of the antisense (guide) strand (the first region defined in the present invention).
Although the method requires a series of steps to be performed in a particular order, it should be appreciated that the method is not limited by the order. For example, some steps may be performed in a different order than described herein. Furthermore, a certain step may occur simultaneously with another step. Moreover, in some cases, not all steps need be performed to implement the methods described herein.
The order of the steps of the method is exemplary, but may be performed in any suitable order, or simultaneously where appropriate. Furthermore, steps may be added or substituted, or individual steps may be deleted from any of the methods, without departing from the scope of the subject matter described herein. Aspects of any of the examples described above may be combined with aspects of any of the other examples described above to form other examples.
It will be appreciated that the above description of the preferred embodiments is by way of example only, and that various modifications may be made by the skilled artisan. The foregoing description includes examples of one or more embodiments. Of course, every conceivable modification or variation of the above-described compounds, compositions, or methods described herein is not described in detail, but many further modifications and permutations of various aspects are possible by those of ordinary skill in the art. Accordingly, the above-described aspects are intended to embrace all such alterations, modifications and variations that fall within the scope of the appended claims.
Examples
The following examples illustrate certain embodiments of the present disclosure, but are not limiting. Further, in the case where specific embodiments are provided, versatility in such specific embodiments can be considered. For example, disclosure of oligonucleotides having specific motifs or modification patterns provides reasonable support for other oligonucleotides having the same or similar motifs or modification patterns.
The synthesis of the RNAi constructs disclosed herein uses synthetic methods known to those skilled in the art, e.g., in
The synthesis methods disclosed in https:// en.wikipedia.org/wiki/oligonucletide_synthis { 16-day cable at 2022, month 2 }, the methods disclosed on this website are incorporated herein by reference. The only difference from the synthesis disclosed in this reference is that GalNac phosphoramides immobilized on a support are used in the first synthesis step.
Example 1:
Materials and methods
Cell culture:
HepG2 (ATCC cat.8501430) cells were passaged every two weeks in EMEM medium, which included 10% FBS, 20mM L-glutamine, 10mM HEPES pH7.2, 1mM sodium pyruvate, 1 XMEM nonessential amino acids, and 1 XPen/Strep (EMEM complete medium).
APOC3 target recognition and duplex preparation:
By bioinformatic analysis of the human APOC3mRNA sequence, as shown in RefSeq sequence ID nm—000040, it is specifically contemplated that the constructs described herein should target APOC3mRNA, irrespective of splice variants and isoforms. 376 targets were selected for synthesis as asymmetric duplex (14 nucleotide sense strand, 19 nucleotide antisense strand). The compound was dissolved in molecular biology grade water to 50uM, annealed by heating at 95 ℃ for 5 minutes, and then gradually cooled to room temperature.
APOC 3-prescreening:
on the day of transfection, hepG2 cells were collected by pancreatin digestion and counted and plated at a density of 10,000 cells per well onto 96-well tissue culture plates with 50uL of complete EMEM medium containing 20% fbs per well. The cells were allowed to stand for 4 hours by
RNAiMax (ThermoFisher) transfected 2 pmoles of APOC3 double strand, 3 replicates per group. Briefly, 8pmoles of duplex were diluted in 100uL OptiMEM and gently mixed with 0.8uLRNAiMax in 100uLOptiMEM to form 200uL of total complex. 50uL RNAiMax complexes were added to each corresponding set of triplicate wells of HepG2 cells, respectively, and the concentration of double strand in the final mixture was 20nM, 100uL in volume, with EMEM/OptiMEM of 50/50 and FBS of 10%.
After 72 hours of transfection, cells were collected and isolated RNA was extracted using PureLink Pro total RNA purification kit (ThermoFisher, 12173011A) according to the manufacturer's instructions. The collected total RNA was tested for APOC3 expression by TAQMANQPCR using Luna Universal Probe One-Step RT-qPCR kit (NEB, E3006). Two independent qPCR assays were performed for each sample, multiplexed with one common GAPDH VIC probe (thermo fisher, 4326317E) using two independent APOC3-Taqman probe sets. Thermal cycling and data acquisition were performed using Applied Biosystems QuantStudio real-time PCR system. Based on the results of the preliminary screening, a subset of 77 oligomeric compounds was selected that exhibited at least 70% target knockdown when evaluated using either probe. These 77 compounds are defined by items 3 and 4 (clauses) above.
