Products and compositions

Abstract

The present invention provides nucleic acid products that interfere with or inhibit expression of APOC3 and AGT genes, and methods of treating subjects using the nucleic acids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 434,688, filed on December 22, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing submitted electronically in ST.26 (XML) format, the entire contents of which are incorporated herein by reference. The XML file was created on December 6, 2023, is named 4690_0082I_SL.xml, and is 505,772 bytes in size.

[0005] field

[0006] The present disclosure relates to products, compositions, and methods of making and using the same. Specifically, the present disclosure relates to nucleic acid products that modulate (particularly interfere with or inhibit) the expression of APOC3 and AGT genes. Thus, the disclosed embodiments may include methods for reducing the expression of APOC3 and AGT mRNA and protein in humans or animals. The disclosed embodiments also include methods for treating, preventing, and / or ameliorating diseases associated with APOC3 and AGT.

[0007] background

[0008] Apolipoprotein 3 (APOC3)

[0009] Triglycerides are the primary form of fat stored in the human body, providing energy when needed. Each triglyceride molecule consists of a glycerol backbone esterified with three fatty acids. Triglycerides can be consumed directly from the diet or produced by the body from excess carbohydrates and other nutrients and transported to various tissues via the bloodstream. However, to survive the aqueous environment of the bloodstream, triglycerides must be transported within lipoproteins. Lipoproteins are spherical groups of molecules whose outer shell protects the triglyceride cargo, enabling it to be delivered to tissues for storage or breakdown for energy production.

[0010] Hypertriglyceridemia (elevated levels of triglycerides in the blood) is a recognized disease in its own right and is a causative factor or contributory factor in the development or progression of a range of diseases. For example, in part due to its association with elevated low-density lipoprotein ("LDL") cholesterol levels, hypertriglyceridemia is associated with the development of atherosclerotic cardiovascular disease ("ASCVD") and related metabolic and other diseases.

[0011] A more comprehensive list of diseases associated with elevated triglyceride levels is given in the Examples disclosed further below.

[0012] Apolipoprotein C3 ("APOC3") is secreted by the liver and small intestine and is present on the surface of triglyceride-rich lipoproteins (e.g., very low-density lipoproteins), such as low-density lipoproteins (VLDL) and chylomicrons. APOC3 is involved in the negative regulation of lipid catabolism, especially triglyceride catabolism, and the clearance of very low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) from the blood.

[0013] One of APOC3's functions is to inhibit lipoprotein lipase and hepatic lipase, two enzymes involved in breaking down triglycerides into glycerol and free fatty acids, leading to elevated triglyceride levels. APOC3 dysregulation can lead to chronically elevated triglyceride levels in the blood, potentially triggering or signaling cardiovascular disease, including vascular inflammation.

[0014] Angiotensinogen (AGT)

[0015] The AGT gene encodes angiotensinogen, which is an upstream component of the renin angiotensin-aldosterone system (RAAS) that regulates blood pressure and the balance of body fluids and electrolytes. Angiotensinogen is mainly secreted by the liver and is a precursor of angiotensin peptides, angiotensin I and angiotensin II. Angiotensinogen is converted into angiotensin I by active renin, and then into angiotensin II by angiotensin converting enzyme (ACE) (Gribouval et al., Human Mutation (Hum. Mutat.) 2012, 33:316-26). Angiotensin II is a peptide hormone that causes vasoconstriction, leading to increased blood pressure. Angiotensin II also stimulates the production of aldosterone, which promotes the absorption of salt and water by the kidneys. The increase in body fluids also leads to increased blood pressure.

[0016] Hypertension and related diseases may be caused by dysregulation of RAAS and AGT gene expression. Dysregulation of angiotensin II, particularly excessive angiotensin II production, can lead to hypertension, increase oxidative stress, and promote inflammation, hypertrophy, and fibrosis in the heart, kidneys, and arteries, ultimately leading to left ventricular fibrosis, arterial remodeling, and glomerulosclerosis.

[0017] Hypertension is the most prevalent and controllable disease in developed countries, affecting 20-50% of the adult population. It is a major risk factor for numerous diseases, disorders, and health problems, including chronic kidney disease, stroke, myocardial infarction, heart failure, conditions such as aneurysms (e.g., aortic aneurysm), peripheral arterial disease, heart damage (e.g., cardiomegaly or hypertrophy), and other cardiovascular-related diseases, disorders, or conditions. In 2017, guidelines for the diagnosis, prevention, and treatment of hypertension were revised to advocate for further lowering of blood pressure to reduce the risk of developing these conditions (e.g., see Reboussin et al. 2017 ACC / AHA / AAPA / ABC / ACPM / AGS / APhA / ASH / ASPC / NMA / PCNA Guideline for the Prevention, Detection, Evaluation, and Management of Hypertension in Adults: A Report of the American College of Cardiology / American Heart Association Task Force on Clinical Practice Guidelines. Journal of the American College of Cardiology, 2017 Nov 7. pii:S0735-1097(17):41517-8. doi:10.1016 / j.jacc.2017.11.004; and Wheeldon et al. (Id. at 41519-1. doi:10.1016 / j.jacc.2017.11.006).

[0018] Dysregulated expression of APOC3 and AGT genes leads to concurrent diseases

[0019] Hypertension and other AGT-related diseases may also co-occur with APOC3-related diseases, such as dyslipidemia, more specifically hypertriglyceridemia, hyperchylomicronemia, and ASCVD.

[0020] Treatment of Hypertriglyceridemia and Hypertension Currently, common treatments for hypertriglyceridemia include statins, such as rosuvastatin and simvastatin, and fibrates, such as fenofibrate. Statins are not suitable for some patients due to their side effects and intolerance.

[0021] Despite the availability of a wide range of antihypertensive medications for the treatment of hypertension, more than two-thirds of participants require multiple antihypertensive medications from different classes. The increased number and variety of medications can lead to increased side effects, which can reduce patient adherence to the medication regimen (and blood pressure control). Furthermore, some studies have suggested a potential association between long-term antihypertensive medication use and worsening renal function, with studies finding that antihypertensive medications used to control blood pressure can also affect renal function independently of their effects on blood pressure (Tomlinson et al. (2013) PLoS ONE 8(11) Article ID e78465; SPRINT Study Group (2015) NEJ1U373(22):2103-2116, ClinicalTrials.gov ID, NCT01206062; Kidney Disease: Improving Global Outcomes (KDIGO) CKD Working Group (2013) Kidney Int Suppl 3:1-150; Kamaroff et al. (2018) Hindawi International J Chron Dis Article ID 1382705 I https: / / doi.Org / 10.l155 / 2018 / 1382705). In addition to antihypertensive medications, renal denervation, baroreceptor activation therapy, dietary changes, and lifestyle modifications may reduce hypertension and its associated diseases (Paulis et al., Nat Rev Cardiol, 2012, 9:276-285). Currently approved treatments for hypertension are limited because a considerable proportion of hypertensive patients fail to achieve adequate blood pressure control. Drugs such as angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) that target part of the RAAS pathway have limited effectiveness in inhibiting this pathway (Nobakht et al., Nature Reviews Nephrology, 2011, 7:356-359). In addition, certain antihypertensive drugs, such as angiotensin-converting enzyme (ACE) inhibitors, are contraindicated in hypertensive patients with kidney disease because they may impair renal function.

[0022] Therefore, there is a need to provide improved treatments for hypertriglyceridemia and / or hypertension and the conditions that may arise therefrom.

[0023] Summary

[0024] The following aspects are non-limiting.

[0025] According to a first aspect, the present invention relates to a nucleic acid construct.

[0026] composition:

[0027] a first nucleic acid portion that is at least partially complementary to at least a first portion of RNA transcribed from the APOC3 gene;

[0028] a second nucleic acid portion that is at least partially complementary to at least a second portion of RNA transcribed from the AGT gene;

[0029] (c) is a third nucleic acid portion that is at least partially complementary to the first nucleic acid portion of (a) so as to form a first nucleic acid duplex region therewith;

[0030] (d) a fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion of (b) so as to form a second nucleic acid duplex region therewith.

[0031] According to a second aspect, the present disclosure relates to a composition comprising the nucleic acid construct according to the first aspect, and a physiologically acceptable excipient.

[0032] According to a third aspect, the present disclosure relates to a pharmaceutical composition comprising the nucleic acid construct according to the first aspect.

[0033] According to a fourth aspect, the present disclosure relates to a nucleic acid construct according to the first aspect, for use in human or veterinary medicine or therapy.

[0034] According to a fifth aspect, the present disclosure relates to a nucleic acid construct according to the first aspect, for use in a method for treating, ameliorating and / or preventing a disease or disorder.

[0035] According to a sixth aspect, the present invention relates to a method for treating a disease or disorder comprising administering a nucleic acid construct according to the first aspect to a subject in need of treatment.

[0036] According to a seventh aspect, the present disclosure relates to the use of the nucleic acid construct according to the first aspect for use in research as a tool for gene function analysis. According to an eighth aspect, the present disclosure relates to the use of the nucleic acid construct according to the first aspect in the manufacture of a medicament for treating a disease or condition. According to an eighth aspect, the present disclosure relates to the use of the nucleic acid construct according to the first aspect in the preparation of a medicament for treating a disease or disorder. Advantageous and / or exemplary features of the construction according to the present invention are as follows:

[0037] 1) They contain multiple (2 or more) at least partially double-stranded factors capable of triggering RNA interference, bound together into a single nanostructure primarily through complementary (Watson-Crick) interactions;

[0038] 2) Optionally, the constructs can be constructed using other (e.g., covalent binding) and / or various ligands can be added (e.g., delivery / targeting moieties such as GalNAc and / or other carbohydrates, cholesterol, peptides, or small molecules, optionally connected via a linker);

[0039] 3) The construct primarily contains chemically modified nucleotides (e.g., 2'F, 2'OMe, LNO, PNA, MOE, BNA, PMO, phosphorothioate, phosphorodithioate, etc.), primarily (but not exclusively) to increase resistance to nucleases;

[0040] 4) These constructs contain "fragile" components (e.g., chemical linkers, unmodified nucleotides, etc.), which cause the constructs to break down when exposed to certain biological environments (e.g., exposure to extracellular and / or intracellular fluids); specific examples may include (but are not limited to): a) cleavage of the oligonucleotide backbone by nucleases at the site of unmodified nucleotides; b) cleavage of chemical bonds due to changes in pH (e.g., in endosomes);

[0041] 5) decomposes and releases components (e.g., the at least partially double-stranded factor capable of triggering RNA interference) upon exposure to the specific biological environment to regulate (upregulate or downregulate, advantageously downregulate) the expression of the target gene in the cell / organism;

[0042] 6) The constructs can be used to modulate, advantageously downregulate or silence gene expression, study gene function, or treat various diseases associated with the target gene to be downregulated.

[0043] Effects achieved by the disclosed nucleic acid constructs

[0044] The nucleic acid construct of the present invention, comprising an APOC3-targeting antisense strand and an AGT-targeting antisense strand, can simultaneously reduce the expression of APOC3 and AGT in an effective manner.

[0045] Furthermore, it has been demonstrated that antisense strands against APOC3 in the form of mxRNA (miniature short hairpin structures; see below for a more comprehensive definition) perform surprisingly well when part of the muRNA nucleic acid constructs disclosed herein (interfering RNA comprising sequences directed against different target mRNAs and / or different regions of a given target mRNA; see below for a more comprehensive definition).

[0046] Advantageously, certain differences may exist between mxRNA and muRNA, including (i) sequence length, particularly the length of the sense strand, and (ii) modifications, including 2' modifications and phosphate modifications.

[0047] In terms of length, the preferred design for mxRNAs is 14-5-14. This means the antisense sequence is 14 + 5 = 19 nucleotides long, with the five 3'-terminal nucleotides forming the loop in the mxRNA. Advantageously, the sense strand is only 14 nucleotides long. The total length of this type of mxRNA is 14 + 5 + 14 = 33 nucleotides.

[0048] In contrast, a preferred muRNA design is 15-4-15. This means that on the first strand of the muRNA, there is an antisense sequence directed against a region of the first target mRNA that is 15+4=19 nucleotides long, and a sense sequence directly adjacent to it that is identical to a region of a second target mRNA or a different region of the same target mRNA, with the sense sequence preferably being 15 nucleotides in length. Mutatically, the same applies to the second strand of the muRNA, which contains an antisense sequence directed against a second mRNA target or a different region of the same target mRNA, and a sense sequence that is complementary to the antisense sequence on the first strand of the muRNA. In terms of total length, each strand of this preferred muRNA has a length of 15+4+15=34 nucleotides.

[0049] Therefore, if both mxRNA antisense and sense sequences are to be used in a muRNA, the antisense sequence can be used without modifying its nucleobase sequence. Regarding the sense sequence, given the preference for 14-nucleotide sense sequences in mxRNAs, it is best to add an extra nucleotide to the sense sequence when using an mxRNA as the basis for designing a 15-4-15 muRNA. Specifically, the extra nucleobase at position 15 must be added to the 5' end of the sense sequence and must be complementary to the nucleobase at position 15 of the antisense sequence directed to the same target mRNA or region thereof. Positions are always calculated in a 5' to 3' direction.

[0050] Regarding modifications, the following points are worth noting. Modification at the 2' position is a means of stabilizing the compounds of the disclosed embodiments (including muRNAs) and mxRNAs (which can serve as a basis for muRNA design). The same applies to phosphorothioates, which are also a stabilization method, and are particularly suitable for regions of mxRNAs and muRNAs that do not participate in base pairing or are located at the ends or termini of their respective chains. Simultaneously, the design of muRNAs also facilitates their decomposition in the cellular environment, producing two different short RNAs that can be loaded separately into the RISC complex. In the aforementioned muRNAs, a favorable position for achieving this decomposition is position 19 of the corresponding antisense sequence. Therefore, when designing a muRNA based on an mxRNA, if the nucleoside at position 19 of the mxRNA antisense sequence is a 2'-modified nucleoside, this modification can be omitted, resulting in a 2'-unmodified nucleoside with an OH group at the 2' position. Otherwise, for the muRNAs of the disclosed embodiments, it is advantageous to use an alternating pattern of 2'-F and 2'-OMe modifications, starting with a 2'-OMe modification at the 5' end.

[0051] As for phosphorothioates, they typically occur at the first two internucleoside junctions at either end of any strand, as well as between ring nucleosides (in the case of mxRNA) or nucleosides in the bulge (in the case of muRNA). Thus, phosphorothioates can be used to link nucleosides 14 to 19 in an mxRNA, for a total of six phosphorothioates in the ring of the designed mxRNA. As described above, because the particularly preferred muRNA follows the 15-4-15 design described above, the positions of phosphorothioates in the central region of each strand of the muRNA may differ from those in the parental mxRNA ring, which preferably follows a 14-5-14 design.

[0052] Specifically,

[0053] The dominant position of phosphorothioate in the central part of muRNA is at positions 15 to 18 of each strand of the muRNA. Since muRNA is cleaved after administration, the newly formed (unformed) ends of the cleavage products are also advantageously protected. For this reason, not only the nucleosides at the muRNA bulge (positions 15 to 18), but also the two nucleosides after the fragile nucleotide at position 19 (the two nucleosides are located at positions 20 and 21) are also linked by phosphorothioate.

[0054] When referring to the position of the phosphorothioate, it is understood that a phosphorothioate at position n means that the phosphorothioate is linked to the nucleosides at positions n and n+1 in a 3' to 5' linkage.

[0055] In summary, while components of mxRNA can be used in the context of muRNA, the aforementioned modifications are advantageous. As shown in the examples, these modifications do not compromise activity. Thus, the performance of an antisense region in the context of mxRNA is surprisingly predictive of its performance in the context of muRNA.

[0056] In summary, the nucleic acid construct according to the present invention can solve the above technical problems, as evidenced by the examples.

[0057] Furthermore, it was surprisingly discovered that the aforementioned effects can be achieved by using oligomeric compounds according to the disclosed embodiments to inhibit the expression of APOC3 and AGT genes in the form of microRNA (muRNA) constructs having a reduced number of nucleosides (e.g., 34 nucleosides per strand) compared to conventional small interfering RNA (siRNA) molecules, which, considering that two conventional siRNAs must be used to inhibit two target genes, have a greater total number of nucleosides. This difference can, for example, make the synthesis of muRNA molecules more cost-effective and more efficient because fewer units are required.

[0058] For certain oligomeric compounds in the form of muRNA constructs for inhibiting the expression of the APOC3 and AGT genes, it was surprisingly found that the above effects could be achieved by using a short sense strand in the muRNA that is 14 or 15 nucleosides in length, which is shorter than the sense strand length in conventional siRNA molecules.

[0059] The effects and technical advantages obtained by inhibiting the expression of APOC3 and AGT using the oligomeric compounds of the present invention will become more apparent in the detailed description and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 The concentration dependence of AGT inhibition in vitro is demonstrated for constructs of the present disclosure as well as a construct targeting an unrelated mRNA (TMPRSS6; negative control).

[0062] Figure 2 The concentration dependence of APOC3 inhibition in vitro is demonstrated for the constructs of the present disclosure as well as a construct targeting an unrelated mRNA (TMPRSS6; negative control).

