APOLIPOPROTEIN B ANTAGONIST

Abstract

This disclosure relates to a nucleic acid comprising a double stranded RNA molecule comprising sense and antisense strands and further comprising a single stranded DNA molecule covalently linked to the 3′ end of either the sense or antisense RNA part of the molecule wherein the double stranded inhibitory RNA targets apolipoprotein B in the treatment hypercholesterolemia.

Claims

1. A nucleic acid molecule comprising a first part that comprises a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand; and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, wherein the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule or wherein the 5′ end of the single stranded DNA molecule is covalently linked to the 3′ of the antisense strand of the double stranded inhibitory RNA molecule, characterized in that the double stranded inhibitory RNA comprises a sense nucleotide sequence that encodes a part of the human apolipoprotein B protein and wherein said single stranded DNA molecule comprises a nucleotide sequence that is adapted over at least part of its length to anneal by complementary base pairing to a part of said single stranded DNA to form a double stranded DNA structure.

2. A nucleic acid molecule comprising a first part that comprises a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense strand and an antisense strand; and a second part that comprises a single stranded deoxyribonucleic acid (DNA) molecule, wherein the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule or wherein the 5′ end of the single stranded DNA molecule is covalently linked to the 3′ of the antisense strand of the double stranded inhibitory RNA molecule, characterized in that the double stranded inhibitory RNA comprises a sense nucleotide sequence that encodes a part of the human apolipoprotein B protein, or polymorphic sequence variant thereof that varies by one, two or three nucleotides, and wherein said single stranded DNA molecule comprises a nucleotide sequence that is adapted over at least part of its length to anneal by complementary base pairing to a part of said single stranded DNA to form a double stranded DNA structure.

3. The nucleic acid molecule according to claim 1, wherein the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the sense strand of the double stranded inhibitory RNA molecule.

4. The nucleic acid molecule according to claim 1, wherein the 5′ end of said single stranded DNA molecule is covalently linked to the 3′ end of the antisense strand of the double stranded inhibitory RNA molecule.

5. The nucleic acid molecule according to claim 1, wherein said single stranded DNA molecule comprises the nucleotide sequence TCACCTCATCCCGCGAAGC (SEQ ID NO: 1).

6. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule is between 18 and 29 nucleotides in length.

7. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence selected from the group consisting of: SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 and 57.

8. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule comprises an antisense nucleotide sequence selected from the group consisting of: 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110.

9. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence selected from the group consisting of: 111, 113, 115, 117, 119, 121, 123 and 125.

10. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule comprises an antisense nucleotide sequence selected from the group consisting of: 112, 114, 116, 118, 120, 122, 124 and 126.

11. The nucleic acid molecule according to claim 1, wherein double stranded inhibitory RNA molecule comprises a sense nucleotide sequence selected from the group consisting of: SEQ ID NO: 7, SEQ ID NO: 36, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115 and SEQ ID NO: 119.

12. The nucleic acid molecule according to claim 1, wherein said double stranded inhibitory RNA molecule comprises an antisense nucleotide sequence selected from the group consisting of: SEQ ID NO: 60, SEQ ID NO: 72, SEQ ID NO: 89, SEQ ID NO: 100, SEQ ID NO: 108, SEQ ID NO: 114 and SEQ ID NO: 118.

13. The nucleic acid molecule according to claim 1, wherein: said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 7 and an antisense nucleotide sequence set forth in SEQ ID NO:60; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 111 and an antisense nucleotide sequence set forth in SEQ ID NO:112; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 117 and an antisense nucleotide sequence set forth in SEQ ID NO:118; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 55 and an antisense nucleotide sequence set forth in SEQ ID NO:108; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 47 and an antisense nucleotide sequence set forth in SEQ ID NO:100; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 36 and an antisense nucleotide sequence set forth in SEQ ID NO:89; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 19 and an antisense nucleotide sequence set forth in SEQ ID NO:72; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 115 and an antisense nucleotide sequence set forth in SEQ ID NO:116; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 113 and an antisense nucleotide sequence set forth in SEQ ID NO:114; said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 119 and an antisense nucleotide sequence set forth in SEQ ID NO:120; or said double stranded inhibitory RNA molecule comprises a sense nucleotide sequence set forth in SEQ ID NO: 113 and an antisense nucleotide sequence set forth in SEQ ID NO:114.

14-23. (canceled)

24. The nucleic acid molecule according to claim 1, wherein said nucleic acid molecule is covalently linked to N-acetylgalactosamine.