APOC 3-two sieves:
Based on the data from the initial screening, the dose curve for the best performing 30 APOC3 double stranded RNAs was tested. HepG2 cells were collected by trypsinization and seeded into 96-well tissue culture plates at 10,000 cells per well, 50uL of EMEM complete medium containing 10% fbs was added per well and allowed to stand for 4 hours, as before. The total complex was brought to 360uL by gently mixing 36pmoles of each double strand and 2.16uLRNAiMax in 180uLOptiMEM to form a transfected complex. Then a double gradient dilution was performed using the base optmem. 50uL of each dilution was added to each of the three replicate wells of HepG2 cells to a final dilution gradient of 50nM to 0.32nM, 100uL volume, EMEM/OptiMEM ratio of 50/50, containing 10% FBS.
After 72 hours of transfection, cells were collected and RNA was isolated using PureLink Pro total RNA purification kit (ThermoFisher, 12173011A) according to the manufacturer's instructions. The collected total RNA was subjected to detection of APOC3 expression by TAQMANQPCR using Luna Universal Probe One-StepRT-qPCR kit (NEB, E3006). qPCR assays were performed once per sample using APOC3Taqman probe set multiplexed with universal GAPDHVIC probes (thermo fisher, 4326317E). Thermal cycling and data acquisition were performed using Applied Biosystems QuantStudio real-time PCR system.
Example 2:
Results
The IC50 values (in nM) of the 30 constructs in the examples are shown in table 4 below.
Sequence ID K/d% of highest concentration IC50
AP277 93.44 3.29
AP337 93.10 4.10
AP028 90.64 4.53
AP343 93.10 4.70
AP369 90.15 4.86
AP366 95.63 5.56
AP274 89.43 5.89
AP367 88.85 5.99
AP336 92.76 6.13
AP332 90.23 6.35
AP293 84.99 6.44
AP373 89.76 6.46
AP280 78.85 6.71
AP221 92.66 6.84
AP334 90.35 6.85
AP286 83.77 6.89
AP149 90.36 7.77
AP193 91.30 7.83
AP328 87.02 7.85
AP175 94.58 8.28
AP262 84.65 8.72
AP254 90.79 9.11
AP185 88.83 9.20
AP328 88.99 9.44
AP271 78.49 9.49
AP137 86.09 9.79
AP225 81.11 10.74
AP167 84.77 11.13
AP297 84.99 13.28
AP191 84.23 14.27
IC50 values in the bit to two bit nanomolar range suggest that many of the constructs described herein have excellent performance.
Example 3
Materials and methods
Cell culture
Human primary hepatocytes (5 donor pools-Sekisui XenoTech, hpch05+) were thawed prior to the experiment and cultured in 1x complete Williams medium (Gibco, a 1217601) with the addition of hepatocyte proliferation supplements (Gibco, CM 3000). To ensure stability of the compound, the FBS concentration was adjusted to the final 2.5% (instead of 5%) according to the production recipe.
1X complete WEM:2.5% FBS, 1. Mu.M dexamethasone, penicillin/streptomycin (100U/mL/100. Mu.g/mL), 4. Mu.g/mL human insulin, 2mM GlutaMAX,15mMHEPES,pH7.4).
Hepatocytes were cultured on 96-well tissue culture plates (Gibco, a 1142803) containing a collagen I (rat tail) coating.
APOC3 compound preparation:
The compound was dissolved in 10mg/mL PBS, heated at 95 ℃ for 5 minutes for annealing, and then rapidly cooled on ice.
APOC3 complex transfection:
On the day of transfection, primary human hepatocytes were thawed in 45ml human OptiThaw (SekisuiXenotech, K8000) and centrifuged at 200g for 5 min. Cells were resuspended in 2xWEM complete medium and counted. Cells were then cultured in 96-well plates containing rat tail Collagen type 1 at a cell concentration of 25,000 cells per well and 50uL of 2x complete WEM per well and allowed to stand for four hours before transfection to attach to 96-well plates.
The compound was further diluted to 2uM in basal WEM. Seven dilutions were performed in the basal WEM from 2uM to 0.000128uM, five times the dilution gradient. In 100uL1x complete WEM, 50uL of each dilution was added to the corresponding triplicate of vaccinated hepatocytes, with a final dilution series of 1uM to 0.000064uM.
72 Hours after transfection, cells were collected and RNA extraction was performed using PureLink Pro a 96 total RNA purification kit (thermosfisher, 12173011 a) according to the manufacturer's instructions. The extracted RNA was tested for APOC3 expression using Luna Universal Probe RT-qPCR kit (NEB, E3006). qPCR was performed once for each sample using an APOC3Taqman probe set (Hs 00906501 _g1-FAM) and a universal GAPDHVIC probe (ThermoFisher, 4326317E). Thermal cycling and data acquisition were performed using Applied Biosystems QuantStudio/3 real-time PCR system.
Table 5: constructs used as positive controls
Note that: vP = vinyl phosphonate; in=2' oh inversion
Results
As can be seen from FIG. 1a, several variants of both the A28 and A277 structures exhibit excellent activity.