[0063] Figure 3(a) and 3(b) The constructs of the present disclosure demonstrate knockdown of AGT mRNA in liver tissue: Figure 3a : The percentages of the PBS group were expressed according to the dose; Figure 3b : Knockdown of AGT mRNA in liver tissue according to the dose.

[0064] Figure 4 (a) and (b) show the knockdown of APOC3 mRNA in liver tissue by the constructs of the present disclosure: Figure 4a : The percentages of the PBS group were expressed according to the dose; Figure 4b : Knockdown of APOC3 mRNA in liver tissues by dose.

[0065] Figure 5 (a) and (b) show the knockdown of AGT protein in plasma by the constructs disclosed herein: Figure 5a : AGT protein level in plasma (percentage relative to the control group); Figure 5b : Dose-dependent reduction in AGT protein in plasma.

[0066] Figure 6 (a) and (b) show the knockdown of APOC3 protein in plasma by the constructs disclosed herein: Figure 6a : APOC3 protein level in plasma (percentage relative to the control group); Figure 6b : Dose-dependent reduction of APOC3 protein in plasma.

[0067] Figure 7(a) shows mRNA levels in liver tissue (AGT-27A vs. control). Duration of response: Week 2: 75% NADIR; Week 4: 70% mRNA KD; Week 8 (21%) and Week 12 (9%), returned to control levels.

[0068] Figure 7(b) shows the sustained response of protein levels in plasma measured using ELISA: Week 2: maximum reduction of 85%; Week 4: reduction of 79%; Week 8: reduction of 58%; Week 12: reduction of 44%.

[0069] FIG8( a ) shows the mRNA levels in liver tissue (AGT and APOC3 compared with the control group); the table following the line graph shows the mRNA KD percentages at weeks 2, 4, 8, and 12.

[0070] Figure 8(b) shows the plasma protein levels (AGT and APOC3 compared with the control group) detected by ELISA; the table following the line graph shows the reduction of plasma proteins at weeks 2, 4, 8, and 12.

[0071] Detailed description

[0072] Provided are embodiments of siRNA molecules, pharmaceutical compositions, and methods for their preparation, administration, delivery, and use in treating AGT and APCO3-related diseases and conditions.

[0073] The following describes further implementations of the disclosed embodiments by way of example only. These examples represent advantageous ways currently known to the applicant to put the present disclosure into practice, although they are not the only ways to achieve this goal.

[0074] For the sake of clarity, it is noted herein that implementations or embodiments labeled "advantageously" or "advantageously" are not intended to limit the scope of the claims, but rather to illustrate alternative embodiments.

[0075] It will be obvious to those skilled in the art that features of different aspects and implementations or embodiments may be appropriately combined.

[0076] definition

[0077] The following definitions apply throughout this disclosure. In many cases, these definitions provide a non-exhaustive list of possible, alternative or advantageous embodiments in addition to the respective definition itself.

[0078] Unless specific definitions are provided, the procedures and techniques of analytical chemistry, synthetic organic chemistry, and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques can be used for chemical syntheses and chemical analyses. For example, some of these techniques and procedures can be found in Sangvi and Cook, eds., Carbohydrate Modifications in Antisense Research, American Chemical Society, Washington, D.C., 1994; Remington's Complete Works of Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, 21st edition, 2005; Stanley T. Crooke, ed., Antisense Drug Technology: Principles, Strategies, and Applications, CRC Press, Boca Raton, Florida; and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference into this application for any purpose. Where permitted, all patents, applications, published applications, and other publications and other data cited throughout this disclosure are incorporated herein by reference in their entirety.

[0079] Unless otherwise stated, the following terms have the following meanings:

[0080] As used herein, "excipient" refers to any compound or mixture of compounds added to the compositions provided herein that is suitable for delivery of the oligomeric compound.

[0081] As used herein, "nucleoside" refers to a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (e.g., nucleotides (present in DNA and RNA) and modified nucleosides. Nucleosides can be linked to a phosphate moiety, which are also referred to as "nucleotides." The structural features and / or length of the oligomeric compounds or nucleic acid constructs disclosed herein are represented by "nucleoside" or "nucleotide."

[0082] As used herein, "chemical modification" or "chemical modification" refers to the chemical differences of a compound compared to its naturally occurring counterpart. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar modifications and base modifications) and internucleoside linkage modifications. For oligonucleotides, chemical modifications include more than just differences in the nucleic acid base sequence.

[0083] As used herein, "furanosyl" refers to a 5-membered ring structure containing four carbon atoms and one oxygen atom.

[0084] As used herein, "naturally occurring sugar moiety" refers to a ribofuranosyl group found in naturally occurring RNA or a deoxyribofuranosyl group found in naturally occurring DNA.

[0085] As used herein, "naturally occurring sugar moieties" are also referred to as "unmodified sugar moieties." Specifically, as used herein, "naturally occurring sugar moieties" or "unmodified sugar moieties" have -H (DNA sugar moieties) or -OH (RNA sugar moieties) at the 2' position of the sugar moiety, and more particularly have -H (DNA sugar moieties) at the 2' position of the sugar moiety.

[0086] As used herein, "sugar moiety" refers to a naturally occurring sugar moiety or a modified sugar moiety in a nucleoside. As used herein, "modified sugar moiety" refers to a substituted sugar moiety or a sugar surrogate.

[0087] As used herein, a "substituted sugar moiety" refers to a furanosyl group that has been substituted. Substituted sugar moieties include, but are not limited to, furanosyl groups that contain substituents at the 2'-position, 3'-position, 5'-position, and / or 4'-position. Certain substituted sugar moieties are bicyclic sugar moieties.

[0088] As used herein, a "2'-substituted sugar moiety" refers to a furanosyl group comprising a substituent other than Hor OH at the 2'-position. Unless otherwise indicated, a 2'-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2'-substituent of the 2'-substituted sugar moiety is not

[0089] forms a bridge with another atom of the furanose ring).

[0090] As used herein, "MOE" refers to -OCH2CH2OCH3.

[0091] As used herein, "2'-F nucleosides" refer to nucleosides containing a fluorine sugar group at the 2' position. Unless otherwise indicated, the fluorine in 2'-F nucleosides is located at the ribose position (replacing the OH group of the natural ribose sugar). Duplexes of uniformly modified 2'-fluorinated (ribose) oligonucleotides hybridized to RNA strands are not substrates for RNase H, while analogs retain RNase H activity.

[0092] As used herein, term " sugar substitute " refers to the structure that does not include furanosyl and can replace the natural sugar part of nucleoside, so that gained nucleoside subunit can be linked together and / or connected to other nucleosides to form oligomeric compounds that can hybridize with complementary oligomeric compounds.Such structure includes comprising the ring (for example, 4,6 or 7 rings) that is different from furanosyl atomic number; The oxygen atom of furanosyl is replaced with non-oxygen atom (for example, carbon, sulphur or nitrogen); Or change atomic number and replace oxygen atom simultaneously.Such structure can also include and replace described in sugar moiety identical substituent (for example, 6 yuan of carbocyclic bicyclic sugar) sugar substitute (alternatively including other substituents).Sugar substitute also includes more complicated sugar substitute (for example, the non-cyclic system of peptide nucleic acid).Sugar substitute includes but is not limited to morpholino, cyclohexenyl and cyclohexitol.

[0093] " bicyclic sugar moiety " used herein refers to a modified sugar moiety comprising 4 to 7 rings (including but not limited to furanosyl), which comprises atoms connecting two 4 to 7 rings to form a second ring, thereby forming a bicyclic structure. In certain embodiments, the 4 to 7 rings are sugar rings. In certain embodiments, the 4 to 7 rings are furanosyl. In some such embodiments, the bridge connects the 2'-carbon and 4'-carbon of the furanosyl.

[0094] As used herein, "nucleotide" refers to a nucleoside group that also includes a phosphate bond. As used herein, "linked nucleosides" may or may not be linked by a phosphate bond, and therefore includes, but is not limited to, "linked nucleotides." As used herein, "linked nucleosides" refers to nucleosides linked in a continuous sequence (i.e., without other nucleosides between the linked nucleosides).

[0095] As used herein, "nucleobase" refers to a group of atoms that can be linked to a sugar moiety to produce a nucleoside that can be incorporated into an oligonucleotide, wherein the group of atoms is complementary to another oligonucleotide or nucleic acid. Nucleobases can be naturally occurring or modified.

[0096] As used herein, the term "unmodified nucleobase" or "naturally occurring nucleobase" refers to the naturally occurring heterocyclic nucleobases of RNA or DNA: purine bases

[0097] adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C) and uracil (U).

[0098] As used herein, "modified nucleobase" refers to any non-naturally occurring nucleobase.

[0099] As used herein, "modified nucleoside" refers to a nucleoside that comprises at least one modified chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides may comprise modified sugar moieties and / or modified core bases. As used herein, "bicyclic nucleosides" or "BNAs" refer to nucleosides that comprise bicyclic sugar moieties.

[0100] As used herein, "locked nucleic acid nucleoside" or "LNA" refers to a nucleoside comprising a bicyclic sugar moiety containing a 4'-CH2-O-2' bridge.

[0101] As used herein, "2'-substituted nucleosides" refer to nucleosides that contain a substituent other than H or OH at the 2' position of the sugar moiety. Unless otherwise indicated, 2'-substituted nucleosides are not bicyclic nucleosides.

[0102] As used herein, "deoxynucleoside" refers to a nucleoside moiety comprising a 2'-H furanose group, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase, or may comprise an RNA nucleobase (e.g., uracil).

[0103] As used herein, "oligonucleotide" refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and / or unmodified deoxyribonucleosides (DNA) and / or one or more modified nucleosides.

[0104] As used herein, "modified oligonucleotide" refers to an oligonucleotide comprising at least one modified nucleoside and / or at least one modified internucleoside linkage. Advantageous modifications of the internucleoside linkage are those that confer increased stability compared to naturally occurring phosphodiester linkages. "Stability" specifically refers to stability against hydrolysis, including enzymatic hydrolysis, including exo- and endo-enzymes.

[0105] Advantageous locations for such modified internucleoside linkages include the ends and hairpin loops of the single-stranded oligomeric compounds of the disclosed embodiments. For example, the internucleoside linkages between the first and second nucleosides and the second and third nucleosides from the 5' end can be modified, and / or the internucleoside linkages between the first and second nucleosides and the second and third nucleosides from the 3' end can be modified. In addition, the bond connecting the 3' terminal nucleoside and the ligand (e.g., GalNAc) can also be modified.

[0106] As described above, the advantageous position is located in the hairpin loop of the single-stranded oligomeric compound. Specifically, all bonds in the hairpin loop, all bonds except one bond, or most bonds are modified. As used herein, "bonds in the hairpin loop" refers to bonds between nucleosides that do not participate in base pairing. For example, in a hairpin loop consisting of five nucleosides, there are bonds between four nucleosides.

[0107] Nucleosides that do not participate in base pairing. Advantageously, the term "linkage within the hairpin loop" also extends to the linkage connecting the stem and the loop, i.e., the linkage connecting a base-pairing nucleoside and a nucleoside that does not participate in base pairing. According to the disclosed embodiments, two such positions are typically present in the hairpin structure and mxRNA. Most advantageously, the modified internucleoside linkages are located at both ends and in the hairpin loop.

[0108] As used herein, "link" or "linking group" refers to a radical that links two or more other radicals together.

[0109] As used herein, "internucleoside linkage" refers to a covalent bond between adjacent nucleosides in an oligonucleotide.

[0110] As used herein, a "naturally occurring internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.

[0111] As used herein, "modified internucleoside linkages" refer to any internucleoside linkage other than naturally occurring internucleoside linkages. Specifically, "modified internucleoside linkages" as used herein may include modified phosphorus linking groups, such as phosphorothioate or phosphorodithioate internucleoside linkages.

[0112] As used herein, "terminal internucleoside linkage" refers to the linkage between the last two nucleosides of an oligonucleotide or a defined region thereof.

[0113] As used herein, a "phosphorus linking group" refers to a linking group comprising a phosphorus atom, and can include naturally occurring phosphorus linking groups, such as naturally occurring RNA or DNA, for example, a phosphodiester linking group, or a modified phosphorus linking group not typically found in naturally occurring RNA or DNA, such as a phosphorothioate or phosphorodithioate linking group. Thus, a phosphorus linking group can include, but is not limited to, phosphodiester, phosphorothioate, phosphorodithioate, phosphonate, methylphosphonate, phosphoramidate, thiophosphoramidate, thionoalkylphosphonate, phosphotriester, thionoalkylphosphotriester, and boranophosphate.

[0114] As used herein, an "internucleoside phosphorus linker" refers to a phosphorus linker that directly links two nucleosides.

[0115] As used herein, "oligomeric compound" refers to a polymer structure comprising two or more substructures. In certain embodiments, the oligomeric compound comprises an oligonucleotide, such as a modified oligonucleotide. In certain embodiments, the oligomeric compound further comprises one or more conjugated groups and / or terminal groups and / or ligands. In certain embodiments, the oligomeric compound consists of an oligonucleotide. In certain embodiments, the oligomeric compound comprises a main chain of one or more connected monomeric sugar moieties, wherein each connected monomeric sugar moiety is directly or indirectly connected to a heterocyclic base moiety. In certain embodiments, the oligomeric compound may also comprise a monomeric sugar moiety that is not connected to a heterocyclic base moiety, thereby providing an abasic site. The oligomeric compound can be defined solely based on the nucleobase sequence, i.e., by specifying a sequence of A, G, C, U (or T). In this case, the structure of the sugar-phosphate backbone is not particularly limited and may or may not include modified sugars and / or modified phosphates. On the other hand, the oligomeric compound can be defined more comprehensively, i.e., not only specifying the nucleobase sequence, but also specifying the main chain structure, particularly the modified state of the sugar (unmodified, 2'-OMe modified, 2'-F modified, etc.) and / or the modified state of the phosphate. mxRNA is a non-limiting example of an oligomeric compound.

[0116] As used herein, "nucleic acid construct" or "construct" refers to an assembly of two or more (eg, four) oligomeric compounds, which in the context of the first aspect of the disclosed embodiments are referred to as "parts."

[0117] The oligomeric compounds can be linked to each other by covalent bonds, such as phosphodiester bonds present in naturally occurring nucleic acids or modified forms thereof disclosed herein, and / or by non-covalent bonds, such as hydrogen bonds, favorable hydrogen bonds between nucleobases, such as Watson-Crick base pairing. In certain embodiments, it is advantageous for the construct to comprise four oligomeric compounds, wherein the first and fourth compounds or moieties and the second and third compounds or moieties are covalently linked to each other, thereby generating two nucleic acid strands that are bound to each other by hydrogen bonds. Due to the covalent linkage, strictly speaking, the result is two compounds, i.e., two strands. The complementarity between the strands can be complete, but is not necessarily so. Specifically, exemplary embodiments provide an antisense region that targets APOC3 mRNA and is covalently linked to a sense region identical to a region of AGT mRNA, and the antisense region that is complementary to the sense region is covalently linked to a sense region that is complementary to the antisense region targeting APOC3 mRNA. Since the antisense and sense regions of the parental single-targeting RNA molecules do not need to be of the same length, and advantageously, they are of different lengths, with the antisense portion being longer than the sense portion, an advantageous construct of the disclosed embodiments comprises a central region in which the 3' regions of the antisense portions of the parental single-targeting RNA molecules face each other. In this region, there is typically no or only partial base pairing, but complete complementarity is not excluded. Otherwise, when the antisense and sense portions of the respective parental RNA molecules face each other, there is complementarity, preferably complete complementarity, or there are one or two mismatches. muRNA is a non-limiting example of a nucleic acid construct.

[0118] The term "chain" has its art-established meaning and refers to a plurality of interconnected nucleosides. The linker is not particularly limited, but includes phosphodiester and variants thereof as described herein. A chain can also be considered as a plurality of linked nucleotides, in which case the linker will be a covalent bond.

[0119] As used herein, "terminal group" refers to one or more atoms connected to the 3' end or 5' end (also referred to as the "terminus") of an oligonucleotide. In certain embodiments, the terminal group includes one or more terminal group nucleosides, while a "terminal nucleoside" has only one nucleotide at the corresponding end (5' end or 3' end).

[0120] As used herein, "conjugate" or "conjugate group" refers to an atom or group of atoms that is bound to an oligonucleotide or oligomeric compound. In certain embodiments, the conjugate group connects a ligand to a modified oligonucleotide or oligomeric compound. Generally, the conjugate group can alter one or more properties of the compound to which it is attached. These include, but are not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, cellular distribution, cellular uptake, charge, and / or clearance properties.

[0121] As used herein, "conjugate linker" or "linker" in the context of a conjugate group refers to a portion of a conjugate group that comprises any atom or group of atoms and covalently links an oligonucleotide to another portion of the conjugate group. In certain 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 certain embodiments, the point of attachment on the oligomeric compound is the 5' oxygen atom of the 5' hydroxyl group of the 5' terminal nucleoside of the oligonucleotide. In certain embodiments, the bond used to form the connection with the oligomeric compound is a cleavable bond. In certain such embodiments, such cleavable bond constitutes all or part of the cleavable moiety.