25. A pharmaceutical composition comprising at least one nucleic acid molecule according to claim 1.

26. A method of treating a subject who has or is predisposed to hypercholesterolemia, comprising administering an effective dose of the pharmaceutical composition according to claim 25, thereby treating or preventing hypercholesterolemia.

27. The method according to claim 26, further comprising treating or preventing a disease associated with hypercholesterolemia.

28. The method according to claim 27, wherein said disease associated with hypercholesterolemia is selected from the group consisting of: familial hypercholesterolemia, 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, hyperapobetalipoproteinemia, cerebrovascular atherosclerosis, cerebrovascular disease and venous thrombosis.

29. A pharmaceutical composition comprising at least one nucleic acid molecule according to claim 2.

30. A method of treating a subject who has or is predisposed to hypercholesterolemia, comprising administering an effective dose of the pharmaceutical composition according to claim 30, thereby treating or preventing hypercholesterolemia.

Description

[0106] An embodiment of the invention will now be described by Example only and with reference to the following figures:

[0107] FIGS. 1A and 1B. Graphs illustrating in vivo Activity of GaINAc-conjugated Crook anti-mouse ApoB siRNA compared to control siRNA constructs.

[0108] (FIG. 1A) Plasma ApoB levels (micrograms/ml) from five adult male wild-type C57BL/6 mice, were measured 96 hours following administration of GaINAc-conjugated ApoB Crook siRNA (one treatment group) and compared with the control treatment group administered with saline. Statistical analysis was applied using the two-tailed paired T test algorithm. Results show a substantive reduction in mean plasma ApoB levels in mice treated with GaINAc-conjugated Crook siRNA, compared to control. However, it just fails significance (p=0.11), most likely due to small sample size and variation in ApoB levels between control animals;

[0109] (FIG. 1B) Plasma ApoB levels (micrograms/ml) from five adult male wild-type C57BL/6 mice, were measured 96 hours following administration of GaINAc-conjugated ApoB Crook siRNA (one treatment group) and compared with the control treatment group, administered with siRNA construct unconjugated (without GaINAc) ApoB Crook siRNA. Statistical analysis was applied using the two-tailed paired T test algorithm. Results show a highly significant reduction in plasma ApoB levels in this GaINAc-conjugated Crook siRNA treatment group when compared to control unconjugated siRNA with Crook (P=0.00435832).

[0110] FIGS. 2A-2J. In vitro screen of 40 custom duplex Crook siRNAs (C1-C40) listed in Table 2. Graphs illustrate relative knock down of ApoB mRNA expression in HepG2 cells by each of the 40 crook siRNAs. Individual graphs present data from each siRNA sense and antisense pair; C1-C20 (sense strand); C21-40 (antisense strand) as shown in Table 2. Each of 40 crook siRNA molecules were reverse transfected into HepG2 cells (in quadruplicate) at five doses (100 nM, 25 nM, 6.25 nM, 1.56 nM and 0.39 nM) using the conditions identified in the assay development phase. 72 hours post transfection, cells were lysed and ApoB mRNA levels determined by duplex RT-qPCR. In order to calculate the relative knockdown of ApoB for each siRNA at each concentration, expression was first normalised to housekeeping reference gene GAPDH mRNA expression and then to the average ApoB expression across the five doses of the corresponding NEG control (Sense or Antisense). However, it should be noted that a decrease in ApoB expression was observed for the NEG sense control and so the knockdown of ApoB for siRNAs C1-C20 may be underestimated. ApoB knockdown efficiency of all 40 siRNAs are described in Table 3. C13 and C23 siRNAs show a knock-down efficiency greater than 85% (at 25 nM).

[0111] Table 3 was compiled from the in vitro ApoB mRNA expression data (FIG. 2 (a-d)) and shows ranking of Crook siRNAs (C1-C40) with highest knockdown performers at the top of the table. C13 and C23 siRNAs show a knock-down efficiency greater than 85% (at 25 nM).

Materials and Methods

[0112] In Vivo Activity of GaINAc-Conjugated Crook Anti-Mouse ApoB siRNA

[0113] A triantennary GaINAc conjugate was attached to the passenger strand of the Crook-siRNA via phosphoramidate linkage in order to improve selective siRNA delivery to the liver.

[0114] Unconjugated (without GaINAc) and conjugated (GaINAc) versions of ApoB Crook-siRNA described below, were administered to adult male wild-type (WT) C57BL/6 mice by intravenous (IV) and sub-cutaneous (SC) routes, respectively, to investigate the relative plasma and tissue exposure. In addition, control GaINAc-conjugated unmodified siRNA (without Crook) consruct was compared.