As can be seen from fig. 1b, all molecules have excellent activity.
Example 4
Study protocol
The following protocol entitled "mxRNA guide for male human liver uPA-SCID mouse non-GLP candidate screening study" was drafted before animal experiments and study completion, thus using future time. However, since the above-described studies have been completed entirely, in the following description of the study scheme, each time "in the future" is used, it should be regarded as "in the past".
Purpose of investigation
The purpose of this non-GLP study was to evaluate the dose and duration response effect of two selected mxRNA primers on candidate GALNAC SIRNA constructs targeting APOC3 using the human liver uPA-SCID mouse model. Mice survived 14 days and 42 days after subcutaneous injection of the compound.
Plasma and serum were collected prior to dissection. At dissection, 3 liver biopsies (2 mm) from each animal were stored separately in vials containing RNAlater, snap frozen and stored at-80 ℃.3 more liver biopsies (2 mm) were taken, frozen in the same vial and stored at-80 ℃.
Compliance with
This non-GLP study will not be performed in accordance with the good laboratory specification (GLP) regulations of the U.S. food and drug administration (21 CFR part 58).
Compliance with animal welfare
The procedures described and carried out below will be carried out in accordance with the guidelines for laboratory animal care and use of the animal and plant quarantine agency of the United states department of agriculture, animal welfare and/or standard procedures.
The protocol has been reviewed and approved by the IACUC committee of the experimental facility.
Study schedule
Adaptation/quarantine end date: not less than 5 days
Baseline program date: without baseline program, program start date is day 0, tentative: waiting 12 months for the test material.
Dissection starts: day 14 and day 42 post-treatment.
End of life cycle study: week 6 after treatment
Preliminary report: sponsors do not require, only provide data
Final report release: does not need
Experimental System information
Animal experiment
Generic name: a mouse
Variety/category: rodent-human liver-uPA-SCID mice
Number of animals (by sex): 36 males, all in the juvenile period
Age range: 14-19 weeks
Body weight range: about 20 g
The mice used in this study were human liver-uPA-SCID mice. Approximately 80% of hepatocytes in each mouse were replaced with human hepatocytes. How a technician makes such a mouse; at least some of these methods were described in P.Meuleman and G.Leroux-Roels in anti Res.2008, 12 months; 80 231-8, which is incorporated herein by reference in its entirety.
The adaptation period is as follows:
duration of time:
All animals were subjected to an acclimation period of at least five days prior to return, during which the overall health of the animals was assessed by the attending veterinarian. Animals that were not enrolled received the corresponding treatment and were further evaluated prior to enrolling. All records of the adaptation period will remain in the study archive.
Animal identification method and location:
Animals will be numbered sequentially. The animal's ear will be perforated to permanently identify each animal. Such methods include punching or scoring the auricle under anesthesia. Or tattooing may be performed on the animal tail. Each animal cage will also be attached with a cage card indicating the animal number, sex, supplier, strain, study responsible and study number.
Study design
Design details
Only one mouse, 36 human liver-uPA-SCID mice, was used in this study. Animals will be grouped by treatment type, dose, and survival. Each animal will be treated by subcutaneous injection of the experimental material. Groups 1A and 1B will have four animals receiving a control dose of PBS. Groups 2A, 2B, 2C, 3A, 3B and 3C will receive one dose (10 or 30 mg/kg) of four animals per dose. All animals will survive for 14 or 42 days. See table 6 below for details.
Table 6: study form
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Prior to dissection, the animals will be deeply anesthetized and a peripheral blood sampling is performed through the vena cava. Each animal was collected as much blood as possible, at least 1.2mL, and evenly distributed into serum and plasma separation tubes. After separation (see section 14.10), the serum will be split equally into two separate vials, as will the plasma (see example below).
1.2ML blood = 0.6mL serum and 0.6mL plasma separation tube
Serum (0.3 mL after separation) =0.15mlx2 bottle
Plasma (0.3 mL after separation) =0.15 mL x2 vial.
The serum and plasma samples were labeled, quick frozen and stored at-80 ℃.
Additional blood beyond a minimum volume of 1.2mL will be placed in the serum separation tube, after treatment, serum will be transferred to the labeled vial and the rodent lipid assay will be performed with refrigeration at 4 ℃.
Note that: serum and plasma will be used to measure proteins, taking care to avoid hemolysis or clot formation.
At dissection, three biopsies of 2mm each from left, middle and right lobes were placed in different vials, soaked in RNAlater for 15 minutes, quick frozen and stored at-80 ℃. Three biopsy sections of 2mm were taken from the left, middle and right lobes, placed in a vial, quick frozen and stored at-80 ℃. The remaining livers will be quick frozen and stored in 10mL conical tubes at-80 ℃.