[0122] In certain embodiments, the conjugate group comprises a cleavable portion (e.g., a cleavable bond or a cleavable nucleoside) and a ligand portion, which may comprise one or more ligands, such as a carbohydrate cluster portion, such as an N-acetylgalactosamine cluster portion, also known as "GalNAc". In certain embodiments, the carbohydrate cluster portion is identified by the number and identity of the ligands. For example, in certain embodiments, the carbohydrate cluster portion comprises 2 GalNAc groups. For example, in certain embodiments, the carbohydrate cluster portion comprises 3 GalNAc groups, which is particularly advantageous. In certain embodiments, the carbohydrate cluster portion comprises 4 GalNAc groups. Such ligand portions are identified by cleavable portions, such as cleavable bonds or cleavable nucleosides. The ligands can be arranged in a linear or branched structure, such as a bi-antennary or tri-antennary structure. A favorable carbohydrate cluster has the following structural formula:

[0123]

[0124] wherein one, two or three phosphodiester bonds in the structural formula may also be replaced by phosphorothioate bonds.

[0125] As used herein, "cleavable moiety" refers to a moiety that is capable of cleaving under physiological conditions. In certain embodiments, the cleavable moiety is cleaved within a cell or subcellular compartment (e.g., an endosome or lysosome). In certain embodiments, the cleavable moiety is cleaved by an endogenous enzyme (e.g., a nuclease). In certain embodiments, the cleavable moiety comprises an atomic group having one, two, three, four, or more cleavable bonds. In certain embodiments, the cleavable moiety is a phosphodiester bond.

[0126] As used herein, "cleavable bond" refers to any chemical bond that is capable of being broken.

[0127] As used herein, "carbohydrate cluster" refers to a compound having one or more carbohydrate residues attached to a linking group.

[0128] As used herein, "modified carbohydrate" refers to a carbohydrate having one or more chemical modifications relative to a naturally occurring carbohydrate.

[0129] As used herein, "carbohydrate derivative" refers to any compound that can be synthesized using a carbohydrate as a starting material or intermediate.

[0130] As used herein, "carbohydrate" refers to naturally occurring carbohydrates, modified carbohydrates, or carbohydrate derivatives. Carbohydrates are biological molecules comprising carbon (C), hydrogen (H), and oxygen (O) atoms. Carbohydrates can include monosaccharides, disaccharides, trisaccharides, tetrasaccharides, oligosaccharides, or polysaccharides, such as one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties. A particularly preferred carbohydrate is N-acetylgalactosamine.

[0131] As used herein, a "chain" refers to an oligomeric compound composed of linked nucleosides.

[0132] As used herein, "single-stranded" or "single-stranded" refers to an oligomeric compound comprising linked nucleosides in a continuous sequence without breaks therebetween. Such a single strand may include sufficient self-complementary regions to form a stable self-duplex in a hairpin structure.

[0133] As used herein, "hairpin" refers to a single-stranded oligomeric compound that includes a duplex formed by base pairing between self-complementary sequences in the strands, and in opposite directions.

[0134] As used herein, "hairpin loop" refers to an unpaired loop formed by the hybridization of self-complementary sequences, in the shape of a hairpin, and connected by nucleosides. The resulting structure is circular or U-shaped.

[0135] Specifically, short hairpin RNA (also known as shRNA) comprises a double-stranded region and a loop connecting the duplex region. The end of the duplex region without a loop can be a blunt end or can have a 3' and / or 5' overhang. Blunt-ended constructs are preferred. The term "shRNA" is more general than "mxRNA" defined below and can include compounds in which the loop is not formed by or is not entirely formed by a portion of the antisense strand. Specifically, shRNA comprises an antisense strand, also known as a guide strand, a sense strand that is complementary to the target RNA region, and a sense strand (i.e., a passenger strand) that is substantially complementary to the antisense strand. More specifically, the antisense strand and the sense strand in the shRNA are directly connected, for example, by a phosphate or phosphorothioate connection, or by a nucleoside connection via a third portion that forms a loop, which means that the 3' end of the antisense strand is covalently bonded to several other groups. This direct connection does not contain a gap or a gap.

[0136] As used herein, "directionality" refers to the end-to-end chemical orientation of an oligonucleotide based on the chemical convention for numbering the carbon atoms of the sugar moiety, meaning that the 5' end is defined by the 5' carbon of the sugar moiety and the 3' end is defined by the 3' carbon of the sugar moiety. In a double-stranded or double-stranded oligonucleotide, the strands extend in opposite 5' to 3' directions to allow base pairing between them.

[0137] As used herein, "duplex" (or abbreviated as "dup") refers to two or more complementary regions or strands of one or more oligonucleotides that are hybridized together through non-covalent, sequence-specific interactions. Most commonly, hybridization in a duplex occurs between the nucleobases adenine (A) and thymine (T), and / or (A) adenine and uracil (U), and / or guanine (G) and cytosine (C). A duplex can be part of a single-stranded structure where self-complementarity results in hybridization, or as a result of hybridization between corresponding strands in a double-stranded construct.

[0138] As used herein, "double-stranded" or "double-stranded" refers to a pair of oligomeric compounds that are hybridized to each other. In certain embodiments, the double-stranded oligomeric compound includes a first and a second oligomeric compound.

[0139] 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 (eg, splicing, polyadenylation, addition of a 5'-cap), and translation.

[0140] As used herein, "transcription" or "transcription" refers to the first of several steps in DNA transcription. In which a DNA target sequence is copied into RNA (especially mRNA) by RNA polymerase. During transcription, RNA polymerase reads the DNA sequence and produces a complementary, antiparallel RNA sequence, called the primary transcript.

[0141] As used herein, "target sequence" refers to a sequence to which an oligomeric compound tends to hybridize to produce the desired activity with respect to APOC3 or AGT expression. An oligonucleotide has sufficient complementarity to its target sequence to allow hybridization under physiological conditions.

[0142] As used herein, "nucleobase complementarity" or "complementarity" when referring to nucleobases refers to nucleobases that can base pair 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 certain embodiments, complementary nucleobases refer to nucleobases in an oligomeric compound that can pair with the nucleobases of its target sequence. For example, if a nucleobase at a certain position in an oligomeric compound can form a hydrogen bond with a nucleobase at a certain position in a target sequence, the hydrogen bond formed between the oligomeric compound and the target sequence is considered to be complementary at that base pair. Bases containing certain modifications may retain the ability to pair with corresponding bases and therefore still have base complementarity.

[0143] As used herein, "non-complementary" with respect to nucleobases refers to a pair of nucleobases that do not form hydrogen bonds with each other.

[0144] As used herein, "complementarity" with respect to oligomeric compounds (eg, linked nucleosides, oligonucleotides) refers to the ability of such oligomeric compounds or a region thereof to hybridize to a target sequence or a region of the oligomeric compound itself through nucleobase complementarity.

[0145] The complementary oligomeric compounds do not have to have nucleobase complementarity at every nucleoside. Rather, some mismatches can be tolerated. In certain embodiments, the complementary oligomeric compounds or regions are complementary at least at 70% of the nucleobases (at least 70% complementary). In certain embodiments, the complementary oligomeric compounds or regions are complementary at least at 80%. In certain embodiments, the complementary oligomeric compounds or regions are complementary at least at 90%.

[0146] The compounds or regions are at least 90% complementary. In certain embodiments, the complementary oligomeric compounds or regions are at least 95% complementary. In certain embodiments, the complementary oligomeric compounds or regions are 100% complementary.

[0147] In a preferred embodiment, the antisense portion is completely or 100% complementary to the corresponding sense portion. In the case where the sense portion is shorter than the corresponding antisense portion, such complete complementarity is understood to mean the same number of base pairs as the number of nucleotides in the sense portion.

[0148] With regard to the complementarity of the antisense portion to the homologous mRNA, the complementarity is preferably perfect or has one mismatch, the mismatch preferably being located at the 5' end of the antisense portion.

[0149] As used herein, "self-complementary" refers to an oligomeric compound that can fold on itself to form a duplex by hybridization of the nucleobases of the internal complementary strand regions. Depending on the distance and / or length between the strand regions, the compound may form a hairpin loop, a junction, a bulge, or an internal loop.

[0150] As used herein, "mismatch" refers to the inability of a nucleobase of an oligomeric compound to pair with a nucleobase at a corresponding position in a target sequence when the oligomeric compound is aligned with the target sequence and / or a self-complementary region of the oligomeric compound, or to pair with a nucleobase at a corresponding position in the oligomeric compound itself when the oligomeric compound hybridizes due to self-complementarity.

[0151] As used herein, "hybridization" refers to the pairing of complementary oligomeric compounds (e.g., an oligomeric compound and its target sequence). Although not limited to a specific mechanism, the most common mechanism of pairing involves hydrogen bonding between complementary nucleobases, which can be Watson-Crick hydrogen bonding, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogen bonding.

[0152] As used herein, "specifically hybridizes" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site.

[0153] As used herein, "fully complementary" with respect to an oligomeric compound or region thereof means that each nucleobase of the oligomeric compound or region thereof is capable of pairing with a nucleobase of a complementary nucleic acid target sequence or a self-complementary region of the oligomeric compound. Thus, a fully 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.

[0154] As used herein, "percent complementarity" refers to the percentage of nucleobases in an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases in an oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.

[0155] As used herein, "percent identity" refers to the number of nucleobases in a first nucleic acid that are the same type of nucleobase as the corresponding position in a second nucleic acid (independent of chemical modification) divided by the total number of nucleobases in the first nucleic acid.

[0156] As used herein, "modulation" refers to a change in the quantity or quality of a molecule, function, or activity compared to the quantity or quality of the molecule, function, or activity before modulation. For example, modulation includes an increase (stimulation or induction) or decrease (inhibition or reduction) in gene expression.

[0157] As used herein, "modification type" in reference to a nucleoside or "type" of nucleoside refers to the chemical modification of the nucleoside, including modified and unmodified nucleosides. Thus, unless otherwise indicated, a "nucleoside having a first type of modification" can be an unmodified nucleoside.

[0158] As used herein, "differently modified" refers to chemical modifications or chemical substitutions that are different from one another, including no modification. Thus, for example, a MOE nucleoside and an unmodified naturally occurring RNA nucleoside are "differently modified," even though the naturally occurring nucleoside is unmodified. Similarly, DNA and RNA oligonucleotides are "differently modified," even though both are naturally occurring unmodified nucleosides. Nucleosides that are identical but contain different nucleic acid bases do not have different modifications. For example, a nucleoside containing a 2'-OMe modified sugar moiety and an unmodified adenine nucleobase and a nucleoside containing a 2'-OMe modified sugar moiety and an unmodified thymidine nucleobase do not have different modifications.

[0159] As used herein, "the same type of modification" refers to modifications that are identical to one another, including the absence of modification. Thus, for example, two unmodified RNA nucleosides have the "same type of modification" even if the RNA nucleosides are unmodified. Such nucleosides having the same type of modification may contain different nucleobases.

[0160] As used herein, "a region" or "regions" or "portion" refers to a plurality of linked nucleosides having a function or feature as defined herein, particularly with reference to the claims and definitions provided herein. Typically, such a region or portion comprises 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 antisense region as defined herein consists essentially of 18 to 20 linked nucleosides.

[0161] The nucleosides and sense regions defined herein consist essentially of 13 to 16 linked nucleosides, notably, according to the first aspect of the disclosed embodiments, the first and second portions are antisense portions, while the third and fourth portions are sense portions.

[0162] As used herein, "pharmaceutically acceptable carrier or diluent" refers to any substance suitable for administration to an animal. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.

[0163] As used herein, "substituent" and "substituent group" refer to an atom or group that replaces an atom or group in a specified parent compound. For example, a substituent of a modified nucleoside refers to any atom or group that is different from the atom or group found in a natural nucleoside (for example, a modified 2'-substituent refers to any atom or group other than H or OH at the 2'-position of a nucleoside). The substituent group may be protected or unprotected. In certain embodiments, the compounds of the present disclosure have substituents at one or more positions of the parent compound. The substituent may also be further substituted by other substituents and may be connected to the parent compound directly or through a linking group (such as oxygen, alkyl or hydrocarbon group). Such substituents may exist as modifications on the sugar moiety, particularly the substituent at the 2' position of the sugar moiety. Unless otherwise indicated, groups that may be used as substituents include, but are not limited to, one or more of halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl, carboxyl, alkoxy, alkoxyalkylene and amino substituents. Certain substituents described herein may represent modifications that are directly attached to the sugar moiety ring (e.g., a halogen, such as fluorine, directly attached to the sugar ring), or indirectly attached to the sugar moiety ring via an oxygen linking atom, which is itself directly attached to the sugar moiety (e.g., an alkoxyalkylene, such as methoxyvinyl, attached to an oxygen atom, generally providing a MOE substituent as described herein, attached to the 2' position of the sugar moiety).

[0164] As used herein, "alkyl" refers to a saturated straight or branched chain alkyl group. A monovalent C1-6 hydrocarbon group, with methyl being the most preferred alkyl group, can serve as a substituent at the 2' position of the saccharide moiety. The alkyl group is typically attached to the oxygen linking atom at the 2' position of the saccharide. Thus, according to the disclosed embodiments, a -Oalkyl substituent, such as an -OCH3 substituent, is generally provided on the saccharide moiety of the oligomeric compound. This is well understood by those skilled in the art.

[0165] As used herein, "alkylene" refers to a saturated straight or branched chain divalent hydrocarbon radical of the general formula -CnH2n-, wherein n is 1 to 6. Methylene or ethylene are preferred alkylene radicals.

[0166] As used herein, "alkenyl" refers to a linear or branched, unsaturated, monovalent C2-6 hydrocarbon group, with ethenyl or propenyl being the most preferred alkenyl groups, which can serve as substituents at the 2' position of the saccharide moiety. As is well known in the art, the unsaturation of an alkenyl group refers to the presence of at least one carbon-carbon double bond. According to the disclosed embodiments, the alkenyl group is generally attached to the oxygen linking atom at the 2' position of the saccharide, thereby generally providing an -O-alkenyl substituent on the saccharide moiety of the oligomeric compound, such as an -OCH2CH=CH2 substituent. This is well understood by those skilled in the art.

[0167] As used herein, "alkynyl" refers to a straight or branched unsaturated C2-6 hydrocarbon group, with ethynyl being the most preferred alkynyl group as a 2'-substituent on the sugar moiety. As is well known in the art, the unsaturation present in an alkynyl group refers to the presence of at least one carbon-carbon triple bond. The alkynyl group is typically attached to the oxygen linking atom at the 2' position of the sugar, thereby generally forming an -O alkynyl substituent on the sugar moiety of the oligomeric compound according to the disclosed embodiments. This is well understood by those skilled in the art.

[0168] As used herein, a "carboxyl group" is a group having the general formula -CO2H.

[0169] As used herein, "acyl" refers to a group formed by removing the hydroxy group from a carboxyl group as defined herein and has the general formula -C(O)-X, wherein X is typically a C1-6 alkyl group.

[0170] As used herein, "alkoxy" refers to a group formed by an alkyl group (e.g., a C1-6 alkyl group) and an oxygen atom, wherein the oxygen atom is used to connect the alkoxy group to the parent molecule (e.g., the 2' position of a sugar moiety), or to other groups, such as alkylene as defined herein. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, and tert-butoxy. As used herein, the alkoxy group may optionally contain other substituents.

[0171] As used herein, alkoxyalkylene refers to an alkoxy group, as defined herein, attached to an alkylene group, as defined herein, wherein the oxygen atom of the alkoxy group is attached to the alkylene group, which in turn is attached to the parent molecule. The alkylene group is typically attached to the oxygen atom at the 2' position of the sugar, and thus, according to the disclosed embodiments, a -Oalkylenealkoxy substituent is generally provided on the sugar portion of the oligomeric compound, such as an -OCH2CH2OCH3 substituent. This is well understood by those skilled in the art and is generally referred to as an MOE substituent.

[0172] As used herein, "amino group" includes primary, secondary and tertiary amino groups.

[0173] As used herein, "halo" and "halogen" refer to an atom selected from fluorine, chlorine, bromine and iodine.

[0174] As used herein, the term "mxRNA" is specifically understood to be WO2020 / 044186 A2, which is incorporated herein by reference in its entirety. Specifically, mxRNA is a hairpin-shaped RNA molecule consisting of an antisense portion (also referred to as a guide strand) and a sense portion (also referred to as a passenger strand). mxRNA comprises a duplex region and a hairpin loop, wherein the length of the mxRNA is about 34 nucleotides. The duplex region comprises a region in which a portion of the antisense portion and substantially the entire sense portion (usually 14 or 15 nucleotides per chain) are base-paired. The hairpin loop connects the two regions of the duplex, i.e., the antisense region and the sense region, by, for example, a phosphate or thiophosphate linker (i.e., covalently linked), and the antisense portion is generally about 18 to 20 nucleotides in length, thus constituting an antisense double-stranded region and a loop. The antisense portion is a portion of a loop that further connects the sense portion and the antisense portion to form a second chain of the loop.

[0175] The term "angiotensinogen," or the abbreviation "AGT," also known as SERPINA 8 or ANHU, is a commonly used term that refers to a protein produced by the liver. It is a component of the renin-angiotensin-aldosterone system (RAAS) and is converted by renin into angiotensin I when released into the bloodstream. The identifier for human AGT mRNA in the RefSeq database is NM_000029. Angiotensinogen is expressed and produced in the liver by the angiotensin gene, or "AGT gene."

