Dual Silencing

Abstract

A nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference t

Description

Field of the Disclosure The disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference either the same gene or different genes to enhance silencing thereby modulating gene expression. Background to the Disclosure A technique to specifically ablate gene function is through the introduction of double stranded inhibitory RNA, also referred to as small inhibitory or interfering RNA (siRNA), into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically, but not exclusively, derived from exons of the gene which is to be ablated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA. Typically, RNA silencing utilises a single siRNA species directed to a single gene to silence expression. It is known to link siRNAs to enable the silencing of two or more genes. For example, WO2016205410 discloses oligonucleotides linked together directly, via functional end-substitutions, or indirectly by way of a linking agent. The oligonucleotide can be bound directly to a linker. Such bonding can be achieved, for example, through use of 3'-thionucleosides. WO2018145086 discloses oligonucleotides in the form of a multimeric oligonucleotide having monomeric subunits of oligonucleotide joined by covalent linkers to decrease clearance due to glomerular filtration. WO2013040429A1 discloses multi-oligomeric complexes that comprise two or more targeting oligonucleotides linked together by a cleavable linker. WO2015113922 discloses oligo oligonucleotides conjugates where two or more antisense oligonucleotides are covalently linked by physiologically labile linkers, and to a biocleavable functional group such as a conjugate group. WO2010141511 discloses bivalent or multivalent nucleic acid molecules or complexes of nucleic acid molecules having two or more target-specific regions, in which the target-specific regions are complementary to a single target gene at more than one distinct nucleotide site, and / or in which the target regions are complementary to more than one target gene or target sequence. WO2017188707 discloses a dicer substrate RNA nanostructure exhibiting enhanced gene silencing effects. The RNA nanostructure includes a plurality of the same or different RNAi sequences in a single RNA nanostructure. The use of oligomeric nanostructures comprising more than one siRNA or antisense molecule is known in the art. There is a desire to provide alternative and simpler approaches to the delivery of more than one siRNA to a cell with the objective of silencing the expression of one or more gene targets to obtain enhanced therapeutic effects. Cardiovascular disease associated with hypercholesterolemia is a common condition and results in heart disease and a high incidence of death and morbidity and can be a consequence of poor diet, obesity or an inherited dysfunctional gene. For example, mutations in Low Density Lipoprotein Receptor (LDL-receptor) or apolipoprotein B (ApoB) as in familial hypercholesterolemia. Cholesterol is essential for membrane biogenesis in animal cells. The lack of water solubility means that cholesterol is transported around the body in association with lipoproteins. Apolipoproteins form together with phospholipids, cholesterol and lipids which facilitate the transport of lipids such as cholesterol, through the bloodstream to the different parts of the body. Lipoproteins are classified according to size and can form HDL (High-density lipoprotein), LDL (Low-density lipoprotein), IDL (intermediate-density lipoprotein), VLDL (very low-density lipoprotein) and ULDL (ultra-low-density lipoprotein) lipoproteins. Familial hypercholesterolemia is an orphan disease and results from elevated levels of LDL cholesterol (LDL-C) in the blood. The disease is an autosomal dominant disorder with both the heterozygous (350-550mg / dL LDL-C) and homozygous (650-1000mg / dL LDL-C) states resulting in elevated LDL-C. The heterozygous form of familial hypercholesterolemia is around 1:500 of the population. The homozygous state is much rarer and is approximately 1:1,000,000. The normal levels of LDL-C are in the region 130mg / dL. Hypercholesterolemia is particularly acute in paediatric patients which if not diagnosed early can result in accelerated coronary heart disease and premature death. If diagnosed and treated early the child can have a normal life expectancy. In adults, high LDL-C, either because of mutation or other factors, is directly associated with increased risk of atherosclerosis which can lead to coronary artery disease, stroke or kidney problems. Lowering levels of LDL-C is known to reduce the risk of atherosclerosis and associated conditions. LDL-C levels can be lowered initially by administration of statins which block the de novo synthesis of cholesterol by inhibiting the HMG-CoA reductase. Some subjects can benefit from combination therapy which combines a statin with other therapeutic agents such as ezetimibe, colestipol or nicotinic acid. However, expression and synthesis of HMG-CoA reductase adapts in response to the statin inhibition and increases over time, thus the