Study design changes
As research progresses, modifications may be made to the present solution. IACUC approval is required for changes to the regimen that may negatively impact the safety of the study or subject.
Animal inclusion and exclusion criteria
Any animals deemed unhealthy in a veterinary pre-test will be excluded from the study and replaced with a backup animal if any. For surviving animals that find death or foment after treatment, replacement can be done by study protocol modifications if there are spare animals.
Animal handling
At the end of the study, animals will be euthanized.
Route of administration
Subcutaneous injection. The injection dose was 200uL.
Results
Figure 3 highlights the dose-response effect of reduced percentage of APOC3mRNA and APOC3 protein levels in plasma in liver tissue of animals treated with different mxRNA constructs as compared to control animals on day 14.
Further, the following is an additional description with respect to fig. 3:
a28 (14-4) mF-10=A28 (14-4) mF 10mg/kg dose group
A28 (14-4) mF-30=A28 (14-4) mF 30mg/kg dose group
A277 (12-5) -10=a277 (12-5) 10mg/kg dose group
A277 (12-5) -30=a277 (12-5) 30mg/kg dose group
Figure 4 highlights the dose-response effect of the average percent reduction in triglycerides and total cholesterol in serum in animals treated with different APOC-3 targeting mxRNA constructs compared to control animals on day 14.
Figures 5a and 5b highlight the sustained effect of average percent reduction in APOC3 mRNA and APOC3 protein levels in plasma in liver tissue of animals treated with different APOC3 targeting mxRNA (10 mg/kg) constructs compared to control animals on day 14 (week 2) and week 6. Further, note that in these figures, one outlier of the a277 (12-5) group has been excluded.
Figures 6a and 6b highlight the sustained effect of average percent reduction in serum Triglycerides (TGs) and Total Cholesterol (TC) on day 14 (week 2) and week 6, compared to control animals, for animals treated with the different APOC3 targeting mxRNA (10 mg/kg) constructs. From these figures, it can be seen that one outlier of the A277 (12-5) group has been excluded.
Summary of results
A28 (14-4) mF APOC3 targeting mxRNA construct:
At week 2, the APOC3mRNA inhibition rate was 88% compared to the control, and was maintained at 78% at week 6.
Plasma APOC3 levels were reduced by 90% compared to the control group at week 6, and maintained at 85% at week 6.
Serum triglyceride levels were reduced by 32% at week 2 and by 41% at week 6 compared to the control group.
At week 2, serum total cholesterol levels were reduced by 43% compared to the control, and maintained at 33% at week 6.
A277 (12-5) APOC3 targeting mxRNA construct:
At week 2, the APOC3mRNA inhibition rate was 56% compared to the control, and was maintained at 42% at week 6.
Plasma APOC3 levels were reduced by 83% compared to the control group at week 6, and maintained at 84% at week 6.
Serum triglyceride levels were reduced by 8% at week 2 and by 52% at week 6 compared to the control group.
At week 2, serum total cholesterol levels were reduced by 36% compared to the control, but this reduction was lost at week 6.
Conclusion(s)
A28 (14-4) the mF construct had excellent activity, and 98% of the target protein was down-regulated after 2 weeks of administration at a dose of 30 mg/kg. In addition, in the case of the optical fiber,
A28 (14-4) the mF construct maintained excellent activity (protein down-regulation) at both weeks 2 and 6 at a dose of 10 mg/kg.
Example 5
Longer observations were made of the effect of compound A28 (14-4) mF (also known as STP 125G) following the protocol detailed in example 4. The outline of this extended study is shown in FIG. 7. The corresponding results are shown in fig. 8a and 8b (mRNA and protein knockouts of APOC3, respectively), and in fig. 9a and 9b (triglyceride and total cholesterol levels).
Several aspects are notable:
Shan Jiliang 10mg/kg was sufficient to knock out mRNA and protein within six weeks, and rebound occurred slowly at the end of the study.
Not only triglycerides (fat levels in the blood are mainly thought to be associated with APOC 3), but also total cholesterol is inhibited.
In evaluating the latter findings, the characteristics of the mice used in the study must be taken into account. Fig. 10 shows that it is estimated that 20% to 25% of cells in the humanized liver remain murine (mouse) cells. A28A (14-4) mF did not target mouse APOC3. Thus, non-silenced mouse APOC3 resulted in observed elevated triglyceride and total cholesterol levels. Thus, the down-regulation of both blood lipids in the human system is expected to exceed the results observed in this study.

Claims (94)

1. An oligomeric compound capable of inhibiting APOC3 expression, said compound comprising a first region comprising at least a linked nucleoside, said region having at least a first nucleobase sequence complementary to at least a portion of RNA transcribed from an APOC3 gene, said first nucleobase sequence selected from the group consisting of: the sequences of tables 1a and 2a (SEQ ID NOs:1 to 391), preferably the portions have a length of at least 18 nucleotides.