[0176] The term "apolipoprotein C3" (abbreviated as "APOC3") has its generally accepted meaning. Human APOC3 mRNA is identified in the RefSeq database as NM_000040. It is secreted by the liver and small intestine. It is present in triglyceride-rich lipoproteins, including very low-density lipoproteins (VLDL) and chylomicrons. It is involved in the negative regulation of lipid catabolism, particularly triglyceride catabolism, as well as in VLDL, LDL, and HDL lipoproteins. The molecular function of APOC3 is the inhibition of lipoprotein lipase and hepatic lipase.

[0177] As used herein, the term "muRNA" or "polyRNA" includes a nucleic acid construct comprising one or more (usually two) RNA sequences, i.e., a first and a second nucleic acid, fragments targeting different regions of AGT mRNA; or targeting a region of AGT mRNA and an mRNA region of another target molecule (preferably APOC3 in this case). The targeting RNA sequence is also referred to as the "antisense strand" or "guide strand," and the corresponding passenger strands, i.e., the third and fourth nucleic acid fragments complementary to the first and second parts, respectively, are also included in the nucleic acid construct. Specifically, such muRNAs are designed so that after in vivo administration, they are decomposed and release the first and second nucleic acid parts. A specific example of such muRNA is shown below, wherein (1) is the first nucleic acid part, (2) is the third nucleic acid part complementary to (1), (3) is the second nucleic acid part complementary to the fourth nucleic acid part, (5) is an unstable linker, and (6) is a ligand, all of which are explained below.

[0178]

[0179] Further miniaturization by shortening the sense region resulted in a bulge in the center of the molecule, where the 3'-terminal regions of the two antisense regions face each other:

[0180]

[0181] In the figure above, "GN" indicates the GalNAc moiety and "SBS" indicates a fragile site that can be realized as a nucleoside with an unmodified sugar.

[0182] It should also be 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 ends of one or both chains, provided that there is sufficient complementarity to maintain hybridization under physiologically relevant conditions. Alternatively, the oligomeric compounds described herein may have at least one blunt end. Preferably, both ends are blunt.

[0183] Herein, the word "comprising" is used to indicate including the specified method steps or elements, but these steps or elements do not constitute an exclusive list, so additional steps or elements may be present.

[0184] Furthermore, to the extent that the word “including” is used in the detailed description or the claims, it is intended to be interpreted in an inclusive manner similar to the word “comprising” as it is interpreted when used as a transitional word in the claims.

[0185] Non-limiting aspects and embodiments

[0186] muRNA nucleic acid constructs

[0187] According to a first aspect, the disclosed embodiments relate to a nucleic acid construct comprising at least:

[0188] a first nucleic acid portion that is at least partially complementary to at least a first portion of RNA transcribed from the APOC3 gene;

[0189] a second nucleic acid portion that is at least partially complementary to at least a second portion of RNA transcribed from the AGT gene;

[0190] a third nucleic acid portion that is at least partially complementary to the first nucleic acid portion of (a) so as to form a first nucleic acid duplex region therewith;

[0191] a fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion of (b) so as to form a second nucleic acid duplex region therewith.

[0192] The construct is designed such that, following in vivo administration, the construct disassembles to produce at least first and second discrete nucleic acid targeting molecules that target the portions of the RNA transcribed from the target genes of (a) and (b), respectively;

[0193] wherein (i) the first nucleic acid targeting molecule is capable of regulating the expression of the target gene of (a) and comprises or is derived from at least the first nucleic acid portion of (a); and (ii) the second nucleic acid targeting molecule is capable of regulating the expression of the target gene of (b) and comprises or is derived from the second nucleic acid portion of (b).

[0194] The construct can be designed to disassemble such that the first and second discrete nucleic acid targeting molecules are each processed by independent RNAi-induced silencing complexes.

[0195] Sequence characteristics, unstable functionality and structural features of RNA molecules

[0196] The construct according to the first aspect and preceding embodiments thereof may comprise at least one labile functionality such that upon in vivo administration said construct is degraded to produce said at least first and second discrete nucleic acid targeting molecules.

[0197] The labile functionality may comprise one or more unmodified nucleotides. In particular, the one or more unmodified nucleotides of the labile functional group represent one or more cleavage sites in the construct, whereby upon in vivo administration, the construct is cleaved at the one or more cleavage sites, thereby producing the at least first and second discrete nucleic acid targeting molecules. In particular, the cleavage sites may be located in the construct, respectively, such that upon cleavage of the first discrete nucleic acid, the nucleic acid targeting molecule comprises or is derived from the first nucleic acid duplex region, and the second discrete nucleic acid targeting molecule comprises or is derived from the second nucleic acid duplex region. Advantageously, the first discrete nucleic acid targeting molecule comprises or consists of the first nucleic acid portion of (a) and the third nucleic acid portion of (c), and / or the second discrete nucleic acid targeting molecule comprises or is composed of the

[0198] (b) the second nucleic acid portion and (d) the fourth nucleic acid portion.

[0199] In certain embodiments

[0200] (a) the first nucleic acid portion has a base sequence selected from SEQ ID NOs: 1 to 30;

[0201] (b) the second nucleic acid portion has a base sequence selected from SEQ ID NOs: 61 to 90

[0202] (c) the third nucleic acid portion has a base sequence selected from SEQ ID NOs: 31 to 60; and / or

[0203] (d) The fourth nucleic acid portion has a base sequence selected from SEQ ID NOs: 91 to 120.

[0204] These sequences have shown excellent performance in the context of molecules used to trigger RNA interference and target a single target; for example, PCT / US2022 / 34965 (for APOC3 targeting molecules) and US 63 / 407,353 (for AGT targeting molecules). These two patent documents are incorporated herein by reference. Since the inventors surprisingly discovered that the outstanding performance of single-targeting molecules (such as mxRNAs) can be transferred to dual-targeting molecules (such as muRNAs), any further sequences, especially the antisense sequences disclosed in the above patent documents, can serve as the basis for designing muRNAs of the disclosed embodiments.

[0205] As described above, the third and fourth nucleobase sequences, insofar as they have a length of 14 nucleobases, are advantageously extended by one nucleotide (at the 5' end of the third and fourth sequences, following the guidelines given above). When used in a degradable nucleic acid construct according to the disclosed examples, compounds that have been shown to be active as single-targeting molecules in APOC3 inhibition have surprisingly high activity.

[0206] In certain such embodiments, the first nucleic acid moiety of (a) can be linked directly or indirectly as a primary structure to the fourth nucleic acid moiety of (d).

[0207] In certain embodiments, the second nucleic acid portion of (b) can be directly or indirectly linked to the third nucleic acid portion of (c) as a primary structure. In certain embodiments, the first nucleic acid portion has a base sequence of SEQ ID NO:3.

[0208] In certain embodiments, the second nucleic acid portion has a base sequence selected from SEQ ID NO: 61, 66, 67, 73, 68 and 76.

[0209] In certain embodiments, the third nucleic acid portion has the nucleobase sequence of SEQ ID NO:33.

[0210] In certain embodiments, the fourth nucleic acid portion has a base sequence selected from the group consisting of SEQ ID NOs: 91, 96, 97, 103, 98, and 106. As described above, the sense sequences of the third and fourth portions are preferably 15 nucleotides in length. If the length of the aforementioned entry in the sequence listing is 14 nucleotides, an additional nucleotide should be added to the 5' end of the corresponding portion, the additional nucleotide being complementary to the 15th nucleotide of the corresponding antisense portion (the third portion being the first portion and the fourth portion being the second portion).

[0211] In certain embodiments, the construct may further comprise 1 to 8 additional nucleic acid portions, each of which is at least partially complementary to 1 to 8 additional RNA portions transcribed from one or more target genes, which may be identical or different to each other and / or identical or different to the target genes defined in (a) and / or (b), wherein each of the 1 to 8 additional nucleic acid portions forms an additional duplex region with a corresponding passenger nucleic acid portion to which the passenger nucleic acid portion is at least partially complementary. Specifically, the second nucleic acid portion and the 1 to 8 additional nucleic acid portions of (b) may be directly or indirectly linked to the selected passenger nucleic acid portion as respective primary structures.

[0212] In certain embodiments, the direct or indirect linkage can be represented by (i) an internucleotide bond, (ii) an internucleotide nick, or (iii) a nucleic acid linker portion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, the nucleic acid linker preferably being single-stranded. Advantageously, the linkage can be direct, thereby generating one or more continuous chains.

[0213] In certain embodiments, there may be some complementarity between the first nucleic acid portion of (a) and the second nucleic acid portion of (b), or between the third nucleic acid portion of (c) and the fourth nucleic acid portion of (d). Preferably, the complementarity is:

[0214] (i) may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pairs, advantageously 2, 3, 4 or 5 base pairs; and / or

[0215] (ii) may be located between the first nucleic acid portion of (a) and the second nucleic acid portion of (b).

[0216] In certain embodiments, the internucleotide bond may comprise at least one of the one or more unmodified nucleotides, wherein advantageously cleavage may occur at the 3' position of the (at least one) unmodified nucleotide.

[0217] In certain embodiments, the first nucleic acid portion of (a), and / or the second nucleic acid portion of (b), and / or the third nucleic acid portion of (c), and / or the fourth nucleic acid portion of (d) are 7 to 25 nucleotides in length, respectively. Optionally, the first nucleic acid portion of (a) and / or

[0218] The second nucleic acid portion of (b) may be 18 to 21 nucleotides in length, preferably 18 to 20 nucleotides, even more preferably 19 nucleotides in length. In a preferred embodiment, the first nucleic acid portion of (a) and the second nucleic acid portion of (b) are 19 nucleotides in length. Further preferably, the third nucleic acid portion of (c) and / or the fourth nucleic acid portion of (d) have 11 to 20 nucleotides, more preferably 13 to 16 nucleotides, even more preferably 14 or 15 nucleotides, and most preferably 15 nucleotides.

[0219] In certain embodiments, the first nucleic acid portion of (a) and the second nucleic acid portion of (b) may have a length of 19 nucleotides, and the third nucleic acid portion of (c) and the fourth nucleic acid portion of (b) may have a length of 15 nucleotides.

[0220] In certain embodiments, the unmodified nucleotide is located at any one of positions 18 to 25, more preferably at any one of positions 18 to 21, and / or the 3' terminal position of the first nucleic acid portion of (a) and / or the second nucleic acid portion of (b).

[0221] In certain embodiments, the unmodified nucleotide is at position 19.

[0222] In certain embodiments, the first nucleic acid portion of (a) and the second nucleic acid portion of (b) may have a length of 19 nucleotides, and the third nucleic acid portion of (c) and the fourth nucleic acid portion of (b) may have a length of 15 nucleotides, and the unmodified nucleoside is located at position 19 of the first nucleic acid portion of (a) and the second nucleic acid portion of (b).

[0223] In certain embodiments, the nucleic acid linker portion can be 1 to 8 nucleotides in length, advantageously 2 to 7 or 3 to 6 nucleotides in length, more advantageously about 4 or 5 nucleotides in length, and most advantageously 4 nucleotides in length.

[0224] In certain embodiments, one or more of all duplex regions may independently have a length of 10 to 19 bases, more advantageously 13 to 19 bases, and even more advantageously 13, 14, or 15 bases, most advantageously 15 base pairs, optionally with one mismatch within the duplex region.

[0225] In certain embodiments, the nucleic acid construct can be blunt-ended.

[0226] In certain embodiments,

[0227] (a) the first nucleic acid portion; and / or

[0228] (b) the second nucleic acid portion; and / or

[0229] (c) a third nucleic acid portion; and / or

[0230] (d) a fourth nucleic acid portion; and / or

[0231] where present, 1 to 8 additional nucleic acid moieties as previously defined herein; and / or

[0232] The passenger nucleic acid portion, where present, is as previously defined herein; possibly having overhangs.

[0233] In certain embodiments, the target RNA can be mRNA or another RNA molecule.

[0234] In certain embodiments, the construct comprises two strands, wherein the nucleobase sequences of the first and second strands are SEQ ID NOs: 130 and 131, SEQ ID NOs: 132 and 133, SEQ ID NOs: 134 and 135, SEQ ID NOs: 136 and 137, SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147, respectively. The sequences cited herein are shown in Tables 1 and 2 below. The sequence names in Table 1 indicate whether the sequence shown is the sense strand or the antisense strand. In particularly preferred embodiments, taking into account information about modifications to the sugar-phosphate backbone, the constructs of the disclosed embodiments are shown in Table 2 below. Particularly advantageous are the constructs labeled Apo28-AGT27 A and Apo28-AGT27. The properties of all constructs in Table 2 are demonstrated in the Examples.

[0235] Table 1

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] Table 2: Preferred muRNAs in the Examples disclosed herein. Each construct comprises two chains as shown below.

[0242]

[0243]

[0244]

[0245] [mN], N is any nucleoside, representing 2'-OMe; [fN], N is any nucleoside, representing 2'-F; [rN], N is any nucleoside, representing 2'-OH; [Ps] represents a phosphorothioate connecting two adjacent nucleosides; [3XGalNAc] represents

[0246] The following ligands, where the chain to which the ligand binds is shown in square brackets:

[0247]

[0248] ligand

[0249] The nucleic acid construct according to the second aspect and the preceding embodiments may further comprise one or more ligands.

[0250] In certain embodiments, the first nucleic acid portion of (a), and / or the second nucleic acid portion of (b), and / or the third nucleic acid portion of (c), and / or the fourth nucleic acid portion of (d), and / or (if present) the 1 to 8 additional nucleic acid portions as previously defined herein, and / or the passenger nucleic acid portion as previously defined herein, respectively, may have a 5' to 3' directionality, thereby defining their 5' and 3' regions.

[0251] In certain embodiments, one or more ligands may be conjugated to the 3' region, advantageously the 3' end, of any of: (i) the third nucleic acid moiety of (c), and / or (ii) the fourth nucleic acid moiety of (d), and / or, where present, (iii) the passenger nucleic acid moiety as previously defined herein.

[0252] In certain embodiments, one or more ligands may bind at one or more regions intermediate to the 5' and 3' regions of said nucleic acid portion, advantageously said third nucleic acid portion of (c), and / or said fourth nucleic acid portion of (d), and / or said passenger nucleic acid portion as previously defined herein.

[0253] In certain embodiments, one or more ligands may be conjugated to the 5' region, advantageously the 5' end, of any of the nucleic acid moieties.

[0254] In certain embodiments, the one or more ligands can be any cell targeting moiety, such as a lipid, carbohydrate, aptamer, vitamin, and / or a peptide that binds to a specific target on a cell membrane or cell surface. In a preferred embodiment, the one or more carbohydrates can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, or a polysaccharide. In a more preferred embodiment, the one or more carbohydrates can comprise one or more hexose moieties. In particular, the one or more hexose moieties can be one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties, and / or one or more mannose moieties. The hexose moiety can comprise two or three N-acetylgalactosamine moieties. Specifically, the hexose moiety can comprise three N-acetylgalactosamine moieties.

[0255] In certain embodiments, the one or more ligands can be connected in a linear configuration or a branched configuration. Advantageously, the one or more ligands can be connected in a bi-antenna or tri-antenna configuration, or in a single ligand configuration based on different positions.

[0256] Advantageously, the ligand may have the following structure:

[0257]

[0258] Internucleoside linkage

[0259] According to the second aspect, the nucleotide construct of the disclosed embodiments may comprise one or more phosphorothioate or phosphorodithioate internucleotide linkages.

[0260] In certain embodiments, the nucleic acid construct may comprise from 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.

[0261] In certain embodiments, the nucleic acid construct may comprise one or more phosphorothioate or phosphorodithioate internucleotide linkages at one or more 5' and / or 3' regions of the first nucleic acid portion of (a), and / or the second nucleic acid portion of (b), and / or the third nucleic acid portion of (c), and / or the fourth nucleic acid portion of (d), and / or the 1 to 8 additional nucleic acid portions as previously defined herein, and / or the passenger nucleic acid portion as previously defined herein.

[0262] In certain embodiments, the nucleic acid construct may comprise a phosphorothioate or phosphorodithioate internucleotide linkage between at least two adjacent nucleotides of the nucleic acid linker portion as previously defined herein.

[0263] In certain embodiments, the nucleic acid construct can comprise phosphorothioate or phosphorodithioate internucleotide linkages between each adjacent nucleotide present in the nucleic acid linker portion.

[0264] In certain embodiments, the nucleic acid construct may comprise phosphorothioate or phosphorodithioate internucleotide linkages linking:

[0265] (a) and the first nucleic acid portion and the nucleic acid linker portion as previously defined herein; and / or

[0266] (b) the second nucleic acid moiety and the nucleic acid linker moiety as previously defined herein; and / or

[0267] (c) and the nucleic acid linker moiety as previously defined herein; and / or

[0268] (d) and the nucleic acid linker moiety as previously defined herein; and / or

[0269] from 1 to 8 additional nucleic acid moieties as previously defined herein and a nucleic acid linker moiety as further defined herein; and / or

[0270] The passenger nucleic acid portion as previously defined herein and the nucleic acid linker portion as further defined herein.