[0115] In Vivo ApoB siRNA Constructs

[0116] For in vivo silencing of Apo B is a mouse the sequence below was used (corresponds to C10 in table 2 below. This was chosen because a similar sequence had been successful previously in vivo (Soutshek et al Nature 2004; 432:173-178).

Crook-siRNA 21mer-dsRNA Construct (1): Anti-Mouse ApoB-GaINAc

[0117] Sense Strand SEQ ID NO: 47 (Passenger SEQ ID NO: 1):

TABLE-US-00001 5′-GUCAUCACACUGAAUACCAAU-d(tcacctcatcccgcgaagc)- 3′-[Tri-GalNAC]

[0118] Antisense Strand SEQ ID NO: 100:

TABLE-US-00002 5′-AUUGGUAUUCAGUGUGAUGAC-3′

Structure of Final GaINAc Conjugate:

[0119] ##STR00001##

Crook-siRNA 21mer dsRNA Construct (2): Unconjugated Anti-Mouse ApoB

[0120] Sense Strand SEQ ID NO: 47 (Passenger SEQ ID NO: 1)

TABLE-US-00003 5′-GUCAUCACACUGAAUACCAAU-d(tcacctcatcccgcgaagc)-3

[0121] Antisense Strand (Guide)

TABLE-US-00004 (SEQ ID NO: 100) 5′-AUUGGUAUUCAGUGUGAUGAC-3

[0122] The rationale for dose selection was based on the following information published in the scientific literature:

[0123] The GaINAc conjugated siRNA is dosed subcutaneously at 5 mg/kg which is expected to produce the required level of gene silencing where the ED.sub.80 of structurally related siRNAs have been reported as 2.5 mg/kg (Soutschek et al., 2004). These structurally related siRNAs were tolerated up to 25 mg/kg, single administration, in the mouse (Soutschek et al., 2004).

[0124] The unconjugated version of the sponsor's siRNA is administered at 50 mg/kg IV. This 10-fold increase in the IV compared to the SC dose is due to the unconjugated siRNA being less effective at targeting the liver. Additionally, it is reported by Soutschek et al (2004) that lower levels of RNA are measured in the liver following IV compared to SC administration. It is stated that slower release of the siRNA from the SC depot leads to prolonged exposure increasing the potential for receptor-ligand interactions and greater uptake into the tissue. Similar related siRNA has been well tolerated by mice at up to 50 mg/kg IV administered on 3 consecutive days (Nair et al. 2014). As a precaution a 15 minutes observation period is left between dosing the 1.sup.st animal IV to determine if the test substance causes any adverse effects before the remaining animals are dosed.

[0125] The mouse is the species of choice because it is used as one of the toxicology species in the safety testing of the test substance. The mouse also possesses a very similar metabolic physiology to humans in relation to the therapeutic target of the Crook-siRNA preparations (ApoB). There is a considerable amount of published data available which are acceptable to the regulatory authorities for assessing the significance to man of data generated in this species.

Animals

[0126] Sufficient C57BL/6 mice were obtained from an approved source to provide 20 healthy male animals (5 mice per treatment group). Animals are in the target weight range of 20 to 30 g at dosing. Mice are uniquely numbered by tail marking. Numbers are allocated randomly. Cages are coded by cards giving information including study number and animal number. The study room is identified by a card giving information including room number and study number. On receipt, all animals were examined for external signs of ill health. Unhealthy animals where be excluded from the study. The animals were acclimatized for a minimum period of 5 days. Where practicable, without jeopardizing the scientific integrity of the study, animals were handled as much as possible. A welfare inspection was performed before the start of dosing to ensure their suitability for the study.

[0127] The mice were kept in rooms thermostatically maintained at a temperature of 20 to 24° C., with a relative humidity of between 45 and 65%, and exposed to fluorescent light (nominal 12 hours) each day. Temperature and relative humidity are recorded on a daily basis. The facility is designed to give a minimum of 15 air-changes/hour. Except when in metabolism cages or recovering from surgery, mice were housed up to 5 per cage according to sex, in suitable solid floor cages, containing suitable bedding.

[0128] Cages conform to the ‘Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014). In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and polycarbonate tunnels. The supplier provided certificates of analysis for each batch of blocks used. All animals will be allowed free access to 5LF2 EU Rodent Diet 14%. The diet supplier provided an analysis of the concentration of certain contaminants and some nutrients for each batch used. All animals were allowed free access to mains water from bottles attached to the cages. Periodic analysis of the mains supply is undertaken.