beneficial effects are only temporary or limited after statin resistance is established. The disclosure relates to isolated nucleic acid molecules comprising at least two double stranded inhibitory ribonucleic acid (RNA) molecules adapted to silence by RNA interference either the same gene or different genes to enhance silencing thereby modulating gene expression. Statements of Invention According to an aspect of the invention there is provided a nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a gene to be silenced and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand; and ii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense and an antisense strand designed with reference to a different or the same a gene to be silenced as set forth in i) above and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules. In a preferred embodiment of the invention said a single stranded deoxyribonucleic acid (DNA) is linked to said first or second double stranded inhibitory (RNA) molecule wherein said linkage is selected from the group: i) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense stand of said second double stranded RNA molecule; ii) 5’ sense strand of said first double stranded inhibitory RNA and 3’ sense strand of said second double stranded RNA molecule; iii) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense strand of said second inhibitory RNA molecule; iv) 5’ sense strand of said first double stranded inhibitory RNA and the 3’ antisense strand of said second double stranded inhibitory RNA molecule; v) 3’ sense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule; vi) 3’ sense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule; vii) 3’ sense strand of said first double stranded inhibitory RNA and the 5’ antisense strand of said second double stranded inhibitory RNA molecule; viii) 3’ sense strand of said first double stranded inhibitory RNA and the 3’ antisense of said second double stranded inhibitory RNA molecule; ix) 5’ antisense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule; x) 5’ antisense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule; xi) 5’ antisense strand of said first double stranded inhibitory RNA and the 5’ antisense strand of said second double stranded inhibitory RNA molecule; xii) 5’ antisense strand of said first double stranded inhibitory RNA and the 3’ antisense strand of said second double stranded inhibitory RNA molecule; xiii) 3’ antisense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule; xiv) 3’ antisense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule; xv) 3’ antisense strand of said first double stranded inhibitory RNA and the 3’ antisense of said second double stranded inhibitory RNA molecule; and xvi) 3’ antisense strand of said first double stranded inhibitory RNA and the 5’ antisense of said second double stranded inhibitory RNA molecule. In a preferred embodiment of the invention said the first and second gene to be silenced is the same gene. In an alternative preferred embodiment of the invention said first and second gene to be silenced are different genes. In a preferred embodiment of the invention said first and second double stranded inhibitory (RNA) molecules comprise different nucleotide sequences. In a preferred embodiment of the invention said first or second gene to be silenced is the Apo B gene. In a preferred embodiment of the invention said Apo B gene comprises a nucleotide sequence set forth in SEQ ID NO: 1 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In an alternative preferred embodiment of the invention said first or second gene to be silenced is PCSK9. In a preferred embodiment of the invention said PCSK9 gene comprises a nucleotide sequence set forth in SEQ ID NO: 2 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In a further alternative embodiment of the invention said first or second gene to be silenced is lipoprotein A. In a preferred embodiment of the invention said lipoprotein A gene comprises a nucleotide sequence set forth in SEQ ID NO: 3 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In a further alternative embodiment of the invention said first or second gene to be silenced is angiotensinogen. In a preferred embodiment of the invention said angiotensinogen gene comprises a nucleotide sequence set forth in SEQ ID NO: 4 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In a further alternative preferred embodiment of the invention said first or second gene to be silenced is ApoCIII. In a preferred embodiment of the invention said Apo Clll gene comprises a nucleotide sequence set forth in SEQ ID NO: 5 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In a further alternative preferred embodiment of the invention said first or second gene to be silenced is DGAT2. In a preferred embodiment of the invention said DGAT2 gene comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length. In a preferred embodiment of the invention said first or second gene to be silenced is selected from the group consisting of: Apo B, PCSK9, Lp(a), APOCIII, angiotensinogen, and DGAT2. In a preferred embodiment of the invention said first gene is Apo B and