[0271] siRNA modification

[0272] The RNA molecules of the disclosed embodiments can be conjugated (e.g., at the 5' or 3' end of their sense or antisense strands) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye, cholesterol, etc.), or unconjugated. Modifying an RNA agent in this manner can improve cellular uptake or enhance the cell-targeting activity of the resulting RNA agent derivative compared to the corresponding unconjugated RNA agent, can be used to track the RNA agent derivative in cells, or improve the stability of the RNA agent derivative compared to the corresponding unconjugated RNA agent.

[0273] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single-stranded or double-stranded form. The term encompasses nucleic acids containing backbone residues or connections of known nucleotide analogs or modifications that are synthetic, naturally occurring, and non-naturally occurring, having similar binding properties to reference nucleic acids and being metabolized in a manner similar to reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphorodithioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, methylribonucleotides, fluororibonucleotides, peptide nucleic acids (PNAs), and unlocked nucleic acids (UNAs; e.g., see Jensen et al., Proceedings of the Nucleic Acids Symposium, Vol. 52: 133-134) and their derivatives.

[0274] As used herein, "nucleotide" refers to nucleotides known in the art, including natural bases (standard bases) and modified bases known in the art. Such bases are generally located at the 1' position of the nucleotide sugar portion. Nucleotides generally comprise a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified at the sugar, phosphate, and / or base moieties (also interchangeably referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, etc., see, e.g., Usman and McSwiggen, Chapter 30: Catalytic RNA (Ribozymes) as Drugs, Annual Reports of Medicinal Chemistry, Vol. 30: 285-294, 1995; Eckstein et al., International PCT Publication No. WO 92 / 07065; Usman et al., International PCT Publication No. WO 93 / 15187; and Uhlman and Peyman, Antisense Oligonucleotides: A New Therapeutic Principle, Chemical Reviews, Vol. 4: 543-584, 1990). Several examples of modified nucleic acid bases are known in the art, as summarized by Limbach et al. Abstract: "Modified Nucleosides of RNA", Nucleic Acids Research, Vol. 22 (Issue 12): pp. 2183-2196, 1994. Examples of base modifications that can be introduced into nucleic acid molecules include hypoxanthine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine), propargyl, etc. (Burgin et al., Biochemistry 35: 14090, 1996; Uhlman and Peyman, supra). A modified base refers to a nucleotide base other than adenine, guanine, cytosine, and uracil at the 1' position or its equivalent.

[0275] Modified nucleotides or modified residues used herein refer to the presence of one or more modifications on a nucleotide, typically non-natural modifications, where the modified nucleotide is located on a nucleoside, base, pentose ring, or phosphate group, but modifications may also include natural modifications produced by enzymes that modify nucleotides (e.g., methyltransferases). Non-natural modifications of nucleotides include 2' modifications, such as 2'-methoxy (2'-OMe), 2'-methoxyethoxy, 2'-fluoro (2'-F), 2'-allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA or other bicyclic or "bridged" nucleoside analogs, and 2'-O-(N-methylcarbamate) or those containing base analogs.

[0276] As used herein, amino modification refers to 2'-NH2 or 2'-O--NH2, which may be further modified, or unmodified. Such modification groups are described, for example, in U.S. Patent No. 5,672,695 to Eckstein et al. and U.S. Patent No. 6,248,878 to Matulic-Adamic et al. "Modified nucleotides" in the embodiments disclosed herein may also include nucleotide analogs as described above.

[0277] With respect to the ribonucleic acid molecules disclosed herein, these agents may provide patterned modifications on one or both strands of a double-stranded ribonucleic acid (RNA). As used herein, "alternating position" modifications refer to a defined length of an RNA chain where every other nucleotide is a modified nucleotide, or where there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between each modified nucleotide (e.g., 5'-MNMNMN-3'; 3'-MNMNMN-5'; wherein M is a modified nucleotide and N is an unmodified nucleotide). According to the position numbering convention, the modification pattern begins at the first nucleotide position at the 5' or 3' end. The pattern of modified nucleotides in alternating positions can run the entire length of the chain, but in certain embodiments, includes at least 4, 6, 8, 10, 12, 14 nucleotides, respectively, of which at least 2, 3, 4, 5, 6, or 7 modified nucleotides are included. The modification of the alternating position pairs represents a pattern, i.e., over a defined length of the RNA chain, two consecutive modified nucleotides are separated by two consecutive unmodified nucleotides. The modification pattern starts from the first nucleotide position (e.g., 5'MMNNMMNNMMNN-3'; 3'-MMNNMMNNMMNN-5'; wherein M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at the 5' or 3' end and is carried out according to the position numbering rules described herein. The modified nucleotide pattern of the alternating position can run through the entire chain, but preferably comprises at least 8, 12, 16, 20, 24, 28 nucleotides, respectively, wherein at least 4, 6, 8, 10, 12 or 14 modified nucleotides are contained, respectively. These modification patterns are for reference only, and those skilled in the art will appreciate that other patterns may be used. In certain embodiments, the first and second oligonucleotide sequences of the siRNA are present on separate oligonucleotide chains, which can be and are typically chemically synthesized. In some embodiments, both chains comprise 19 nucleotides. These molecules can be fully complementary and have blunt ends, or they can have dTdT overhangs on one or both chains. In certain embodiments, the siRNA strands are of different lengths, wherein the 3' end of one strand (the sense strand) has a blunt end, while the 3' end of the other strand (the antisense strand) has a 3' overhang. The siRNA may also contain one or more deoxyribonucleic acid (DNA) base substitutions.

[0278] In the nucleic acid construct according to the second aspect of the disclosed embodiment, at least one nucleotide of at least one of the following may be modified:

[0279] (a) the first nucleic acid portion; and / or

[0280] (b) the second nucleic acid portion; and / or

[0281] (c) a third nucleic acid portion; and / or

[0282] (d) a fourth nucleic acid portion; and / or

[0283] where present, 1 to 8 further nucleic acid moieties as defined above; and / or

[0284] where present, a passenger nucleic acid moiety as defined above; and / or

[0285] Where present, a nucleic acid linker moiety as further defined above.

[0286] In an advantageous embodiment, one or more of the odd-numbered coding nucleotides starting from the 5' region may be modified, and / or one or more of the even-numbered coding nucleotides starting from the 5' region may be modified, wherein typically the modification of the even-numbered coding nucleotides is a second modification that is different from the modification of the odd-numbered coding nucleotides:

[0287] the first nucleic acid portion of (a); and / or

[0288] (b) the second nucleic acid portion; and / or

[0289] (c) a third nucleic acid portion; and / or

[0290] (d) a fourth nucleic acid portion; and / or

[0291] where present, 1 to 8 further nucleic acid moieties as defined above; and / or

[0292] where present, a passenger nucleic acid moiety as defined above; and / or

[0293] In certain embodiments, one or more odd-numbered coding nucleotides starting from the 3' region of the third nucleic acid portion of (c) may be modified by a modification that is different from the modification of the odd-numbered coding nucleotides starting from the 5' region of the first nucleic acid portion of (a); and / or

[0294] One or more odd-numbered coding nucleotides from the 3' region of the fourth nucleic acid portion of (d) may be modified by a modification that is different from the modification of the odd-numbered coding nucleotides from the 5' region of the second nucleic acid portion of (b); and / or

[0295] One or more odd-numbered nucleotides from the 3' region of the passenger nucleic acid portion as previously defined herein, if present, may be modified by a modification that is different from the modification of the odd-numbered nucleotides from the 5' region of the 1 to 8 additional nucleic acid portions as previously defined herein; and / or wherein one or more nucleotides of the nucleic acid linker portion as further previously defined herein, if present, may be modified by a modification that is (i) different from the modification of the adjacent nucleotides from the 3' region of the first nucleic acid portion as previously defined herein; and / or (ii) different from the modification of the adjacent nucleotides from the 3' region of the second nucleic acid portion as previously defined herein; and / or different from the modification of the adjacent nucleotides from the 3' region of the 1 to 8 additional nucleic acid portions as previously defined herein (if present).

[0296] In certain embodiments, one or more even-numbered nucleotides from the 3' end of the following regions: (i) the third nucleic acid portion of (c), and / or (ii) the fourth nucleic acid portion of (d), and / or (iii) the passenger nucleic acid portion as previously defined herein (if present) may be modified with a modification that is different from the modification of the odd-numbered nucleotides from the 3' end of these respective portions.

[0297] In certain embodiments, at least one or more modified even-numbered nucleotides of (i) (a) the first nucleic acid portion, and / or (ii) (b) the second nucleic acid portion, and / or (iii) (if present) the 1 to 8 additional nucleic acid portions as previously defined herein, may be adjacent to at least one or more differently modified odd-numbered nucleotides of these respective portions.

[0298] In certain embodiments, at least one or more modified even-numbered nucleotides in the third nucleic acid portion of (i) (c), and / or the fourth nucleic acid portion of (ii) (d), and / or (iii) (if present) the passenger nucleic acid portion as previously defined herein, may be adjacent to at least one or more differently modified odd-numbered nucleotides in these respective portions.

[0299] In certain embodiments, multiple adjacent nucleotides of (i) (a) the first nucleic acid portion, and / or (ii) (b) the second nucleic acid portion, and / or (iii) (if present) 1 to 8 further nucleic acid portions as previously defined herein may be modified by the same modification.

[0300] In certain embodiments, multiple adjacent nucleotides of the third nucleic acid portion of (i) (c), and / or the fourth nucleic acid portion of (ii) (d), and / or (iii) if present) the passenger nucleic acid portion as previously defined herein may be modified by the same modification.

[0301] In certain embodiments, the plurality of adjacent commonly modified nucleotides may be 2 to 4 adjacent nucleotides, advantageously 3 or 4 adjacent nucleotides.

[0302] In certain embodiments, the plurality of adjacent commonly modified nucleotides may be located in the 5' region of the third nucleic acid portion of (i)(c), and / or the 5' region of the fourth nucleic acid portion of (ii)(d), and / or (iii) (if present) the 5' region of the passenger nucleic acid portion described hereinabove.

[0303] In certain embodiments, as further defined herein, a plurality of adjacent co-modified nucleotides may be located in a nucleic acid linker portion. In certain embodiments, the one or more modified nucleotides of the first nucleic acid portion of (a) may not be co-modified in the corresponding nucleotides of the third nucleic acid portion of (c) in the first duplex region; and / or the one or more modified nucleotides of the second nucleic acid portion of (b) may not be co-modified in the corresponding nucleotides of the fourth nucleic acid portion of (d) in the second duplex. And / or, where present, as defined herein, the one or more modified nucleotides of the 1 to 8 additional nucleic acid portions may not have a co-modification that is present in the corresponding nucleotides of the corresponding passenger strand nucleic acid portion of the corresponding duplex region.

[0304] In certain embodiments, the one or more modified nucleotides of the first nucleic acid portion (a) may be shifted by at least one nucleotide relative to the commonly modified nucleotides of the third nucleic acid portion (c); and / or, the one or more modified nucleotides of the second nucleic acid portion (b) may be shifted by at least one nucleotide relative to the commonly modified nucleotides of the fourth nucleic acid portion (d); and / or,

[0305] Where present, the modified nucleotides of the 1 to 8 additional nucleic acid portions may be shifted by at least one nucleotide relative to the common modified nucleotides of the passenger strand nucleic acid portion, as defined herein before.

[0306] In certain embodiments, the modification and / or modifications may each and individually be a sugar, phosphate, or base modification.

[0307] In certain embodiments, the modification can be selected from nucleotides having a 2' modified sugar; conformationally constrained nucleotide (CRN) sugars, such as locked nucleic acids (LNA), (S)-constrained ethyl bicyclic nucleic acids and constrained ethyl (cEt), tricyclic DNA; morpholinos, unlocked nucleic acids (UNA), glycol nucleic acids (GNA), D-hexitol nucleic acids (HNA) and cyclohexene nucleic acids (CeNA). In advantageous embodiments, the 2' modified sugar can be selected from 2'-O-alkyl modified sugars, 2'-O-methyl modified sugars, 2'-O-methoxyethyl modified sugars, 2'-O-allyl modified sugars, 2'-C-allyl modified sugars, 2'-deoxy modified sugars such as 2'-deoxyribose, 2'-F modified sugars, 2'-arabinose-fluorine modified sugars, 2'-O-benzyl modified sugars, 2'-amino modified sugars, 2'-O-methyl-4-pyridine modified sugars.

[0308] In certain embodiments, the base modification may be any of an abasic nucleotide and a nucleotide containing a non-natural base.

[0309] In certain embodiments, at least one modification may be a 2'-O-methyl modification in the ribose moiety.

[0310] In certain embodiments, at least one modification can be a 2'-F modification of the ribose moiety.

[0311] In certain embodiments, the first nucleic acid portion (i)(a); and / or, the second nucleic acid portion (ii)(b); and / or, where present, the nucleotide at any one of positions 2 and 14 downstream of the first nucleotide of the 5' region of the 1 to 8 additional nucleic acid portions (iii), as defined herein before, may not contain a 2'-O-methyl modification in the ribose sugar.

[0312] In certain embodiments, the third nucleic acid portion (i)(c); and / or, the fourth nucleic acid portion (ii)(d); and / or, where present, one, two or all three nucleotides in the passenger strand (iii) as defined herein before; the positions of which correspond to the first nucleic acid portion (i)(a); and / or, the second nucleic acid portion (ii)(b); and / or, where present, any one of the nucleotides at positions 11 to 13 downstream of the 1 to 8 further nucleic acid portions (iii) as defined herein before, respectively, may not contain a 2'-O-methyl modification in the ribose moiety.

[0313] In certain embodiments, the first nucleic acid portion (i)(a); and / or, the second nucleic acid portion (ii)(b); and / or, where present, 1 to 8 additional nucleic acid portions (iii) as defined herein before, located nucleotides 2 and 14 downstream; may comprise a 2'-F modification in the ribose moiety.

[0314] In certain embodiments, the third nucleic acid portion (i)(c); and / or, the fourth nucleic acid portion (ii)(d); and / or, where present, one, two or all three nucleotides in the passenger strand (iii) as defined herein before; whose positions correspond to the first nucleic acid portion (i)(a); and / or, the second nucleic acid portion (ii)(b); and / or, where present, any one of the nucleotides 11 to 13 downstream of the 1 to 8 additional nucleic acid portions (iii) as defined herein before; may contain a 2'-F modification in the ribose moiety.

[0315] In certain embodiments, all remaining nucleotides may comprise a 2'-O-methyl modification or a 2'-F modification in the ribose moiety, advantageously with the exception of labile linked unmodified nucleotides as defined herein. Advantageously, the remaining nucleotides may comprise a 2'-O-methyl modification in the ribose moiety.

[0316] In certain embodiments, the one or more (advantageously one) unmodified nucleotides represent any nucleotide of the nucleic acid linker portion as further defined herein before, advantageously adjacent to the following part of the nucleic acid linker portion as further defined herein before: the third nucleic acid portion (i)(c); and / or, the fourth nucleic acid portion (ii)(d); and / or, where present, the passenger strand (iii) as defined herein before.

[0317] In certain embodiments, the 3' terminal positions of the first and third nucleic acids may be partially replaced with unmodified nucleotides.

[0318] In certain embodiments, the nucleic acid construct may comprise at least one vinylphosphonate modification, e.g., in the 5' region of the first nucleic acid portion (i)(a); and / or, the second nucleic acid portion (ii)(b); and / or, where present, 1 to 8 additional nucleic acid portions (iii) as previously defined herein.

[0319] In certain embodiments, one or more nucleotides of

[0320] a first nucleic acid portion (a); and / or

[0321] a second nucleic acid portion (b); and / or

[0322] a third nucleic acid portion (c); and / or

[0323] a fourth nucleic acid portion (d); and / or

[0324] where present, 1 to 8 further nucleic acid moieties as defined herein before; and / or

[0325] where present, the portion of the Passenger Chain as defined herein before;

[0326] It can be an inverted nucleotide and can be linked to an adjacent nucleotide via the 3' carbon of a nucleotide and the 3' carbon of an adjacent nucleotide, and / or it can be an inverted nucleotide and can be linked to an adjacent nucleotide via the 5' carbon of a nucleotide and the 5' carbon of an adjacent nucleotide.

[0327] In certain embodiments, the inverted nucleotide can be linked to an adjacent nucleotide through a phosphate group via a phosphodiester bond; or can be linked to an adjacent nucleoside through a phosphorothioate group; or can be linked to an adjacent nucleoside through a phosphorodithioate group.

[0328] In certain embodiments, modifications between strands within the construct comprise an alternating modification pattern, advantageously with odd-numbered nucleosides substituted with fluorine and even-numbered nucleotides replaced with -OMe.

[0329] Compositions and pharmaceutical compositions including muRNA oligomer constructs

[0330] According to a second aspect, the disclosed embodiments relate to a composition comprising the nucleic acid construct according to the first aspect and a physiologically acceptable excipient.

[0331] According to a third aspect, the disclosed embodiments relate to a pharmaceutical composition comprising a nucleic acid construct constructed according to the first aspect.

[0332] The pharmaceutical composition may further include pharmaceutically acceptable excipients, diluents, antioxidants and / or preservatives.