2. The oligomeric compound of claim 1, further comprising a second region comprising at least a linking nucleoside, the region having a second nucleobase sequence of at least a partially complementary pair to the first nucleobase sequence, the second nucleobase sequence selected from the group consisting of: the sequences of tables 1c and 2c (SEQ ID NOs:401 to 791), preferably, the moiety has a length of at least 8, 9, 10 or 11 nucleosides, more preferably at least 10 nucleosides.
3. The oligomeric compound according to claim 1 or 2, the first nucleobase sequence being selected from the following sequences or a part ：SEQ ID NOs：175,293,262,297,277,366,337,254,274,286,137,149,280,343,225,221,185,373,121,281,331,367,296,28,345,328,339,278,271,212,223,369,276,332,300,341,334,138,193,340,31,167,275,191,336,90,346,219,283,213,23,24,285,347,370,206,282,342,272,303,220,209,29,89,291,117,372,218,368,148,217,128,338,171,94,324 and 299 thereof.
4. The oligomeric compound of claim 3, wherein the second nucleobase sequence is selected from the group consisting of seq id nos. ：SEQ ID NOs：575,693,662,697,677,766,737,654,674,686,537,549,680,743,625,621,585,773,521,681,731,767,696,428,745,728,739,678,671,612,623,769,676,732,700,741,734,538,593,740,431,567,675,591,736,490,746,619,683,613,423,424,685,747,770,606,682,742,672,703,620,609,429,489,691,517,772,618,768,548,617,528,738,571,494,724 and 699.
5. The oligomeric compound according to any of claims 1-4, wherein the first nucleobase sequence is selected from the group consisting of seq id nos. ：SEQ ID Nos：277,337,28,343,369,366,274,367,336,332,293,373,280,221,334,286,149,193,328,175,262,254,185,328,271,137,225,167,297 and 191, or a portion thereof.
6. The oligomeric compound of claim 5, wherein the second nucleobase sequence is selected from the group consisting of seq id nos. ：SEQ ID Nos：677,737,428,743,769,766,674,767,736,732,693,773,680,621,734,686,549,593,728,575,662,654,585,728,671,537,625,567,697 and 591.
7. The oligomeric compound according to any of claims 1-6, wherein the first nucleobase sequence is selected from the group consisting of: SEQ ID NOs: 28. 277, 336, 337, 366, 367 and 369.
8. The oligomeric compound of claim 7, wherein the second nucleobase sequence is selected from the group consisting of: SEQ ID NOs: 428. 677, 736, 737, 766, 767 and 769.
9. The oligomeric compound according to any of claims 1-8, the first region of linked nucleosides consisting essentially of 18 to 35, preferably 18 to 20, more preferably 18 or 19, more preferably 19 linked nucleosides.
10. The oligomeric compound according to any of claims 2-9, the second region of linked nucleosides consisting essentially of 10 to 35, preferably 10 to 20, more preferably 10 to 16, more preferably 10 to 15 linked nucleosides.
11.The oligomeric compound according to any of claims 2 to 10,which comprises at least one complementary duplex region that comprises at least a portion of said first nucleoside region directly or indirectly linked to at least a portion of said second nucleoside region,wherein optionally said duplex region has a length of 10 to 19,12 to 19,12 to 15 base pairs,or 14 base pairs,wherein optionally there is one mismatch within said duplex region.
The oligomeric compound according to any of claims 2-10, comprising at least one complementary double stranded region comprising at least a portion of a first nucleoside region directly or indirectly linked to at least a portion of a second nucleoside region, wherein preferably the double stranded region is 10 to 19 base pairs in length, more preferably 10 to 19 base pairs, more preferably 12 to 15 base pairs, 12-15 base pairs, or 14 base pairs in length, wherein preferably there is a mismatch within the double stranded region.
12. The oligomeric compound of claim 11, each of said first and second nucleoside regions having 5 'to 3' directionality, thereby defining their 5 'and 3' regions, respectively.
13. The oligomeric compound of claim 12, wherein the 5 'region of the first nucleoside region is directly or indirectly linked to the 3' region of the second nucleoside region, such as by complementary base pairing, wherein preferably the 5 'terminal nucleoside of the first nucleoside region base pairs with the 3' terminal nucleoside of the second nucleoside region.
14. The oligomeric compound of claim 12 or 13, wherein the 3 'region of the first nucleoside region is directly or indirectly linked to the 5' region of the second nucleoside region, wherein preferably the first nucleoside region is directly covalently linked to the second nucleoside region, e.g. by a phosphate, phosphorothioate or phosphorodithioate.