said second gene is PCSK9. In a preferred embodiment of the invention said first gene is Apo B and said second gene is angiotensinogen. In a preferred embodiment of the invention said first gene is Apo B and said second gene is ApoCiII. In a preferred embodiment of the invention said first gene is Apo B and said second gene is DGAT2. In a preferred embodiment of the invention said first gene is Apo B and said second gene is Lp(a). In a preferred embodiment of the invention said first gene is PCSK9 and said second gene is angiotensinogen. In a preferred embodiment of the invention said first gene is PCSK9 and said second gene is Apo Clll. In a preferred embodiment of the invention said first gene is PCSK9 and said second gene is DGAT2. In a preferred embodiment of the invention said first gene is PCSK9 and said second gene is Lp(a). In a preferred embodiment of the invention said first gene is angiotensinogen and said second gene is Apo Clll. In a preferred embodiment of the invention said first gene is angiotensinogen and said second gene is DGAT2. In a preferred embodiment of the invention said first gene is angiotensinogen and said second gene is Lp(a). In a preferred embodiment of the invention said first gene is Apo Clll and said second gene is DGAT2. In a preferred embodiment of the invention said first gene is Apo Clll and said second gene is Lp(a). In a preferred embodiment of the invention said first gene is DGAT2 and said second gene is Lp(a). In a preferred embodiment of the invention said single stranded DNA comprises the nucleotide sequence CGAAGCGCCCTACTCCACT. Preferably said complementary single stranded and DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG. In a preferred embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises or consists of natural nucleotides. In an alternative preferred embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises modified nucleotides and / or modified sugar(s). In a preferred embodiment of the invention said modified nucleotides / sugars are selected from the group: a 3 '-terminal deoxy- thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, 2'-C-alkyl-modified nucleotide, 2' -hydroxly- modified nucleotide, a 2'-methoxyethyl modified nucleotide, a 2'-0- alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising phosphorodithioate (PS2), a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5’- phosphate, and a nucleotide comprising a 5 ‘-phosphate mimic, for example a 5’-vinyl phosphate, a nucleotide comprising a 2’-deoxy-2’-fluro and a 2’ methyl sugar base. In a preferred embodiment of the invention said first or and / or second double stranded inhibitory (RNA) molecule comprises at least one modified nucleotide wherein said modification is 2'-deoxy-2'-fluoro. In a preferred embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises at least one modified nucleotide wherein said modification is 2'-O-methyl. In a further preferred embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises at least one phosphorothioate linkage. In a further preferred embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises at least one 5'-vinyl phosphate. In an embodiment of the invention said first and / or said second double stranded inhibitory (RNA) molecule comprises at least one modified sugar. A sugar modification includes a modified version of the ribosyl moiety, such as -O-modified RNA such as 2'-0-alkyl or 2'-0-(substituted)alkyl e.g. 2'-0-methyl, T-0-(2-cyanoethyl), 2'-0-(2-methoxy)ethyl (2-MOE), 2'-0-(2-thiomethyl)ethyl, 2'-0-butyryl, -O-propargyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl, e.g. 2'-0-(2-chloroethoxy)methyl (MCEM), -O- (2,2-dichloroethoxy)methyl (DCEM); 2'-<3-a I koxy carbonyl e.g. T-0-[2- (methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), T-0-[2-(N,N- dimethylcarbamoyl)ethyl] (DCME); 2'-halo e.g. 2'-F, FANA (2'-F arabinosyl nucleic acid); carbasugar and azasugar modifications; 3 '-O-alkyl e.g. 3'-0-methyl, 3 '-O-butyryl, V-O- propargyl and their derivatives. In a preferred embodiment of the invention said nucleic acid molecule is covalently linked to A / -acetylgalactosamine. In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense part of said inhibitory RNA or the sense part of said inhibitory RNA. In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid. In a further embodiment of the invention said N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic. In a further embodiment of the invention N-acetylgalactosamine is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid and is linked to either the antisense strand or the sense strand of said double stranded inhibitory RNA molecule of said second nucleic. Preferably, N-acetylgalactosamine is linked to the 3’ terminus is of said sense RNA. In an alternative embodiment of the invention N-acetylgalactosamine is linked to the 5’ terminus of said sense RNA. In an alternative preferred embodiment of the invention said N-acetylgalactosamine is linked to the 3’ terminus of said antisense RNA. In a preferred embodiment of the invention N-acetylgalactosamine is monovalent. In a preferred embodiment of the invention