[0333] The oligomeric compound according to the first aspect and / or the construct according to the second aspect may be the sole pharmaceutically active agent. Alternatively, the pharmaceutical composition further comprises one or more further pharmaceutically active agents. Advantageously, the further pharmaceutically active agent is selected from the following options:

[0334] Vossipa; wupanosan; statins such as rosuvastatin and simvastatin; fibrates such as fenofibrate; compounds that lower low-density lipoprotein cholesterol such as statins and ezetimibe, and / or drugs that reduce hypertension, wherein the further pharmaceutically active agent can be selected from the group consisting of diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, alpha2 receptor agonists, renin inhibitors, alpha-blockers, peripherally acting adrenergic agents, selective D1 receptor partial agonists, non-selective alpha adrenergic antagonists, synthetic steroidal antimineralocorticoid agents; combinations of any of the foregoing; and a hypertension therapeutic agent formulated as a multi-drug combination, more optionally, an angiotensin II receptor antagonist selected from the group consisting of losartan, valsartan, olmesartan, eprosartan and azilsartan Determining the efficacy of the siRNA molecule

[0335] Depending on the specific target, one or more siRNA sequences targeting one or more proteins, and the dosage of the nanoparticle composition, can be observed to result in partial or complete loss of function of the targeted protein. Typical examples include reduced or absent RNA levels, or reduced or absent expression of the target protein or its encoded polypeptide, in at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more of the target cells. Inhibition of target protein levels or expression refers to the absence (or observable decrease) of the target RNA or the protein encoded by the RNA. Specificity refers to the ability to inhibit the RNA of the target protein without significantly affecting other genes in the cell. Inhibition can be confirmed by examining the external properties of the cells or organisms or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, microarray gene expression monitoring, antibody binding, enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence-activated cell analysis (FACS). The inhibitory effect of the siRNA agents of the disclosed embodiments on the target RNA sequence can also be measured based on the effect of such siRNA agents on the development / progression of a disease or condition associated with the target protein (such as tumor formation, growth, metastasis, etc.) in vivo or in vitro. The treatment and / or reduction of tumor or cancer cell levels can include preventing or reducing the growth of tumor or cancer cell levels, or reducing them by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and can also be measured in logarithmic form, for example, by administering the nanoparticle composition to a cell, tissue or subject, a 10-fold, 10 ... 4 times, 105 times, 10 6 times or 107 The subject may be a mammal, such as a human.

[0336] Determination of dose and toxicity

[0337] Toxicity and therapeutic efficacy of the compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD 50 (dose that causes 50% mortality) and ED 50 (The therapeutic dose that is effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as LD 50 / ED 50 Advantageous compounds exhibit high therapeutic indices.

[0338] The data from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. Advantageously, the dosage of the composition should be within the range that contains the ED 50 The dose can vary within this range depending on the dosage form and route of administration used. For the compositions described herein, the therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC values ​​determined in cell culture. 50 (ie, the concentration of the composition that achieves a half-maximal inhibition of symptoms.) Such information can be used to more accurately determine effective doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography (HPLC).

[0339] The therapeutically effective amount of the compositions described herein can be in the range of about 1 pg to 1000 mg. For example, 10, 30, 100, or 1000 picograms, or 10, 30, 100, or 1000 nanograms, or 10, 30, 100, or 1000 micrograms, or 10, 30, 100, or 1000 milligrams, or 1-5 grams of the composition can be administered. Typically, a suitable dosage unit for the compositions described herein is 0.001 to 0.25 mg / kg body weight, or 0.01 to 20 micrograms / kg body weight, or 0.001 to 5 micrograms / kg body weight, or 1 to 500 nanograms / kg body weight, or 0.01 to 10 micrograms / kg body weight, or 0.10 to 5 micrograms / kg body weight, or 0.1 to 2.5 micrograms / kg body weight per subject per day. The pharmaceutical composition can be administered once daily or in a dosage unit comprising two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In this case, the siRNA contained in each sub-dose must be reduced accordingly to achieve the total daily dosage unit. The dosage unit can also be formulated as a single dose for several consecutive days, for example using a conventional sustained-release formulation that can continuously and stably release siRNA over several days. Sustained-release formulations are known in the art. In this embodiment, the dosage unit comprises a corresponding multiple of the daily dose. Regardless of the dosage form, the pharmaceutical composition must contain a sufficient amount of siRNA to inhibit the expression of the target gene in the treated animal or human. The composition should be formulated so that the sum of the multiple siRNA units contains a sufficient dose.

[0340] The composition can be administered as a single dose, or one or more times per day, to one or more times per week, including administration every other day. It will be appreciated by those skilled in the art that certain factors may affect the dosage and duration required to effectively treat a subject, including but not limited to the severity of the disease or condition, previous treatment, the general health and / or age of the subject, and the presence of other diseases. In addition, treatment of a subject with a therapeutically effective amount of a composition as described herein may include a single treatment, or advantageously, may include a series of treatments.

[0341] As used herein, a "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount of a siRNA composition sufficient to produce the intended pharmacological, therapeutic, or preventive effect. The phrases "pharmacologically effective amount," "therapeutically effective amount," or "effective amount" all refer to the amount of the composition that produces the intended pharmacological, therapeutic, or preventive effect. For example, if a particular clinical treatment is considered effective when a measurable indicator associated with a disease or condition is reduced by at least 30%, then the therapeutically effective amount of the drug used to treat that disease or condition is the amount sufficient to reduce that indicator by at least 30%.

[0342] The pharmaceutical compositions described herein, suitably formulated, can be administered by means known in the art, such as parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, respiratory (aerosol), rectal, vaginal, and topical (including oral and sublingual) administration. Advantageously, the pharmaceutical compositions are administered by intravenous or parenteral infusion or injection.

[0343] Pharmaceutical composition and administration method thereof

[0344] The nanoparticle composition can be further formulated into a pharmaceutical composition using methods known in the art. The composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral routes, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may contain the following ingredients: a sterile diluent, such as water for injection, physiological saline, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate; and an agent for regulating osmotic pressure, such as sodium chloride or glucose. The pH value can be adjusted using an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0345] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (such as water-soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid for easy syringability. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyols (such as mannitol, trehalose, sorbitol), sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions can be prepared by incorporating the active compound into the composition.

[0346] Sterile injectable solutions are prepared by adding the active compound in the desired amount to a selected solvent, along with one or more of the ingredients listed above, as needed, followed by sterile filtration. Dispersions are typically prepared by adding the active compound to a sterile vehicle containing a basic dispersion medium and the other ingredients listed above. Preferred methods for preparing sterile powders for sterile injectable solutions are vacuum drying and freeze drying, which yield a powder of the active ingredient and any other desired ingredients from a previously sterile-filtered solution.

[0347] The composition can also be prepared by a carrier to protect the compound from rapid clearance in the body, such as a controlled release formulation, including implants and microcapsule delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such preparations can be prepared using standard techniques, and related materials can also be commercially available from Alza and Nova Pharmaceuticals. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as described in U.S. Patent No. 4,522,811.

[0348] Treatment

[0349] The compositions can be administered as described above and, advantageously, can be delivered systemically or intratumorally. The compositions can be administered as a monotherapy (i.e., not in combination with other treatments) or as part of a combination therapy regimen that includes one or more additional drugs. Advantageously, as described below, the compositions can be used as part of a combination therapy regimen that includes an effective amount of at least one additional chemotherapeutic agent.

[0350] Diseases treated by muRNA oligomer compounds and further applications

[0351] According to a fourth aspect, the disclosed embodiments relate to a nucleic acid construct according to the first aspect, for use in human or veterinary medicine or therapy;

[0352] According to a fifth aspect, the disclosed embodiments relate to the nucleic acid construct according to the first aspect, for use in a method of treating, ameliorating and / or preventing a disease or disorder.

[0353] The disease or disorder is a disease or disorder associated with APOC3 and / or AGT, or a disease or disorder requiring reduction of APOC3 and / or AGT expression.

[0354] Specifically, the disease or condition is selected from a disease or condition associated with APOC3, or a disease or condition requiring a decrease in APOC3 expression level, and the disease or condition is advantageously selected from dyslipidemia, including mixed dyslipidemia, hyperchylomicronemia, familial hyperchylomicronemia; hypertriglyceridemia, advantageously severe hypertriglyceridemia and / or hypertriglyceridemia with a triglyceride level greater than 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; pancreatitis, including acute pancreatitis; and / or hypertension, critical hypertension, essential hypertension, secondary hypertension, isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, Hypertension, resistant hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, unstable hypertension; hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypotension, glomerulosclerosis, aortic coarctation, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD); impaired glucose tolerance, type 2 diabetes and metabolic syndrome.

[0355] According to a sixth aspect, the disclosed embodiments relate to a method of treating a disease or condition comprising administering a nucleic acid construct according to the first aspect to an individual in need of treatment.

[0356] The nucleic acid construct can be administered to the individual subcutaneously or intravenously.

[0357] According to a seventh aspect, the disclosed embodiment relates to a use of the nucleic acid construct according to the first aspect as a tool for studying gene function in research.

[0358] According to an eighth aspect, the disclosed embodiments relate to use of the nucleic acid construct according to the first aspect in the preparation of a medicament for treating a disease or disorder.

[0359] It should also be noted that the scope of the disclosed embodiments encompasses sequences corresponding to the above-disclosed sequences, wherein the 5'-terminal nucleoside of the antisense strand (guide strand) can comprise any nucleobase that may be present in an RNA molecule, in other words, can be any of adenine (A), uracil (U), guanine (G), or cytosine (C). Furthermore, the scope of the disclosed embodiments also encompasses sequences corresponding to the above-disclosed sequences, wherein the 3'-terminal nucleoside of the sense strand (passenger strand) can comprise any nucleobase that may be present in an RNA molecule, in other words, can be any of adenine (A), uracil (U), guanine (G), or cytosine (C), but ideally is a nucleobase that is complementary to the 5'-terminal nucleobase of the antisense strand (guide strand). Specific examples include SEQ ID NOs: 142 and 143, 144 and 145, and 146 and 147, wherein these sequences have been modified at the aforementioned positions compared to SEQ ID NOs: 103 and 131, 132 and 133, and 134 and 135.

[0360] Although these methods are shown and described as a series of actions that are performed in a particular order, it is to be understood and appreciated that these methods are not limited by this order. For example, certain actions may occur in a different order than described herein. Additionally, one action may occur simultaneously with another action. Furthermore, in some cases, not all actions may be required to implement the methods described herein.

[0361] The order of the steps of the methods described herein is exemplary, but the steps may be performed in any suitable order, or simultaneously where appropriate. Furthermore, steps may be added or substituted, and individual steps may be deleted, from any method without departing from the scope of the subject matter described herein. Aspects of any of the above examples may be combined with aspects of any other example to form further examples.

[0362] It should be understood that the above description of preferred embodiments is merely illustrative and that various modifications may be made by those skilled in the art. The above description covers examples of one or more embodiments. Of course, for the purposes of describing the above aspects, it is not possible to describe every conceivable modification and variation of the above-described compounds, compositions, or methods, but those skilled in the art will recognize that there are many further modifications and permutations of the various aspects. Therefore, the described aspects are intended to cover all such variations, modifications, and variations that fall within the scope of the appended claims.

[0363] example

[0364] The following examples illustrate some specific embodiments of the present disclosure, but are not intended to limit them. In addition, while providing specific embodiments, the inventors have considered the general application of these specific embodiments. For example, disclosures of oligonucleotides with specific motifs or modification patterns provide reasonable support for other oligonucleotides with identical or similar motifs or modification patterns.

[0365] The synthesis of RNAi constructs according to the disclosed embodiments can be performed using synthesis methods known to those skilled in the art, such as those disclosed at https: / / en.wikipedia.org / wiki / Oligonucleotide_synthesis, which is hereby incorporated by reference in its entirety. The only difference from the synthesis method disclosed in this reference is that a GalNAc phosphoramidite immobilized on a support is used in the first synthesis step.

[0366] Example 1: In vitro assay

[0367] Materials and methods

[0368] Cell culture

[0369] Human primary hepatocytes (5 donors pooled, Sekisui XenoTech, HPCH05+) were thawed immediately before the experiment and cultured in 1x complete Williams medium (Gibco, A1217601) supplemented with a hepatocyte plating supplement (Gibco, CM3000). To ensure compound stability, the fetal bovine serum (FBS) concentration was adjusted from the manufacturer's formula to a final 2.5% (instead of 5%).

[0370] compound; preparation

[0371] A seven-step, five-fold dilution series was prepared for each compound in basal WEM medium, ranging from 2 μM to 0.000128 μM.

[0372] Transfection

[0373] On the day of transfection, human primary hepatocytes were thawed in 45 mL of human OptiThaw (Sekisui XenoTech, K8000) and centrifuged at 200 g for 5 minutes. The cells were resuspended in 2x complete WEM and counted. The cells were then seeded into a 96-well type 1 rat tail collagen plate at a density of 25,000 cells per well, with 50 μL of 2x complete WEM per well, and the cells were allowed to rest and adhere for 4 hours before transfection. After standing, 50 microliters of each dilution was added to each set of three replicate wells seeded with hepatocytes, so that the final dilution series concentration was reduced from 1 μM to 0.000064 μM in 100 microliters of 1x complete WEM.

[0374] qPCR

[0375] 72 hours after transfection, cells were harvested and RNA was extracted using the PureLink Pro 96 Total RNA Purification Kit (ThermoFisher, 12173011A) according to the manufacturer's protocol. ApoC3 and AGT expression was analyzed using TaqMan quantitative PCR (qPCR) using the Luna Universal Probe One-Step RT-qPCR Kit (NEB, E3006). For each sample, the ApoC3 TaqMan probe set (Hs00906501_gl-FAM) or probe (Hs01586213_m1-FAM) was multiplexed with a universal GAPDH VIC probe (ThermoFisher, 4326317E). Thermal cycling and data acquisition were performed using the ABI QuantStudio 3 / 5 Real-Time PCR System.

[0376] result

[0377] The results are shown in Figure 1 and Figure 2 and Tables 3 and 4 below.

[0378] Table 3: AGT knockdown (1000 nM) and IC50 (nM)

[0379] SEQ ID No. Structure Name KD% (1000nM) IC50(nM) 164-165 APO28-AGT52A 82.96 1.955 148-149 APO28-AGT27 88.82 1.867 160-161 APO28-AGT27A 89.09 0.553 162-163 APO28-AGT62A 60.76 13.270

[0380] Table 4: Knockdown effect of APOC3 (1000 nM) and IC50 (nM)

[0381] SEQ ID No. Structure Name KD% (1000nM) IC50(nM) 164-165 APO28-AGT52A 87.10 0.953 148-149 APO28-AGT27 95.50 0.753 160-161 APO28-AGT27A 95.82 0.432 162-163 APO28-AGT62A 94.85 0.477

[0382] The dual-targeting muRNAs tested performed extremely well, inhibiting either target with half-maximal inhibitory concentration (IC50) values ​​in the picomolar to single-digit (and in one case, double-digit) nanomolar range.

[0383] Example 2: Dose-response study evaluating angiotensinogen-apolipoprotein C3 combination (AGT-APOC3; muRNA) in a humanized liver-uPA-SCID mouse model

[0384] Materials and methods

[0385] 1. Research objectives

[0386] The objectives of this non-GLP study in humanized Liver-uPA-SCID mice were to evaluate:

[0387] i. Dose response of N-acetylgalactosamine (GalNAc)-conjugated mxRNA constructs targeting human angiotensinogen (AGT)

[0388] ii. Dose response of N-acetylgalactosamine (GalNAc)-conjugated dual-targeting muRNA constructs targeting human angiotensinogen (AGT) and apolipoprotein C3 (APOC3)

[0389] Compounds will be administered subcutaneously, and mice will survive for up to 14 days.

[0390] Prior to necropsy, blood will be collected for plasma samples. At necropsy, three liver biopsies (2 mm) from each animal will be preserved in separate vials in RNAlater, snap-frozen, and stored at -80°C. Three additional liver biopsies (2 mm) will be taken, snap-frozen in the same vial, and stored at -80°C. The remaining liver will be snap-frozen and stored at -80°C.

[0391] 2. Test system information

[0392] Animal testing

[0393] 2.1.1. Common name: Mouse

[0394] 2.1.2. Species / Type: Rodent - Humanized Liver - uPA-SCID Mouse

[0395] 2.1.3. Number of animals (by sex): 60 males, all naive (primary immunization status)

[0396] 2.1.4. Age range: 14-19 weeks old mice,

[0397] 2.1.5. Weight range: All mice approximately 20 grams

[0398] 3. Study Design

[0399] 3.1. Design details

[0400] This study will use one type of mouse, 60 humanized liver-uPA-SCID mice. Animals will be divided into treatment type, dose and

[0401] Survival groups. Each animal will be treated with the test substance by subcutaneous injection. (Note: Subcutaneous injection is required. If the

[0402] The test article will not work if injected into the muscle area instead of the subcutaneous injection site, or if injected into a vein / bloodstream.

[0403] use).

[0404] • Group 1A will have four animals and receive a control dose of PBS.

[0405] Groups 2A, 2B, and 2C will have 4 / 5 animals and will receive a single dose of (AGT27A)

[0406] 5mg / kg, 10mg / kg and 30mg / kg.