15. The oligomeric compound of any one of claims 1-14 further comprising one or more ligands.
16. The oligomeric compound of claim 15, wherein one or more ligands are conjugated to the second nucleoside region and/or the first nucleoside region.
17. Oligomeric compound according to claims 12 and 16, wherein one or more ligands are conjugated in the 3' region, preferably in the 3' end of the second nucleoside region and/or the first nucleoside region and/or in the 5' end of the second nucleoside region.
18. The oligomeric compound according to any of claims 15-17, the one or more ligands being any cell-directed molecule, such as a lipid, carbohydrate, aptamer, vitamin and/or peptide that binds to a cell membrane or a cell surface specific target.
19. The oligomeric compound of claim 18, the one or more ligands comprising one or more carbohydrates.
20. The oligomeric compound of claim 19, wherein the one or more carbohydrates may be monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides.
21. The oligomeric compound of claim 20, the one or more carbohydrates comprising or consisting of one or more hexose molecules.
22. The oligomeric compound of claim 21, wherein the one or more hexose molecules are one or more galactose molecules, one or more lactose molecules, one or more N-acetyl-galactosamine molecules, and/or one or more mannose molecules.
23. The oligomeric compound of claim 22, the one or more carbohydrates comprising one or more N-acetyl-galactosamine molecules.
24. The oligomeric compound of claim 23 comprising two or three N-acetyl-galactosamine molecules, preferably three.
25. The oligomeric compound according to any of claims 15-24, wherein the one or more ligands are attached to the oligomeric compound in a linear configuration or a branched configuration, preferably to the second nucleoside region thereof.
26. The oligomeric compound of claim 25, wherein the one or more ligands are attached to the oligomeric compound in a two-terminal configuration or a three-terminal configuration.
27. The oligomeric compound of any one of claims 1-26, wherein the compound consists of a first region of linked nucleosides and a second region of linked nucleosides.
28. The oligomeric compound of claim 11, wherein the oligomeric compound comprises a single strand consisting of a first nucleoside region and a second nucleoside region, at least a portion of the first nucleoside region being directly or indirectly linked to at least a portion of the second nucleoside region, thereby forming an at least partially complementary double-stranded region.
29. The oligomeric compound of claim 28 wherein the first nucleoside region has a greater number of linked nucleosides than the second nucleoside region, such that an additional number of linked nucleosides of the first nucleoside region form a hairpin loop linking the first and second nucleoside regions.
30. The oligomeric compound according to claims 12 and 29, wherein a hairpin loop is present in the 3' region of the first nucleoside region.
31. The oligomeric compound of claim 29 or 30, wherein the hairpin loop comprises 4 or 5 linked nucleosides.
32. The oligomeric compound according to any of claims 28-31, wherein the single strand has a sequence selected from the group consisting of SEQ ID NOs 792 to 803,SEQ ID NOs:792, 793, 796, 800 and 803, preferably SEQ ID NOs 796 and 803, or SEQ ID NOs
NO:803。
33. The oligomeric compound of claim 32 wherein the single strand is selected from table 3b, optionally construct a28 (14-4) mF or a277 (12-5).
34. The oligomeric compound of any one of claims 1-33 comprising internucleoside linkages, wherein at least one internucleoside linkage is a modified internucleoside linkage.
35. The oligomeric compound of claim 34, wherein the modified internucleoside linkage is a phosphorothioate or phosphorodithioate internucleoside linkage.
36. The oligomeric compound of claim 35 comprising 1 to 15 phosphorothioate or phosphorodithioate internucleoside linkages.
37. The oligomeric compound of claim 36 comprising 7, 8, 9, or 10 phosphorothioate or phosphorodithioate internucleoside linkages.
38. The oligomeric compound of claim 12 and any of claims 35-37 comprising one or more phosphorothioate or phosphorodithioate internucleoside linkages in the 5' region of the first nucleoside region.
39. The oligomeric compound of claim 12 and any of claims 35-38 having one or more phosphorothioate or phosphorodithioate internucleoside linkages in the 5' region of the second nucleoside region.
40. The oligomeric compound of claim 29 and any of claims 35-39 having phosphorothioate or phosphorodithioate internucleoside linkages between at least two, preferably at least three, more preferably at least four, more preferably at least five adjacent nucleosides of a hairpin loop, depending on the number of nucleosides present in the hairpin loop.
41. The oligomeric compound according to claim 40, wherein phosphorothioate or phosphorodithioate internucleoside linkages between each adjacent nucleoside are present in the hairpin loop.
42. The oligomeric compound according to any of claims 1-41, wherein at least one nucleoside comprises a modified sugar.
43. The oligomeric compound of claim 42, wherein said modified sugar is selected from the group consisting of a 2' modified sugar, a Locked Nucleic Acid (LNA) sugar, an (S) -restricted ethyl bicyclic nucleic acid sugar, a tricyclic-DNA sugar, a morpholino group, an Unlocked Nucleic Acid (UNA) sugar, and an ethylene Glycol Nucleic Acid (GNA) sugar.