N-acetylgalactosamine is divalent. In an alternative embodiment of the invention N-acetylgalactosamine is trivalent. In a preferred embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: AcHN In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: OH In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: In an alternative embodiment of the invention said nucleic acid molecule is covalently linked to a molecule comprising the structure: According to a further aspect of the invention there is provided a medicament comprising a nucleic acid according to the invention. According to a further aspect of the invention there is provided a pharmaceutical composition comprising a nucleic acid molecule according to the invention. In a preferred embodiment of the invention said composition further includes a pharmaceutical carrier and / or excipient. According to a further aspect of the invention there is provided a nucleic acid molecule or a pharmaceutical composition according to the invention for use in the treatment or prevention of a subject that has or is predisposed to hypercholesterolemia. In a preferred embodiment of the invention said use is the treatment or prevention of diseases associated with hypercholesterolemia. In a preferred embodiment of the invention said disease associated with hypercholesterolemia is selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis. When administered the compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers and optionally other therapeutic agents, such as cholesterol lowering agents, which can be administered separately from the nucleic acid molecule according to the invention or in a combined preparation if a combination is compatible. The combination of a nucleic acid according to the invention and the other, different therapeutic agent is administered as simultaneous, sequential or temporally separate dosages. The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, transdermal ortransepithelial. The compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a disease, such as cardiovascular disease, the desired response is inhibiting or reversing the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods. Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of a nucleic acid molecule according to the invention for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by determining regression of cardiovascular disease and decrease of disease symptoms etc. The doses of the nucleic acid molecule according to the invention administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. If a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. It will be apparent that the method of detection of the nucleic acid according to the invention facilitates the determination of an appropriate dosage for a subject in need of treatment. In general, doses of the nucleic acid molecules herein disclosed of between 1nM - 1pM generally will be formulated and administered according to standard procedures. Preferably doses can range from 1nM- 500nM, 5nM-200nM, 10nM-100nM. Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration and the like vary from the foregoing. The administration of compositions to mammals other than humans, (e.g., for testing purposes or veterinary therapeutic purposes), is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a nonhuman primate, cow, horse, pig, sheep, goat, dog, cat or rodent. When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents e.g. statins. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts. Compositions may be combined, if desired, with a pharmaceutically acceptable carrier. The term “pharmaceutically acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “pharmaceutically acceptable carrier” in this context denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate, for example, solubility and / or stability. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. The pharmaceutical compositions may contain suitable buffering agents, including acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives. The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of nucleic acid, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol. Among the acceptable solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA. According to a further aspect of the invention there is provided a method to treat a subject that has or is predisposed to hypercholesterolemia comprising administering an effective dose of a nucleic acid or a pharmaceutical composition according to the invention thereby treating or preventing hypercholesterolemia. In a preferred method of the invention the hypercholesterolemia is familial hypercholesterolemia. In a preferred method of the invention there is provided the treatment or prevention of diseases associated with hypercholesterolemia. In a preferred method of the invention said disease associated with hypercholesterolemia is selected from the group consisting of: stroke prevention, hyperlipidaemia, cardiovascular disease, atherosclerosis, coronary heart disease, aortic stenosis, cerebrovascular disease, peripheral arterial disease, hypertension, metabolic syndrome, type II diabetes, non-alcoholic fatty acid liver disease, non-alcoholic steatohepatitis, Buerger’s disease, renal artery stenosis, hyper-apobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. “Consisting essentially” means having the essential integers but including integers which do not materially affect the function of the essential integers. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise. Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. An embodiment of the invention will now be described by example only and with reference to the following Figures: Figure 1: Nucleic acid sequences of APOB (SEQ ID NO 1), PCSK9 (SEQ ID NO 2), LP(a) (SEQ ID NO 3), Angiotensinogen (SEQ ID NO 4), APOCIII (SEQ ID NO 5), DGAT2 (SEQ ID NO 6). MATERIALS AND METHODS Transfection in primary mouse hepatocytes Duplex siRNAs and heterodimer siRNAs synthesized by Bio-Synthesis (Lewisville, TX) (Table 3 and 5), were resuspended in nuclease-free water (Invitrogen™ AM9932) to generate a stock solution of 10 pM. For serum stability assay, stock siRNAs were incubated in vehicle (nuclease-free water) or in various concentrations (20% - 80%) of human serum (HS) for 2 hours at 37°C. After pre-incubation in serum or vehicle, siRNAs were transfected into primary mouse hepatocytes in triplicates in a 384-well plate (Thermo Scientific™ 164688) at various concentrations ranging from 2.5 nM to 100 nM using 0.15 pL of Lipofectamine RNAiMAX (I nvitrogen™ 13778075) per well. Transfected cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control. Free-uptake assay of GalNAc-conjugated ApoB-mTTR heterodimers in primary mouse hepatocytes Standard siRNA controls (mTTR and ApoB_C3_01) and GalNAc-conjugated ApoB-mTTR heterodimers (Table 7) were synthesized at Bio-Synthesis (Lewisville, TX). GalNAc-conjugated constructs and standard siRNA controls were resuspended in nuclease-free water (Invitrogen™ AM9932) to generate a stock solution of 10 pM. Stock siRNAs were distributed in triplicates in 384-well plates (Thermo Scientific™ 164688) to a final concentration of 4 nM, 25 nM and 100 nM and primary mouse hepatocytes were added at a concentration of 5,000 cells per well for free-uptake assay. After treatment, cells were incubated at 37°C and 5% CO2 for 48 hours. Cells receiving no siRNA treatment were used as control. Duplex RT-qPCR Cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (Invitrogen™ A25603). Briefly, cells were washed with 50pL ice-cold PBS and lysed in 20 pl Lysis solution containing DNase I. Lysis was stopped after 5 minutes by addition of 2 pl STOP Solution for 2 min. For the RT-qPCR analysis, 1 pL of lysate was dispensed per well into a 96-well PCR plate in a 10 pL RT-qPCR reaction volume. RT-qPCR was performed using the TaqMan® 1-Step qRT-PCR Mix from the Cells-to-CT 1-step TaqMan Kit, with TaqMan probes for GAPDH (VIC_PL, Assay Id Mm99999915_g1), mTTR (FAM, Assay Id Mm00443267_m1), ApoB (FAM, Assay Id Mm01545150_m1) or DGAT2 (FAM, Assay Id Mm00499536_m1). RT-qPCR was performed using a QuantStudio 5 thermocycling instrument (Applied BioSystems). Relative quantification was determined using the AACT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (no siRNA treatment). Example 1 As shown in Table 1, when transfected in primary mouse hepatocytes at concentrations of 2.5-100 nM to test efficacy, ApoB_C3_S19 and ApoB_C3_S18 (single siRNAs with hybridized complementary DNA at the 5' end of the sense strand - Table 3) showed knockdown of ApoB comparable to ApoB_C3_OR with no complementary DNA hybridized to the DNA crook at the 5’ end of the sense strand. As for the ApoB-mTTR heterodimer, the mTTR portion showed knockdown of mTTR target comparable to the modified single siRNA mTTR (bottom 2 rows in Table 1) while the ApoB portion showed reduced efficacy (KD range: 64.2% - 76.3%) compared to the single siRNA counterpart ApoB_C3_OR (KD range: 90.1% - 92.8%). To test stability in human serum, all the siRNA constructs were preincubated in human serum for 2 hours and transfected in primary mouse hepatocytes for 48 hours when knockdown of target gene was measured by RT-qPCR. As shown in Table 2, the mTTR portion in the ApoB-mTTR heterodimer was stable across different concentrations of human serum showing KD levels between 87% and 94.5% which were comparable to KD levels of the standard mTTR siRNA (KD between 74.3% and 93.1%). As for the ApoB portion of the ApoB-mTTR heterodimer siRNA, serum stability was high in 20% human serum showing 92.1% and 84.7% KD at 2.5 nM and 25 nM respectively, while KD was reduced after incubation in 80% human serum showing values of 32.5% and 23.9% KD at 2.5 nM and 25 nM respectively. In contrast, the standard ApoB siRNA counterpart ApoB_C3_OR showed stable KD ranging from 82.4% to 97.6% across different serum concentrations (Table 2). Table 1. Efficacy of ApoB-mTTR heterodimer siRNA and standard siRNAs siRNA ID KD levels (%) in no serum preincubation 2.5 nM 10 nM 25 nM 100 nM ApoB_C3_OR 90.1 90.2 90.7 92.8 ApoB_C3_S19 89.0 88.4 88.5 90.6 ApoB_C3_S18 84.6 90.6 91.6 92.0 ApoB-mTTR (ApoB) 76.3 71.5 71.1 64.2 ApoB-mTTR (mTTR) 92.4 94.3 95.3 96.2 mTTR 92.3 94.1 95.9 95.3 Table 2. Serum stability of