[0407] Groups 3A, 3B, and 3C will have 4 / 5 animals and will receive a single dose of (AGT52A), respectively.

[0408] 5mg / kg, 10mg / kg and 30mg / kg.

[0409] Groups 4A, 4B, and 4C will have 4 / 5 animals and will receive a single dose of (AGT27A-A28) at 5 mg / kg, 10 mg / kg, and 30 mg / kg, respectively.

[0410] • Groups 5A, 5B and 5C will have 4 / 5 animals and will receive a single dose of (AGT52A-A28) at 5 mg / kg, 10 mg / kg and 30 mg / kg respectively.

[0411] Animals will survive for 14 days. See Study Table 5 for details.

[0412] Prior to necropsy, animals are deeply anesthetized, and a terminal blood sample is collected via the vena cava. The collected blood is placed in a plasma separator tube. After separation, the plasma sample is divided equally into two aliquots, placed into labeled vials, quickly frozen, and stored at -80°C.

[0413] Note: Plasma will be used for protein detection, so care must be taken to avoid hemolysis or coagulation.

[0414] At autopsy, three 2-mm biopsies were collected from each of the left, middle, and right lobes of the liver. These were placed in separate vials, soaked in RNA later reagent for 15 minutes, then snap-frozen and stored at -80°C. Additionally, one additional 2-mm liver biopsy was collected from each of the left, middle, and right lobes of the liver, placed in the same vial, snap-frozen, and stored at -80°C. The remaining liver tissue was snap-frozen, stored in 10-ml conical tubes, and stored at -80°C.

[0415] Table 5: Research table

[0416]

[0417] AGT27A sequence (SEQ ID NO: 166):

[0418] [5Phos][m A ][Ps][fU][Ps][mA][fG][mA][fA][mG][fA][mA][fA][mA][fG][mG][fU][Ps][mG][Ps][fG][Ps][m G][Ps][fA][Ps][mG][Ps][mA][fC][fC][fU][mU][fU][mU][fC][mU][fU][mC][fU][Ps][mA][Ps][f U ][Ps][3xGalNac]

[0419] 4. Test items and auxiliary materials information

[0420] 4.1. Test drug 1:

[0421] 4.1.1. Identification: AGT27A

[0422] 4.1.2. Manufacturer: Sirna Pharmaceuticals

[0423] 4.1.3. Description: GalNAc-mxRNA targeting human angiotensinogen

[0424] (AGT)mRNA

[0425] 4.1.4. Lot / Batch Number: To be recorded on the Study Material Form.

[0426] 4.1.5. Validity period: will be recorded on the research materials form.

[0427] 4.1.6. Storage temperature: 4℃

[0428] 4.1.7. Biohazard conditions: None

[0429] 4.1.8.SDS*: To be determined

[0430] 4.1.9. Appearance: Transparent liquid

[0431] 4.1.10. Dosage information: See Table 5 of the study

[0432] 4.1.11. Storage of residual samples: None

[0433] 4.2. Test drug 2:

[0434] 4.2.1. Identification: AGT52A

[0435] 4.2.2. Manufacturer: Sirna Pharmaceuticals

[0436] 4.2.3. Description: GalNAc-mxRNA targeting human angiotensinogen (AGT) mRNA

[0437] 4.2.4. Lot / Batch Number: To be recorded on the Study Material Form.

[0438] 4.2.5. Validity period: will be recorded on the research materials form.

[0439] 4.2.6. Storage temperature: 4°C

[0440] 4.2.7. Biohazard conditions: None

[0441] 4.2.8.SDS*: To be determined

[0442] 4.2.9. Appearance: Transparent liquid

[0443] 4.2.10. Dosage information: See Table 5 of the study

[0444] 4.2.11. Storage of residual samples: None

[0445] 4.3. Test drug 3:

[0446] 4.3.1. Identification: AGT27A-A28

[0447] 4.3.2. Manufacturer: Sirnaomics

[0448] 4.3.3. Description: GalNAc-muRNA targeting human angiotensinogen (AGT) and apolipoprotein C3 (APOC3) mRNA

[0449] 4.3.4. Batch number: will be recorded on the research material form

[0450] 4.3.5. Validity period: will be recorded in the research material form

[0451] 4.3.6. Storage temperature: 4℃

[0452] 4.3.7. Biohazard status: None

[0453] 4.3.8.SDS*: To be determined (TBD)

[0454] 4.3.9. Appearance: Clear liquid

[0455] 4.3.10. Dosage Information: See Study Table 5

[0456] 4.3.11. Storage of remaining test items: None

[0457] 4.4. Test drug 4:

[0458] 4.4.1 Identification: AGT52A-A28

[0459] 4.4.2 Manufacturer: Sirnaomics

[0460] 4.4.3 Description: Targeting human angiotensinogen (AGT) and apolipoprotein C3 (APOC3) GalNAc-muRNA of mRNA

[0461] 4.4.4 Batch number: will be recorded in the research material form

[0462] 4.4.5 Validity period: will be recorded in the research material form

[0463] 4.4.6 Storage temperature: 4℃

[0464] 4.4.7 Biohazard Status: None

[0465] 4.4.8SDS*: To be determined (TBD)

[0466] 4.4.9 Appearance: Clear liquid Dosage information: See Study Table 5

[0467] 4.4.10. Storage of residual samples: None

[0468] 5. Necropsy and Tissue Sample Collection Procedure

[0469] NOTE: Each time a tissue sample is collected, a separate tool will be used. To prevent cross contamination, the tissue collection tool must be changed after each tissue sample is collected.

[0470] A 2 mm biopsy specimen was collected from each of the left, middle, and right lobes of the liver. Each biopsy specimen was placed in a 2 ml Eppendorf tube, infused with 1.5 ml of RNAlater reagent, and allowed to soak for 15 minutes. The specimen was then snap-frozen and stored at -80°C. Another 2 mm biopsy specimen was collected from each of the left, middle, and right lobes of the liver. All specimens were placed in the same 2 ml Eppendorf tube, snap-frozen, and stored at -80°C. The remaining liver tissue was snap-frozen, stored in a 10 ml conical tube, and stored at -80°C.

[0471] result

[0472] The results are shown in Figures 3 to 8. Excellent gene knockdown effects were demonstrated at both the mRNA and protein levels.

Claims

1. A nucleic acid construct comprising or consisting of: (a) a first nucleic acid portion that is at least partially complementary to at least a first portion of an RNA transcribed from an apolipoprotein C3 (APOC3) gene; (b) a second nucleic acid portion that is at least partially complementary to at least a second portion of an RNA transcribed from an angiotensinogen (AGT) gene; (c) a third nucleic acid portion that is at least partially complementary to the first nucleic acid portion in (a) so as to form a first nucleic acid double-stranded region therewith; (d) a fourth nucleic acid portion that is at least partially complementary to the second nucleic acid portion in (b) so as to form a second nucleic acid double-stranded region therewith.

2. The construct of claim 1 , wherein the construct is designed such that upon in vivo administration, the construct dissociates to produce at least first and second discrete nucleic acid targeting molecules that target the portion of the RNA transcribed from the target gene in (a) and (b), respectively; Thus, (i) the first nucleic acid targeting molecule is capable of regulating the expression of the target gene in (a) and comprises or is derived from at least the first nucleic acid portion in (a), and (ii) the second nucleic acid targeting molecule is capable of regulating the expression of the target gene in (b) and comprises or is derived from the second nucleic acid portion in (b).

3. The construct of claim 1 or 2, wherein the construct is designed to dissociate such that the first and second discrete nucleic acid targeting molecules are each processed by an independent RNAi-inducing silencing complex.

4. The construct according to any one of claims 1 to 3, further comprising at least one labile functional group, such that upon in vivo administration, the construct is cleaved to produce the at least first and second discrete nucleic acid targeting molecules.

5. The construct of claim 4, wherein the labile functional group comprises one or more unmodified nucleotides.

6. The construct of claim 5, wherein one or more unmodified nucleotides in the labile functional group represent one or more cleavage sites within the construct, such that upon in vivo administration, the construct is cleaved at the one or more cleavage sites, thereby producing the at least first and second discrete nucleic acid targeting molecules.

7. The construct of claim 6, wherein the cleavage sites are respectively located within the construct such that after cleavage, the first discrete nucleic acid targeting molecule comprises or is derived from the first nucleic acid double-stranded region, and the second discrete nucleic acid targeting molecule comprises or is derived from the second nucleic acid double-stranded region.

8. The construct of claim 7, wherein the first discrete nucleic acid targeting molecule comprises or consists of the first nucleic acid portion in (a) and the third nucleic acid portion in (c), and / or the second discrete nucleic acid targeting molecule comprises or consists of the second nucleic acid portion in (b) and the fourth nucleic acid portion in (d).

9. The construct according to any one of claims 1 to 8, wherein (a) the nucleobase sequence of the first nucleic acid portion is selected from SEQ ID NOs: 1 to 30; (b) the nucleobase sequence of the second nucleic acid portion is selected from SEQ ID NOs: 61 to 90; (c) the nucleobase sequence of the third nucleic acid portion is selected from SEQ ID NOs: 31 to 60; and / or (d) The nucleobase sequence of the fourth nucleic acid portion is selected from SEQ ID NOs: 91 to 120.

10. The construct according to any one of claims 1 to 9, wherein the first nucleic acid portion in (a) is directly or indirectly linked to the fourth nucleic acid portion in (d) in the form of a primary structure.

11. The construct according to any one of claims 1 to 10, wherein the second nucleic acid portion in (b) is directly or indirectly linked to the third nucleic acid portion in (c) in the form of a primary structure.

12. The construct according to any one of the preceding claims, wherein the first nucleic acid portion has a nucleobase sequence of SEQ ID NO: 3, and / or the second nucleic acid portion has a nucleobase sequence selected from SEQ ID NO: 61, 66, 67, 73, 68 and 76.

13. The construct according to any one of the preceding claims, wherein the third nucleic acid portion has the nucleobase sequence of SEQ ID NO: 33, and / or the fourth nucleic acid portion has a nucleobase sequence selected from the group consisting of SEQ ID NO: 91, 96, 97, 103, 98 and 106.

14. The construct according to any one of claims 1 to 9, 12 or 13, further comprising 1 to 8, preferably 2, additional nucleic acid portions, each of which is at least partially complementary to an additional 1 to 8 RNA portions transcribed from one or more target genes, the target genes being identical or different to each other and / or identical or different to the target genes defined in (a) and / or (b), and wherein each of the 1 to 8 additional nucleic acid portions forms an additional double-stranded region with a respective complementary nucleic acid portion, each of which is at least partially complementary thereto.

15. The construct of claim 14, wherein the second nucleic acid portion and the 1 to 8 additional nucleic acid portions in (b) are directly or indirectly linked to the selected passenger nucleic acid portion in the form of their respective primary structures.

16. The construct of any one of claims 10, 11 or 15, wherein the direct or indirect linkage represents: (i) an internucleotide bond, or (ii) a nucleic acid linking portion consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides, which is preferably single-stranded.

17. The construct of claim 16(i), wherein the linkage is direct, thereby forming (a) a continuous chain.

18. The construct according to any one of claims 1 to 17, in particular the construct according to claim 16(i), wherein there is a certain complementarity between the first nucleic acid part in (a) and the second nucleic acid part in (b), or between the third nucleic acid part in (c) and the fourth nucleic acid part in (d).

19. The construct of claim 18, wherein the complementarity: (i) is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pairs, preferably 2, 3, 4 or 5 base pairs; and / or (ii) is present between the first nucleic acid portion in (a) and the second nucleic acid portion in (b).

20. The construct according to claim 16(i) to 19, as defined in claim 5, wherein the internucleotide bond involves at least one of the one or more unmodified nucleotides, and cleavage preferably occurs at the 3' position (at least one) of the unmodified nucleotide.

21. according to the construct described in any one in claim 1 to 20, wherein the first nucleic acid part in (a), and / or the second nucleic acid part in (b), and / or the third nucleic acid part in (c), and / or the fourth nucleic acid part in (d) are respectively 7 to 25 nucleotides in length.

22. The construct according to claim 21, wherein the first nucleic acid portion in (a) and / or the second nucleic acid portion in (b) is 18 to 21 nucleotides in length, more preferably 18 to 20 nucleotides in length, most preferably 19 nucleotides in length.

23. The construct of claim 21 or 22, wherein the third nucleic acid portion in (c), and / or the fourth nucleic acid portion in (d) is 11 to 20 nucleotides in length, more preferably 13 to 16 nucleotides in length, further preferably 14 or 15 nucleotides in length, and most preferably 15 nucleotides in length.

24. according to the construct described in any one in claim 21 to 23, wherein said unmodified nucleotide is positioned at any position among the 18th to 25th, more preferably is positioned at any position among the 18th to 21st, and / or is positioned at the 3 ' terminal position of the first nucleic acid part among (a) and / or the third nucleic acid part among (c).

25. The construct of claim 24, wherein the unmodified nucleotide is located at position 19.

26. A construct according to any one of claims 17 to 19 or 21 to 23 as based on claim 16(ii), wherein the nucleic acid linking portion is 1 to 8 nucleotides in length, preferably 2 to 7 or 3 to 6 nucleotides in length, more preferably about 4 or 5 nucleotides, most preferably 4 nucleotides in length.

27. The construct of any one of claims 21 to 26, wherein the length of one or more double-stranded regions is independently 10 to 19 base pairs, more preferably 13 to 19 base pairs, further preferably 13, 14 or 15 base pairs, and most preferably 15 base pairs, wherein one mismatch may optionally be present within the double-stranded region.

28. The construct of any one of claims 1 to 27, further comprising one or more ligands.

29. The construct according to any one of claims 1 to 28, wherein the first nucleic acid part in (a), and / or the second nucleic acid part in (b), and / or the third nucleic acid part in (c), and / or the fourth nucleic acid part in (d), and, where present, the 1 to 8 additional nucleic acid parts as defined in claims 14 and 15, and / or the passenger nucleic acid part as defined in claim 14 or 15, respectively, have a 5' to 3' directionality, thereby defining their 5' and 3' end regions.

30. The construct according to any one of claims 28 or 29, wherein one or more ligands are conjugated at the 3' region, preferably the 3' end, of any of: (i) the third nucleic acid moiety in (c), and / or (ii) the fourth nucleic acid moiety in (d), and / or, if present, (iii) the passenger nucleic acid moiety as defined in claim 14 or 15.

31. The construct according to any one of claims 28 to 30, wherein one or more ligands are conjugated to one or more regions between the 5' and 3' regions of any of the nucleic acid moieties, preferably to the third nucleic acid moiety in (c), and / or the fourth nucleic acid moiety in (d), and / or to the passenger nucleic acid moiety as defined in claim 14 or 15.

32. The construct according to any one of claims 28 to 31, wherein one or more ligands are conjugated to the 5' terminal region, preferably the 5' end, or the 3' terminal region, preferably the 3' end, of any of the nucleic acid moieties.

33. The construct of any one of claims 28 to 32, wherein the one or more ligands are any cell targeting moieties such as lipids, carbohydrates, aptamers, vitamins and / or peptides that bind to a specific target on the cell membrane or cell surface.

34. The construct of claim 33, wherein the one or more carbohydrates can be a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide or a polysaccharide.

35. The construct of claim 34, wherein the one or more carbohydrates comprise one or more hexose moieties.

36. The construct of claim 35, wherein the one or more hexose moieties are one or more galactose moieties, one or more lactose moieties, one or more N-acetylgalactosamine moieties and / or one or more mannose moieties.

37. The construct of claim 36 comprising two or three N-acetylgalactosamine moieties.

38. The construct of any one of claims 28 to 37, wherein the one or more ligands are linked in a linear or branched configuration.

39. The construct of claim 38, wherein the one or more ligands are attached in a bi-antennary or tri-antennary configuration, or in a configuration based on individual ligands at different positions.

40. The construct of claim 37 or 38, wherein the ligand has the structure:

41. according to the construct described in any one in claim 1 to 40, it further comprises one or more phosphorothioate or dithioate internucleotide connections.

42. The construct of claim 41 comprising 1 to 15 phosphorothioate or phosphorodithioate internucleotide linkages.

43. The construct of claim 41 or 42, comprising one or more phosphorothioate or phosphorodithioate internucleotide linkages in one or more 5' and / or 3' regions of the first nucleic acid portion of (a), and / or the second nucleic acid portion of (b), and / or the third nucleic acid portion of (c), and / or the fourth nucleic acid portion of (d), and / or 1 to 8 additional nucleic acid portions as defined in claim 14 or 15, and / or the passenger nucleic acid portion as defined in claim 14 or 15.

44. The construct according to any one of claims 41 to 43, comprising a phosphorothioate or phosphorodithioate internucleotide linkage between at least two adjacent nucleotides of the nucleic acid linker portion as defined in claim 16(ii).

45. The construct of any one of claims 44, comprising phosphorothioate or phosphorodithioate internucleotide linkages between each pair of adjacent nucleotides present in the nucleic acid linker portion.