44. The oligomeric compound according to claim 42, wherein the 2' modified sugar is selected from the group consisting of a 2' -O-methyl modified sugar, a 2' -O-methoxyethyl modified sugar, a 2' -F modified sugar, a 2' -Arabic-fluoro modified sugar, a 2' -O-benzyl modified sugar, and a 2' -O-methyl-4-pyridine modified sugar.
45. The oligomeric compound according to claim 44, wherein at least one modified sugar is a 2' -O-methyl modified sugar.
46. The oligomeric compound of claim 44 or 45 wherein at least one modified sugar is a 2' -F modified sugar.
47. The oligomeric compound of claim 45 or 46 wherein the sugar is ribose.
48. The oligomeric compound of claim 12 and any of claims 45-47 comprising no 2 '-O-methyl modification of the sugar of the nucleoside at any one of positions 2 and 14 downstream of the first nucleoside of the 5' region of the first nucleoside region.
49. The oligomeric compound according to claim 12 and any of claims 45-48, wherein the sugar of the nucleoside of the second nucleoside region, in particular the sugar at any of positions 9 to 11 downstream of the first nucleoside of the 5 'region of the first nucleoside region, particularly sequences a277 (12-5) and a28 (14-4) mF, does not comprise A2' -O-methyl modification.
50. The oligomeric compound according to claim 49, wherein the sugar of the nucleoside at positions 2 and 14 downstream of the first nucleoside of the 5 'region of the first nucleoside region comprises a 2' -F modification.
51. The oligomeric compound according to any of claims 49-51, wherein the sugar of the nucleoside of the second nucleoside region corresponds in position to 1,2 or any nucleoside downstream of position 9 to 11 of the first nucleoside of the 5 'region of the first nucleoside region, containing a 2' -F modification.
52. The oligomeric compound according to claim 50 or 51, wherein the 3 '-terminal position of the second nucleoside region comprises a 2' -F modification.
53. The oligomeric compound according to claim 12 and any of claims 47-52, which is modified with one or more of the odd-numbered and/or even-numbered nucleosides starting from the 5' region of the first nucleoside region, wherein the modification of the even-numbered nucleoside is typically a second modification that is different from the modification of the odd-numbered nucleoside.
54. The oligomeric compound according to claim 53, wherein one or more of the odd numbered nucleosides starting from the 3' region of the second nucleoside region are modified differently than the modification of the odd numbered nucleosides of the first nucleoside region.
55. The oligomeric compound of claim 53 or 54 having one or more even nucleosides starting from the 3' region of the second nucleoside region modified differently than the even nucleosides of the first nucleoside region of claim 54.
56. The oligomeric compound according to any of claims 53-55, wherein at least one or more modified even nucleosides of a first nucleoside region are adjacent to at least one or more differently modified odd nucleosides of the first nucleoside region.
57. The oligomeric compound according to any of claims 53-56, wherein at least one or more modified even nucleosides of the second nucleoside region are adjacent to at least one or more differently modified odd nucleosides of the second nucleoside region.
58. The oligomeric compound according to any of claims 53-57, wherein the sugar of one or more odd numbered nucleosides starting from the 5 'region of the first nucleoside region is a 2' -O-methyl modified sugar.
59. The oligomeric compound according to any of claims 53-58, wherein one or more of the saccharides of the even numbered nucleosides starting from the 5 'region of the first nucleoside region are 2' -F modified saccharides.
60. The oligomeric compound of any of claims 53-60 wherein the sugar of one or more odd numbered nucleosides starting from the 3 'region of the second nucleoside region is a 2' -F modified sugar.
61. The oligomeric compound according to any of claims 53-61, wherein one or more of the saccharides of the even numbered nucleosides starting from the 3 'region of the second nucleoside region are 2' -O-methyl modified saccharides.
62. The oligomeric compound according to any of claims 42-61, wherein the sugar of a plurality of adjacent nucleosides of the first nucleoside region is co-modified.
63. The oligomeric compound of any of claims 42-62 wherein the sugar of a plurality of adjacent nucleosides of the second nucleoside region is co-modified.
64. The oligomeric compound according to any of claims 29 and 53-63, wherein the saccharides of multiple adjacent nucleosides of a hairpin loop are co-modified.
65. The oligomeric compound of any of claims 62-64 having a common modification of a 2' -F modified sugar.
66. The oligomeric compound of any of claims 62-64 having a common modification of a 2' -O-methyl modified sugar.
67. The oligomeric compound according to claim 66, wherein a plurality of adjacent 2' -O-methyl modified sugars are present in at least eight adjacent nucleosides of the first and/or second nucleoside region.