ApoB-mTTR heterodimer siRNA and standard siRNAs siRNA ID KD levels (%) after preincubation in human serum 2.5 nM + 20% HS 25 nM + 20% HS 2.5 nM + 50% HS 25 nM + 50% HS 2.5 nM + 80% HS 25 nM + 80% HS ApoB_C3_OR 97.6 96.8 94.4 95.5 82.4 91.3 ApoB_C3_S19 96.8 95.4 86.5 89.0 59.3 84.7 ApoB_C3_S18 96.6 95.2 90.4 90.4 74.1 83.6 ApoB-mTTR (ApoB) 92.1 84.7 80.0 66.8 32.5 23.9 ApoB-mTTR (mTTR) 87.2 87.4 89.2 87.0 93.9 94.5 | mTTR| 82.9 | 89.3 | 74.3 | 84.0 | 89.2 | 93.1 | Table 3. siRNA and ApoB-mTTR heterodimer siRNA description siRNA ID Sense (5' - 3') Antisense (5' - 3') ApoB_C3_O R CGAAGCGCCCTACTCCACT rG*rG*rUrGrU rArUrG rGrCrU rllrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU ApoB_C3_S1 9 CGAAGCGCCCTACTCCACT rG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA 1 rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU ApoB_C3_S1 8 CGAAGCGCCCTACTCCACT rG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA 2 rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU ApoB-mTTR heterodimer ApoB CGAAGCGCCCTACTCCACTrG*rG*rUr GrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA 3 rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU mTTR AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmAfUmAfA 3 mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA - GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU 1 AGTGGAGTAGGGCGCTTCG (19 nt DNA fragment hybridised with complementary DNA at the 5' end of ApoB_C3_S19) 2 GTGGAGTAGGGCGCTTCG (18 nt DNA fragment hybridised with complementary DNA at the 5' end of ApoB_C3_S18) 3 DNA sequence CGAAGCGCCCTACTCCACT and DNA sequence AGTGGAGTAGGGCGCTTCG are hybridised to form the dual siRNA structure ApoB-mTTR fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rU, rG, rC, rA = RNA bases Example 2 As shown in Table 4, DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers pre-incubated in vehicle showed high level of knockdown (KD) for both target genes DGAT2 and ApoB (KD range: 82.7% - 93%) when transfected in primary mouse hepatocytes at concentration of 25 nM. When pre-incubated in 50% human serum followed by transfection in primary mouse hepatocytes at 25 nM, both DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers showed high KD levels fully comparable to pre-incubation in vehicle (KD range after serum pre-incubation at 25 nM: 75.9% - 89.8%) suggesting stability in 50% human serum. Interestingly, the KD levels of target genes ApoB and DGAT2 showed by DGAT2-ApoB-A and DGAT2-ApoB-B heterodimers when preincubated in vehicle or 50% human serum followed by transfection, were fully comparable to the KD levels of their single siRNA control counterparts, ApoB_C3_OR, ApoB_REVCR, DGAT2_3 at 25 nM (KD range for siRNA controls: 90.2% - 93.7%). A full description of siRNA structures can be found in Table 5. As for GS-mTTR-A and GS-mTTR-B heterodimers, pre-incubation in vehicle showed high levels of KD for both target genes ApoB and mTTR (KD range: 83.2% - 97%) when transfected in primary mouse hepatocytes at concentration of 25 nM. When preincubated in 50% human serum followed by transfection in primary mouse hepatocytes at 25 nM, both GS-mTTR-A and GS-mTTR-B heterodimers showed high KD levels fully comparable to pre-incubation in vehicle (KD range after serum pre-incubation followed by transfection at 25 nM: 87.5% - 96.2%) suggesting high serum stability for these heterodimers. The KD levels of target genes ApoB and mTTR showed by GS-mTTR-A and GS-mTTR-B heterodimers when pre-incubated in vehicle or 50% human serum followed by transfection, were fully comparable to the KD levels of their single siRNA control counterparts mTTR, mTTR-AC, mTTR-ARG, PAR-ApoB, GS-AC and GS-ARG (KD range for siRNA controls: 90.2% - 98.2%). A full description of siRNA structures used here can be found in Table 5. Table 4. Serum stability of heterodimer siRNAs DGAT2-ApoB and GS-mTTR and standard siRNA controls siRNA ID KD levels (%) after pre-incubation in 50% human serum or vehicle 25 nM + vehicle 25 nM + 50% HS ApoB_C3_OR 91.4 92.1 ApoB_REVCR 91.6 90.2 DGAT2_3 93.7 91.8 DGAT2_9 95.3 17.3 DGAT2-ApoB-A (DGAT2) 93.0 86.9 DGAT2-ApoB-A (ApoB) 85.1 75.9 DGAT2-ApoB-B (DGAT2) 90.1 89.8 DGAT2-ApoB-B (ApoB) 82.7 79.4 mTTR 97.8 98.2 mTTR-AC 97.1 97.7 mTTR-ARG 96.8 97.4 PAR-ApoB 92.7 92.3 GS-AC 88.1 91.8 GS-ARG 90.5 90.2 GS-mTTR-A (ApoB) 86.7 89.5 GS-mTTR-A (mTTR) 95.2 96.2 GS-mTTR-B (ApoB) 86.1 87.5 GS-mTTR-B (mTTR) 96.2 96.1 Table 5. Description of control siRNAs and ApoB-mTTR heterodimer siRNAs siRNA ID Sense (5'-3') Antisense (5'-3') ApoB_C3_ OR CGAAGCGCCCTACTCCACT rG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU ApoB_REV CR AGTGGAGTAGGGCGCTTCGrG*rG* rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU DGAT2_3 CGAAGCGCCCTACTCCACTrGrUrC rArll rGrG rG r U rG r U rCrll rG r U rG rG rG r UrUrA TUrArArCrCrCrArCrArGrArCrArCr CrCrArUrGrArC DGAT2_9 rGrUrCrArUrGrGrGrUrGrUrCrUrGrUr GrGrGrUrUrATCACCTCATCCCGCG AAGC rUrArArCrCrCrArCrArGrArCrArCr CrCrArUrGrArC DGAT2-ApoB_A heterodime r CGAAGCGCCCTACTCCACTrG*rU*r CrArUrGrGrGrUrGrU rCrU rGrUrGrGrG rUrllrA rU*rA*rArCrCrCrArCrArGrArCrArC rCrCrArUrG*rA*rC AGTGGAGTAGGGCGCTTCGrG*rG* rllrGrll rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU DGAT2-ApoB_B heterodime r AGTGGAGTAGGGCGCTTCGrG*rU*r CrArUrGrGrGrUrGrUrCrU rGrUrGrGrG rUrUrA rU*rA*rArCrCrCrArCrArGrArCrArC rCrCrArUrG*rA*rC CGAAGCGCCCTACTCCACTrG*rG*r UrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR_AC AGTGGAGTAGGGCGCTTCGfA*mA* fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU mTTR-ARG CGAAGCGCCCTACTCCACTfA*mA*f C mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fllmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU PAR_ApoB fC*mC*fU mGfGmA fCmAfll fUfCmA fGmAfA mCfAmA fGmAfA -GalNAc [VPm U*]fU* mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU GS_AC AGTGGAGTAGGGCGCTTCGfC*mC *fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU* mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU GS_ARG CGAAGCGCCCTACTCCACTfC*mC* fU mGfGmA fCmAfll fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU* mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU GS_ mTTR_A heterodime r CGAAGCGCCCTACTCCACTfC*mC* fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU*mCfUmU fGmUfU mCfUmG mAmAfU mGfUmC fCmAfG mG*mG*mU AGTGGAGTAGGGCGCTTCGfA*mA* fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fllmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU GS_ mTTR_B heterodime r AGTGGAGTAGGGCGCTTCGfC*mC *fU mGfGmA fCmAfU fUfCmA fGmAfA mCfAmA fGmAfA [VPm U*]fU*mCfUmUfGmUfUmCfUmGm AmAfUmGfUmCfCmAfGmG *mG‘mU CGAAGCGCCCTACTCCACTfA*mA*f C mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU Notes: DNA sequence CGAAGCGCCCTACTCCACT and DNA sequence AGTGGAGTAGGGCGCTTCG are hybridised to form the heterodimer siRNAs DGAT2-ApoB and GS_ mTTR. fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rU, rG, rC, rA = RNA bases Example 3 As shown in Table 6, ApoB-mTTR_AG1 and ApoB-mTTR_AG2 heterodimers showed 5 ApoB KD levels of 51.9% and 57% respectively at 100 nM and KD levels of 48% and 46.2% respectively at 25 nM in free-uptake. When primary hepatocytes were treated with the single ApoB siRNA counterpart, ApoB_C3_01, at 100 nM and 25 nM, KD levels were 68.6% and 54.5% respectively suggesting that the heterodimer worked almost as effectively as single siRNA control in silencing target gene ApoB. Overall, ApoB-mTTR 10 heterodimer structures showed to be highly effective in silencing mTTR target in all the ApoB-mTTR heterodimers tested here showing KD levels of mTTR ranging from 88.4% to 97.7% at 25 nM and 100 nM treatment in free-uptake assay. mTTR KD values showed by treatment with heterodimers were similar to KD values obtained with single mTTR siRNA control treatment (KD level at 25 nM and 100 nM: 98.1%). A full description of 5 siRNA structures used can be found in Table 7. Table 6. Efficacy of GalNAc-conjugated ApoB-mTTR heterodimer siRNAs or standard siRNA controls by free-uptake in primary mouse hepatocytes siRNA ID Knockdown levels (%) of target gene by free-uptake 4 nM 25 nM 100 nM mTTR 97.3 98.1 98.1 ApoB_C3_01 23.2 54.5 68.6 ApoB-mTTR_AG1 (mTTR) 96.2 97.4 97.5 ApoB-mTTR_AG1 (ApoB) 37.1 48.0 51.9 ApoB-mTTR_AG2 (mTTR) 73.1 88.7 94.3 ApoB-mTTR_AG2 (ApoB) 29.5 46.2 57.0 ApoB-mTTR_AG3 (mTTR) 94.3 97.2 97.7 ApoB-mTTR_AG3 (ApoB) 10.2 14.2 9.3 ApoB-mTTR_BG1 (mTTR) 95.2 96.4 97.3 ApoB-mTTR_BG1 (ApoB) 12.9 24.8 43.6 ApoB-mTTR_BG2 (mTTR) 78.1 88.4 94.4 ApoB-mTTR_BG2 (ApoB) 20.2 15.8 43.1 ApoB-mTTR_BG3 (mTTR) 94.5 97.2 97.1 ApoB-mTTR_BG3 (ApoB) 4.5 0.0 0.0 Table 7. Standard control siRNAs and GalNAc-conjugated ApoB-mTTR heterodimer siRNA structures siRNA ID Sense (5'-3') Antisense (5'- 3') ApoB-mTTR_AG1 heterodimer CGAAGCGCCCTACTCCACTrG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfll mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_AG2 heterodimer CGAAGCGCCCTACTCCACTrG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrllrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fllmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_AG3 CGAAGCGCCCTACTCCACTrG*rG*rUrGrU rArllrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU heterodimer AGTGGAGTAGGGCGCTTCGfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fllmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG1 heterodimer AGTGGAGTAGGGCGCTTCGrG*rG*rUrGrU rArllrG rGrCrll rllrCrA rArCrC rCrllrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG2 heterodimer AGTGGAGTAGGGCGCTTCGrG*rG*rUrGrU rArllrG rGrCrU rllrCrA rArCrC rCrUrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB-mTTR_BG3 heterodimer AGTGGAGTAGGGCGCTTCGrG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU CGAAGCGCCCTACTCCACTfA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA -GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU ApoB_C3_01 CGAAGCGCCCTACTCCACT rG*rG*rUrGrU rArUrG rGrCrU rUrCrA rArCrC rCrUrG rA -GalNAc rU*rC*rA rGrGrG rUrUrG rArArG rCrCrA rUrArC rArCrC*rU*rU mTTR fA*mA*fC mAfGmU fGmUfU fCfUmU fGmCfU mCfUmA fUmAfA - GalNAc mU*fU*mA fUmAfG mAfGmC fAmAmG mAfAmC fAmCfU mGfUmU*mU*mU Notes: DNA sequence CGAAGCGCCCTACTCCACT and DNA sequence AGTGGAGTAGGGCGCTTCG in the heterodimer structures are hybridised to form the dual siRNA structure ApoB-mTTR fA, fU, fC, fG = 2’-deoxy-2’-fluro mA, mil, mC, mG = 2’ Methyl sugar base A, T, C, G = DNA bases rU, rG, rC, rA = RNA bases GalNAc: 20 09 24