46. ​​The construct of any one of claims 41 to 45, comprising a phosphorothioate or phosphorodithioate internucleotide linkage connecting: (a) and a nucleic acid linker moiety as defined in claim 16(ii); and / or (b) and the nucleic acid linker moiety as defined in claim 16(ii); and / or (c) and the nucleic acid linker moiety as defined in claim 16(ii); and / or (d) and the nucleic acid linker moiety as defined in claim 16(ii); and / or 1 to 8 additional nucleic acid moieties as defined in claim 14 or 15 and a nucleic acid linker moiety as defined in claim 16(ii); and / or A passenger nucleic acid portion as defined in claim 14 or 15 and a nucleic acid linker portion as defined in claim 16(ii).

47. The construct of any one of claims 1 to 46, wherein at least one nucleotide in at least one of the following is modified: (a) a first nucleic acid portion; and / or the second nucleic acid portion of (b); and / or (c) a third nucleic acid portion; and / or (d) a fourth nucleic acid portion; and / or where present, 1 to 8 additional nucleic acid moieties as defined in claim 14 or 15; and / or where present, a passenger nucleic acid moiety as defined in claim 14 or 15; and / or Where present, a nucleic acid linker moiety as defined in claim 16(ii).

48. Construct according to claim 47, wherein one of the following one or more odd-numbered nucleotides from the 5' end are modified, and / or one of the following one or more even-numbered nucleotides from the 5' end are modified, wherein generally the modification of the even-numbered nucleotides is different from the modification of the odd-numbered nucleotides: (a) a first nucleic acid portion; and / or the second nucleic acid portion of (b); and / or (c) a third nucleic acid portion; and / or (d) a fourth nucleic acid portion; and / or where present, 1 to 8 additional nucleic acid moieties as defined in claim 14 or 15; and / or Where present, a passenger nucleic acid moiety as defined in claim 14 or 15.

49. The construct of claim 47 or 48, wherein the modification of one or more odd-numbered nucleotides of the third nucleic acid portion (c) from the 3' end is different from the modification of the odd-numbered nucleotides of the first nucleic acid portion (a) from the 5' end; and / or The modification of one or more odd-numbered nucleotides from the 3' end of the fourth nucleic acid portion of (d) is different from the modification of the odd-numbered nucleotides from the 5' end of the second nucleic acid portion of (b); and / or where present, the modification of one or more odd-numbered nucleotides from the 3′ end of the passenger nucleic acid portion as defined in claim 14 or 15 is different from the modification of the odd-numbered nucleotides from the 5′ end of the 1 to 8 additional nucleic acid portions as defined in claim 14 or 15; and / or Where present, the modification of one or more nucleotides of the nucleic acid linker portion as defined in claim 16(iii) is: (i) different from the modification of the nucleotide adjacent to the 3' end of the first nucleic acid portion of (a); and / or (ii) different from the modification of the nucleotide adjacent to the 3' end of the second nucleic acid portion of (b); and / or different from the modification of the nucleotide adjacent to the 3' end of 1 to 8 additional nucleic acid portions as defined in claim 14 or 15 (where present).

50. The construct of any one of claims 47 to 49, wherein one or more of the following even-numbered nucleotides from the 3' end: the third nucleic acid portion of (i)(c); and / or the fourth nucleic acid portion of (ii)(d); and / or (iii) where present, the passenger nucleic acid portion as defined in claim 14 or 15, are modified differently from the modification of the odd-numbered nucleotides from the 3' end of these corresponding portions.

51. The construct of any one of claims 47 to 50, wherein the first nucleic acid portion of (i) (a); and / or the second nucleic acid portion of (ii) (b); and / or (iii) where present, one to eight additional nucleic acid portions as defined in claim 14 or 15, comprise at least one or more modified even-numbered nucleotides adjacent to at least one or more differently modified odd-numbered nucleotides in the corresponding portions.

52. The construct of any one of claims 47 to 51 , wherein the third nucleic acid portion of (i) (c); and / or the fourth nucleic acid portion of (ii) (d); and / or (iii), where present, the passenger nucleic acid portion as defined in claim 14 or 15, comprises at least one or more modified even-numbered nucleotides adjacent to at least one or more differently modified odd-numbered nucleotides in the corresponding portions.

53. according to the construct described in any one of claim 47 to 52, wherein the first nucleic acid part of (i) (a); and / or the second nucleic acid part of (ii) (b); and / or (iii) when present, a plurality of adjacent nucleotides in 1 to 8 additional nucleic acid parts as defined in claim 14 or 15 are modified in the same manner.

54. The construct of any one of claims 47 to 53, wherein the third nucleic acid portion of (i)(c); and / or the fourth nucleic acid portion of (ii)(d); and / or (iii) where present, a plurality of adjacent nucleotides of the passenger nucleic acid portion as defined in claim 14 or 15, are modified in the same manner.

55. The construct of claim 53 or 54, wherein the plurality of identically modified adjacent nucleotides is 2 to 4 adjacent nucleotides, preferably 3 or 4 adjacent nucleotides.

56. A construct according to claim 55, wherein the plurality of identically modified adjacent nucleotides are located in the third nucleic acid portion of (i)(c); and / or the fourth nucleic acid portion of (ii)(d); and / or (iii) where present, the 5' region of the passenger nucleic acid portion as defined in claim 14 or 15.

57. The construct of any one of claims 53 to 56, wherein a plurality of identically modified adjacent nucleotides are located in the nucleic acid linker portion as defined in claim 16(iii).

58. The construct of any one of claims 47 to 57, wherein the one or more modified nucleotides of the first nucleic acid portion of (a) do not have the same modification in the corresponding nucleotides of the third nucleic acid portion of the first double-stranded region (c); and / or the one or more modified nucleotides of the second nucleic acid portion of (b) do not have the same modification in the corresponding nucleotides of the fourth nucleic acid portion of the second double-stranded region (d); and / or, where present, the one or more modified nucleotides of 1 to 8 additional nucleic acid portions as defined in claim 14 or 15 do not have the same modification in the corresponding nucleotides of the passenger nucleic acid portion of the corresponding double-stranded region.

59. The construct of any one of claims 47 to 58, wherein the one or more modified nucleotides of the first nucleic acid part of (a) are shifted by at least one nucleotide relative to the same modified nucleotide of the third nucleic acid part of (c); and / or the one or more modified nucleotides of the second nucleic acid part of (b) are shifted by at least one nucleotide relative to the same modified nucleotide of the fourth nucleic acid part of (d); and / or, where present, the one or more modified nucleotides of 1 to 8 additional nucleic acid parts as defined in claim 14 or 15 are shifted by at least one nucleotide relative to the same modified nucleotide of the passenger nucleic acid part as defined in claim 14 or 15 (where present).

60. The construct of any one of claims 47 to 59, wherein the one or more modifications are each independently a sugar modification, a phosphate modification, or a base modification.

61. The construct of claim 60, wherein the modification is selected from nucleotides having a 2' modified sugar; conformationally restricted nucleotides; (CRN) sugars such as locked nucleic acid (LNA), (S)-constrained ethyl bicyclic nucleic acid, constrained ethyl (cEt), tricyclic-DNA; morpholino, unlocked nucleic acid (UNA), glycol nucleic acid (GNA), D-hexitol nucleic acid (HNA), and cyclohexene nucleic acid (CeNA).

62. The construct of claim 61, wherein the 2'-modified sugar is selected from the group consisting of 2'-O-alkyl modified sugars, 2'-O-methyl modified sugars, 2'-O-methoxyethyl modified sugars, 2'-O-allyl modified sugars, 2'-C-allyl modified sugars, 2'-deoxy modified sugars (such as 2'-deoxyribose), 2'-F modified sugars, 2'-arabino-fluoride modified sugars, 2'-O-benzyl modified sugars, 2'-amino modified sugars, and 2'-O-methyl-4-pyridine modified sugars.

63. according to the construct described in any one in claim 60 to 62, wherein base is modified to be any one in abasic nucleotide and the nucleotide that comprises non-natural base.

64. The construct of any one of claims 47 to 63, wherein at least one modification is a 2'-O-methyl modification in the ribose moiety.

65. The construct of any one of claims 47 to 64, wherein at least one modification is a 2'-F modification in the ribose moiety.

66. The construct of any one of claims 47 to 65, wherein the ribose moiety of the nucleotides 2 and 14, counting downstream from the first nucleotide from the 5' end of (i) (a); and / or (ii) (b) the second nucleic acid moiety; and / or (iii) (where present) the 1 to 8 additional nucleic acid moieties defined in claim 14 or 15, does not comprise a 2'-O-methyl modification.

67. The construct according to any one of claims 47 to 66, wherein the ribose moiety of one, two or all three nucleotides of the satellite nucleic acid portion as defined in claim 14 or 15, which corresponds in position to the first nucleic acid portion of (i) (a); and / or the second nucleic acid portion of (ii) (b); and / or, if present, the nucleotides at any of positions 11 to 13 counting downstream from the first nucleotide at the 5' end of the 1 to 8 additional nucleic acid portions as defined in claim 14 or 15, does not contain a 2'-O-methyl modification.

68. The construct according to claim 66 or 67, wherein in the first nucleic acid part of (i) (a); and / or the second nucleic acid part of (ii) (b); and / or, (iii) where present, in the 1 to 8 additional nucleic acid parts as defined in claim 14 or 15, the nucleotide at any of the 2nd and 14th positions counting downstream from the first nucleotide comprises a 2'-F modification in its ribose moiety.

69. The construct according to any one of claims 66 to 68, wherein in the third nucleic acid portion of (i) (c); and / or the fourth nucleic acid portion of (ii) (d); and / or, (iii) if present, in the satellite nucleic acid portion as defined in claim 14 or 15, one, two or all three nucleotides corresponding in position to the first nucleic acid portion of (i) (a); and / or the second nucleic acid portion of (ii) (b); and / or, (iii) if present, the nucleotides at any of positions 11 to 13 counting downstream from the 5'-end of the 1 to 8 additional nucleic acid portions as defined in claim 14 or 15, respectively, comprise a 2'-F modification in their ribose moiety.

70. The construct according to any one of claims 65 to 69, wherein the ribose moieties of all remaining nucleotides comprise a 2'-O-methyl modification or a 2'-F modification, advantageously excluding the unmodified nucleotides described in claim 5.

71. The construct of claim 70, wherein the ribose moieties of the remaining nucleotides comprise a 2'-O-methyl modification.

72. The construct according to claim 70 or 71, wherein the one or more, advantageously one, unmodified nucleotides are any nucleotides of the nucleic acid linker portion as defined in claim 16(iii), advantageously a nucleotide of the nucleic acid linker portion as defined in claim 16(iii) that is adjacent to the third nucleic acid portion of (i)(c); and / or the fourth nucleic acid portion of (ii)(d); and / or (iii) (where present) the passenger nucleic acid portion as defined in claim 14 or 15.

73. The construct of any preceding claim, wherein the construct comprises a first and a second strand, the nucleobase sequences of the first and second strands being SEQ ID NOs: 130 and 131, SEQ ID NOs: 132 and 133, SEQ ID NOs: 134 and 135, SEQ ID NOs: 136 and 137, SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147, respectively.

74. The construct of claim 73, wherein the construct is selected from the group consisting of SEQ ID Nos. 148-165.

75. The construct of claim 73 or 74, wherein the 3' terminal position of the first and third nucleic acid portions are substituted with unmodified nucleotides.

76. A construct according to any one of claims 1 to 75 comprising at least one vinylphosphonate modification, e.g., in the first nucleic acid portion of (i) (a); and / or the second nucleic acid portion of (ii) (b); and / or (iii) (where present) at least one vinylphosphonate modification in the 5' region of 1 to 8 additional nucleic acid portions as defined in claim 14 or 15.

77. The construct of any one of claims 1 to 76, wherein the first nucleic acid part of (a); and / or the second nucleic acid part of (b); and / or the third nucleic acid part of (c); and / or the fourth nucleic acid part of (d); and / or (where present) 1 to 8 additional nucleic acid parts as defined in claim 14 or 15; and / or, where present, one or more nucleotides of the passenger nucleic acid part as defined in claim 14 or 15 are inverted nucleotides and are linked to the 3' carbon of the adjacent nucleotide via the 3' carbon of the nucleotide, and / or are inverted nucleotides and are linked to the 5' carbon of the adjacent nucleotide via the 5' carbon of the nucleotide.

78. The construct of claim 77, wherein the inverted nucleotide is linked to the adjacent nucleotide via a phosphate group via a phosphodiester bond; or It is linked to adjacent nucleotides through a phosphorothioate group; or it is linked to adjacent nucleotides through a phosphorodithioate group.

79. The construct of any one of claims 1 to 78 which is blunt ended.

80. The construct of any one of claims 1 to 79, wherein the first nucleic acid portion of (a); and / or the second nucleic acid portion of (b); and / or the third nucleic acid portion of (c); and / or the fourth nucleic acid portion of (d); and / or, where present, 1 to 8 additional nucleic acid portions as defined in claim 14 or 15; and / or, where present, the passenger nucleic acid portion as defined in claim 14 or 15 has an overhang.

81. The construct of any one of claims 1 to 80, wherein the target RNA is an mRNA or other RNA molecule.

82. according to the construct described in any one in claim 17 to 81, the total length of any chain of wherein said construct is 30 to 35 nucleosides, is preferably 33 or 34 nucleosides.

83. A composition comprising the nucleic acid construct of any one of claims 1 to 82 and a physiologically acceptable excipient.

84. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 1 to 82.

85. The pharmaceutical composition of claim 86, further comprising a pharmaceutically acceptable excipient, diluent, antioxidant and / or preservative.

86. The pharmaceutical composition of claim 84 or 85, wherein the construct of any one of claims 1 to 82 is the sole pharmaceutically active ingredient.

87. The pharmaceutical composition of claim 84 or 85, wherein the pharmaceutical composition further comprises one or more other pharmaceutically active ingredients.

88. The pharmaceutical composition of claim 87, wherein the additional pharmaceutically active agent is selected from the group consisting of: fish oil triglycerides (Vascepa), Vupanorsen, statins (such as rosuvastatin and simvastatin) (Simvastatin), fibrates (such as fenofibrate), and drugs that lower low-density lipoprotein cholesterol (LDL- cholesterol) compounds (such as statins and ezetimib). and / or A drug for reducing hypertension, wherein the other pharmaceutically active ingredients can be selected from: diuretics, angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists, beta-blockers, vasodilators, calcium channel blockers, aldosterone antagonists, α2-agonists, renin inhibitors, α-receptor blockers, peripherally acting adrenergic drugs, selective D1 receptor partial agonists, non-selective α-adrenergic antagonists, synthetic drugs, steroidal antimineralocorticoid drugs; any combination of the above drugs; and antihypertensive therapeutic drugs formulated into drug combinations, and can be further selected from angiotensin II receptor antagonists in the group consisting of losartan, valsartan, olmesartan, eprosartan and azilsartan.

89. The pharmaceutical composition of claim 87 or 88, wherein the nucleic acid construct and the other pharmaceutically active ingredients are administered simultaneously or in any order.

90. The nucleic acid construct of any one of claims 1 to 82, for use in human or veterinary medicine or therapy.

91. The nucleic acid construct of any one of claims 1 to 82, for use in a method for treating, ameliorating and / or preventing a disease or condition.

92. The compound and / or construct for use according to claim 91, wherein the disease or condition is a disease or condition associated with APOC3 and / or AGT, or a disease or condition requiring reduction of APOC3 and / or AGT expression.

93. The construct for use of claim 92, wherein the disease or condition is selected from the group consisting of: Diseases or conditions associated with APOC3, or diseases or conditions requiring reduction of APOC3 expression levels, are preferably selected from dyslipidemia (including mixed dyslipidemia); hyperchylomicronemia (including familial hyperchylomicronemia); hypertriglyceridemia, preferably severe hypertriglyceridemia and / or hypertriglyceridemia with a blood triglyceride level greater than 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); pancreatitis (including acute pancreatitis); and / or Hypertension, hypertension, critical hypertension, essential hypertension, secondary hypertension (isolated systolic or diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, resistant hypertension, refractory hypertension, paroxysmal hypertension, renovascular hypertension, Goldblatt hypertension, ocular hypertension, glaucoma, pulmonary hypertension, portal hypertension, systemic venous hypertension, systolic hypertension, fluctuating hypertension); hypertensive heart disease, hypertensive nephropathy, atherosclerosis, arteriosclerosis, vascular disease, diabetic nephropathy, diabetic retinopathy, chronic heart failure, cardiomyopathy, diabetic cardiomyopathy, nocturnal hypotension, glomerulosclerosis, coarctation of the aorta, aortic aneurysm, ventricular fibrosis, heart failure, myocardial infarction, angina pectoris, stroke, kidney disease, renal failure, systemic sclerosis, intrauterine growth restriction (IUGR), fetal growth restriction, obesity, hepatic steatosis / fatty liver disease, nonalcoholic steatohepatitis (NASH), nonalcoholic fatty liver disease (NAFLD); glucose intolerance, type 2 diabetes, and metabolic syndrome.

94. A method of treating a disease or condition comprising administering the nucleic acid construct of any one of claims 1 to 82 to an individual in need of treatment.

95. The method of claim 94, wherein the nucleic acid construct is administered to the individual subcutaneously or intravenously.

96. Use of the nucleic acid construct of any one of claims 1 to 82 as a tool for gene function analysis in research.

97. Use of the nucleic acid construct of any one of claims 1 to 82 in the preparation of a medicament for the treatment of a disease or condition.

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