68. The oligomeric compound according to claim 66, wherein a plurality of adjacent 2' -O-methyl modified sugars are present in three or four adjacent nucleosides of a hairpin loop.
69. The oligomeric compound according to claim 29 and claim 42, wherein the hairpin loop comprises at least one nucleoside having a modified sugar.
70. The oligomeric compound of claim 69, at least one nucleoside of which is adjacent to a nucleoside having a different modified sugar.
71. The oligomeric compound of claim 70 wherein the modified sugar is a 2 '-O-methyl modified sugar and the different modified sugar is a 2' -F modified sugar.
72. The oligomeric compound of any of claims 1-71 comprising one or more nucleosides with unmodified sugar molecules.
73. The oligomeric compound according to claim 72, wherein unmodified sugar is present in the 5' region of the second nucleoside region.
74. The oligomeric compound of claim 29 and claim 72 or 73, wherein unmodified sugar is present in the hairpin loop.
75. The oligomeric compound of any of claims 1-74 wherein one or more nucleosides of the first nucleoside region and/or the second nucleoside region are inverted nucleosides and are linked to an adjacent nucleoside by a3 'carbon of its sugar and a 3' carbon of a sugar of an adjacent nucleoside and/or are linked to an adjacent nucleoside by a 5 'carbon of its sugar and a 5' carbon of a sugar of an adjacent nucleoside.
76. The oligomeric compound of any of claims 1-75, having a blunt end.
77. The oligomeric compound of any of claims 1-75, wherein either the first nucleoside region or the second nucleoside region has overhang.
78. The oligomeric compound according to any of the preceding claims, wherein the first region of the linking nucleoside is selected from table 1b or table 2b, preferably from table 1b having a nucleobase sequence as defined in any of claims 3, 5 or 7.
79. The oligomeric compound according to any of the preceding claims, the second region of the linking nucleoside of which is selected from table 1d or table 2d, preferably from table 1b having a nucleobase sequence as defined in any of claims 4, 6 or 8.
80. A composition comprising the oligomeric compound of any one of claims 1-79 and a physiologically acceptable excipient.
81. A pharmaceutical composition comprising the oligomeric compound of any one of claims 1-79.
82. The pharmaceutical composition of claim 81, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant and/or preservative.
83. The pharmaceutical composition of claim 81 or 82, wherein the oligomeric compound is the only pharmaceutically active agent thereof.
84. The pharmaceutical composition according to claim 83, to be used in the treatment of statin intolerant and/or statin contraindicated patients or individuals.
85. The pharmaceutical composition of claim 81 or 82, further comprising one or more pharmaceutically active agents.
86. The pharmaceutical composition of claim 85, further wherein the pharmaceutically active agent is directed against another oligomeric compound other than an APOC3 target, preferably PCSK9; vascepa; vupanorsen; statin drugs such as rosuvastatin and simvastatin; fibrates, such as fenofibrate; and/or low density lipoprotein cholesterol lowering compounds such as statins and ezetimibe.
87. The pharmaceutical composition of claim 85 or 86, wherein the oligomeric compound and the additional pharmaceutically active agent can be administered simultaneously or in any order.
88. The oligomeric compound according to any of claims 1-79 for use in human or veterinary medicine or therapy.
89. The oligomeric compound according to any of claims 1-79 for use in the treatment, amelioration and/or prevention of a disease or disorder.
90. The compound of claim 89, wherein the disease or disorder is a disease or disorder associated with APOC3, or a disease or disorder in which reduced expression levels of APOC3 are desired, preferably selected from dyslipidemia, including mixed dyslipidemia; hypercholesterolemia, including familial hypercholesterolemia; hypertriglyceridemia, preferably severe hypertriglyceridemia and/or hypertriglyceridemia with triglyceride levels in the blood exceeding 500 mg/dl; inflammation, including low grade inflammation; atherosclerosis; atherosclerotic cardiovascular disease (ASCVD) including Major Adverse Cardiovascular Events (MACE) such as myocardial infarction, stroke and peripheral arterial disease; and pancreatitis, including acute pancreatitis.
91. A method of treating a disease or disorder comprising administering to a subject in need of treatment an oligomeric compound according to any of claims 1-79.
92. The method of claim 91, wherein the oligomeric compound is administered subcutaneously or intravenously.
93. Use of the oligomeric compound of any of claims 1-79 as a tool for gene function analysis in research.
94. Use of an oligomeric compound of any of claims 1-79 in the manufacture of a medicament for treating a disease or disorder.
CN202280055021.6A 2021-06-24 2022-06-24 Products and compositions Pending CN117957321A (en)

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