Claims

1. A nucleic acid molecule comprising:i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a gene to be silenced and wherein there is provided a single stranded deoxyribonucleic acid (DNA) comprising the nucleotide sequence 5’ CGAAGCGCCCTACTCCACT 3’ molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand; andii) a second nucleic acid comprising a double stranded inhibitory (RNA) molecule comprising a sense and an antisense strand designed with reference to a different or the same a gene to be silenced as set forth in i) above and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to either the 5’ or 3’ end of said sense or antisense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules.

2. Then nucleic acid molecule according to claim 1 wherein said a single stranded deoxyribonucleic acid (DNA) is linked to said first or second double stranded inhibitory (RNA) molecule wherein said linkage is selected from the group:i) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense stand of said second double stranded RNA molecule;ii) 5’ sense strand of said first double stranded inhibitory RNA and 3’ sense strand of said second double stranded RNA molecule;iii) 5’ sense strand of said first double stranded inhibitory RNA and 5’ antisense strand of said second inhibitory RNA molecule;iv) 5’ sense strand of said first double stranded inhibitory RNA and the 3’ antisense strand of said second double stranded inhibitory RNA molecule;v) 3’ sense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule;20 09 24vi) 3’ sense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule;vii) 3’ sense strand of said first double stranded inhibitory RNA and the 5’ antisense strand of said second double stranded inhibitory RNA molecule;viii) 3’ sense strand of said first double stranded inhibitory RNA and the 3’ antisense of said second double stranded inhibitory RNA molecule;ix) 5’ antisense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule;x) 5’ antisense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule;xi) 5’ antisense strand of said first double stranded inhibitory RNA and the 5’ antisense strand of said second double stranded inhibitory RNA molecule;xii) 5’ antisense strand of said first double stranded inhibitory RNA and the 3’ antisense strand of said second double stranded inhibitory RNA molecule;xiii) 3’ antisense strand of said first double stranded inhibitory RNA and the 5’ sense strand of said second double stranded inhibitory RNA molecule;xiv) 3’ antisense strand of said first double stranded inhibitory RNA and the 3’ sense strand of said second double stranded inhibitory RNA molecule;xv) 3’ antisense strand of said first double stranded inhibitory RNA and the 3’ antisense of said second double stranded inhibitory RNA molecule; andxvi) 3’ antisense strand of said first double stranded inhibitory RNA and the 5’ antisense of said second double stranded inhibitory RNA molecule.

3. The nucleic acid molecule according to claim 1 or 2 wherein said first and second gene to be silenced are different genes.

4. The nucleic acid molecule according to any one of claims 1 to 3 wherein said first or second gene to be silenced is the ApoB gene.

5. The nucleic acid molecule according to claim 4 wherein said ApoB gene comprises a nucleotide sequence set forth in SEQ ID NO: 1 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

6. The nucleic acid molecule according to any one of claims 1 to 3 wherein said first or second gene to be silenced is PCSK9.20 09 247. The nucleic acid molecule according to claim 6 wherein said PCSK9 gene comprises a nucleotide sequence set forth in SEQ ID NO: 2 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

8. The nucleic acid molecule according to any one of claims 1 to 3 wherein said first or second gene to be silenced is lipoprotein A.

9. The nucleic acid molecule according to claim 8 wherein said lipoprotein A gene comprises a nucleotide sequence set forth in SEQ ID NO: 3 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

10. The nucleic acid molecule according to any one of claims 1 to 3 wherein said first or second gene to be silenced is angiotensinogen.

11. The nucleic acid molecule according to claim 10 wherein said angiotensinogen gene comprises a nucleotide sequence set forth in SEQ ID NO: 4 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

12. The nucleic acid molecule according to claim 11 wherein said first or second gene to be silenced is ApoCIII.

13. The nucleic acid molecule according to claim 12 wherein said ApoCIII gene comprises a nucleotide sequence set forth in SEQ ID NO: 5 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

14. The nucleic acid molecule according to any one of claims 1 to 3 wherein said first or second gene to be silenced is DGAT2.

15. The nucleic acid molecule according to claim 14 wherein said DGAT2 gene comprises a nucleotide sequence set forth in SEQ ID NO: 6 wherein said double stranded inhibitory RNA is 19-23 nucleotides in length.

16. The nucleic acid molecule according to any one of claims 1 to 15 wherein said first or second gene to be silenced is selected from the group consisting of: ApoB, PCSK9, Lp(a), APOCIII, angiotensinogen and DGAT2.20 09 2417. The nucleic acid molecule according to any one of claims 1 to 16 wherein said complementary single stranded DNA comprises the nucleotide sequence AGTGGAGTAGGGCGCTTCG.

18. The nucleic acid molecule according to any one of claims 1 to 17 wherein said first and / or said second double stranded inhibitory (RNA) molecule comprises or consists of natural nucleotides.

19. The nucleic acid molecule according to any one of claims 1 to 17 wherein said first and / or said second double stranded inhibitory (RNA) molecule comprises modified nucleotides and / or modified sugar(s).

20. The nucleic acid molecule according to any one of claims 1 to 19 wherein said first and / or said second double stranded inhibitory (RNA) molecule comprises at least one phosphorothioate linkage and / or phosphorodithioate.

21. The nucleic acid molecule according to any one of claims 1 to 20 wherein said nucleic acid molecule is covalently linked to A / -acetylgalactosamine.

22. The nucleic acid molecule according to claim 21 wherein N-acetylgalactosamine is linked to 3’ terminus of the sense strand of said double stranded inhibitory RNA molecule of said first nucleic acid and is linked to the 3’ terminus of said sense strand of said double stranded inhibitory RNA molecule of said second nucleic acid.

23. A medicament comprising a nucleic acid according to any one of claims 1 to 22.

24. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 22 and further includes a pharmaceutical carrier and / or excipient.

25. A nucleic acid molecule, medicament, or a pharmaceutical composition according to any one of claims 1 to 24 for use in the treatment or prevention of a subject that has or is predisposed to hypercholesterolemia.

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