[0129] All procedures to be carried out on live animals as part of this study will be subject to provisions of United Kingdom National Law, the Animals (Scientific Procedures) Act 1986.

[0130] All animals were examined at the beginning and the end of the working day, to ensure that they are in good health. Any animal, which shows marked signs of ill health, were isolated. Moribund animals or those in danger of exceeding the severity limits imposed by the relevant Home Office License were killed.

Materials and Methods for In Vivo Experiments

Preparation of Formulations

[0131] Test substances were diluted in 0.9% saline to provided concentrations of 25 mg/mL and 0.6 mg/mL for the IV and SC doses of ApoB Crook-siRNA GaINAc-unconjugated and conjugate, respectively. The formulations were gently vortexed as appropriate until the test substances are fully dissolved. The resulting formulation(s) were assessed by visual inspection only and categorised accordingly:

[0132] (1) Clear solution

[0133] (2) Cloudy suspension, no particles visible

[0134] (3) Visible particles

[0135] After use, formulations were stored refrigerated nominally at 2-8° C.

Dosing Details Each animal received either a single IV dose of the ApoB Crook-siRNA—unconjugated or a single SC dose of the ApoB Crook-siRNA GaINAc— conjugate. The IV dose was administered as a bolus into the lateral tail vein at a volume of 2 mL/kg. The SC dose was administered into the subcutaneous space at a volume of 5 mL/kg.

TABLE-US-00005 Group1: GalNAc-conjugated ApoB Crook siRNA 5 mg/kg dose Group 2: Unconjugated (without GalNAc) 50 mg/kg ApoB Crook siRNA dose Group 3: Saline control group

[0136] Body Weights

[0137] As a minimum, body weights were recorded the day after arrival and before dose administration. Additional determinations were made, if required.

Sample Storage

[0138] Samples were uniquely labelled with information including, where appropriate: study number; sample type; dose group; animal number/Debra code; (nominal) sampling time; storage conditions. Samples were stored at <−50° C.

Pharmacokinetic Investigation

Designation of Dose Groups

[0139] Animals were assigned to dose groups as follows:

TABLE-US-00006 Dose Number of Dose level animals Group Dose route Test Substance mg/kg Male A subcutaneous ApoB Crook-siRNA 5 5 GalNAc-conjugate B subcutaneous Saline control 0 5 C Intravenous ApoB Crook-siRNA 50 5 (bolus) unconjugated D Intravenous Saline control 0 5 (bolus)

Blood Sampling

[0140] Serial blood samples of (nominally 100 μL, dependent on bodyweight) were collected by tail nick at the following times: 0, 48- and 96-hours post dose. Animals were terminally anaesthetised using sodium pentobarbitone and a final sample (nominally 0.5 mL) was collected by cardiac puncture.

[0141] Blood samples were collected in to a K2EDTA microcapillary tube (tail nick) or a K2EDTA blood tube (cardiac puncture) and placed on ice until processed. Blood was centrifuged (1500 g, 10 min, 4° C.) to produce plasma for analysis. The bulk plasma was divided into two aliquots of equal volume. The residual blood cells were discarded.

TABLE-US-00007 Scheduled Collection Acceptable Time Time Range  0-15 minutes ±1 minute 16-30 minutes ±2 minutes 31-45 minutes ±3 minutes 46-60 minutes ±4 minutes 61 minutes- ±5 minutes 2 hours 2 hours 1 minute- ±10 minutes 8 hours 8 hours 1 minute- ±15 minutes 12 hours 12 hours ±30 minutes onwards

[0142] Where a scheduled collection time is outside the acceptable range, the actual blood collection time was reported for inclusion in any subsequent PK analysis

Animal Fate

[0143] Animals were anaesthetised via an intraperitoneal injection of Sodium Pentobarbitone prior to terminal blood sampling and sacrificed by perfusion and exsanguination.

[0144] A full body perfusion was performed, all animals were flushed with Heparinised Saline Solution at a rate 4 ml/min for 5 minutes (approximately 20 mL total flush). Death was confirmed by the absence of breathing, heartbeat and blood flow. Animal carcasses were retained for tissue collection.

Tissue Collection

[0145] The liver was removed from all animals (Groups A-D) and placed into a pre-weighed tube. The tissue samples were homogenised with 5 parts RNAlater to 1 part tissue using the UltraTurrax homogenisation probe. The following tissues were excised from animals in ApoB treated groups (Groups A & C) and placed into a pre-weighed pot: [0146] Spleen [0147] Brain [0148] Heart [0149] Lung Lobes [0150] Skin (Inguinal region ca. 25 mm.sup.2)

[0151] Following collection, the external surface of the tissues is rinsed with PBS and gently patted dry using a tissue. Tissues are initially placed on wet ice until weighed and then tissues were snap frozen on dry ice prior to storage. Tissues are stored at <−50° C. (nominally −80° C.).

Immunoassay for APOB

[0152] Plasma ApoB levels were measured via enzyme-linked immunosorbent assay (ELISA) using the commercial mouse ApoB detection kit from Elabscience Biotechnology Inc. (catalogue number E-EL-M0132). Plasma samples were stored at −80° C. prior to analysis, thawed on ice and centrifuged at 13,000 rpm for 5 minutes prior to aliquots being diluted in Assay Buffer and applied to the ELISA plate. The ApoB assay kit uses a sandwich ELISA yielding a colorimetric readout, measured at OD450. Samples from each animal at specific time points (0 hours and 96 hours) were assayed in duplicate and measurements were recorded as micrograms ApoB per ml of plasma based on the standard curve reagents supplied with the kit. All data points were measured with a coefficient of variation <20%.

In vitro Screening of ApoB Crook siRNA

HepG2 Reverse Transfection

[0153] A description of the custom library evaluated in this study is provided in Table 2. Custom duplex siRNAs synthesized by Horizon Discovery were resuspended in UltraPure DNase and RNase free water to generate a stock solution of 10 μM. [0154] Stock siRNAs were dispensed into 4×384-well assay plates. On each assay plate, 10 Custom siRNAs and 3 controls (POS ApoB, NEG sense and NEG antisense) were dispensed to generate five-point four-fold dilution series from a top final concentration in the assay plate of 100 nM. ON-TARGETplus Non-Targeting and ApoB siRNAs controls were dispensed to give a final concentration of 25 nM. [0155] Lipofectamine RNAiMAX (ThermoFisher #13778075) was diluted in OptiMEM media before 10 μL of the Lipfectamine RNAiMAX:OptiMEM solution was added per well to the assay plate. The final volume of RNAiMAX per well was 0.08 μL. [0156] The lipid-siRNA mix was incubated 30 min at room temperature before being added to the cells. [0157] HepG2 cells were diluted in assay media (MEM GlutaMAX (GIBCO) 10% FBS 1% Pen/Strep) before 4,000 HepG2 cells were seeded into each well of the assay plate in 40 μL volume. Quadruplicate technical replicates were seeded per assay condition. [0158] The plates were incubated 72 h at 37° C., 5% CO.sub.2 in a humidified atmosphere, prior to assessment of the cells.

ApoB/GAPDH Duplex RT-qPCR

[0159] 72 h post-transfection, cells were processed for RT-qPCR read-out using the Cells-to-CT 1-step TaqMan Kit (Invitrogen, 4391851C). Briefly, cells were washed with 50 μl cold PBS and then lysed in 20 μl Lysis solution containing DNase I. After 5 min, lysis was stopped by addition of 2 μl STOP Solution for 2 min. [0160] For the RT-qPCR analysis, 3 μl of lysate was dispensed per well into 384-well PCR plate as template in an 11 μl RT-qPCR reaction volume. [0161] RT-qPCR was performed using the ThermoFisher TaqMan Fast Virus1-Step Master Mix (#4444434) with TaqMan probes for GAPDH (VIC #4448486) and ApoB (FAM #4351368). [0162] RT-qPCR was performed using a QuantStudio 6 thermocycling instrument (Applied BioSystems). [0163] Relative quantification (RQ) was determined using the ΔΔCT method, where GAPDH was used as internal control and expression changes normalized to the reference sample (either NEG sense or NEG antisense siRNA treated cells).

Statistics

[0164] For all assays in this project, four technical replicates were obtained for each data point. [0165] Mean and Standard Error of the Mean (SEM) were calculated using Excel or Graphpad Prism. [0166] All graphs were generated using Graphpad Prism.

TABLE-US-00008 TABLE 1 ApoB crook siRNA sequences and corresponding SEQ ID NOs. SEQ SEQ ID Sense ID Antisense Start NO strand base sequence NO strand base sequence NM_009693.2   5 GAGGUGUAUGGCUUCAACCCU  58 AGGGUUGAAGCCAUACACCUC  403   6 AGGUGUAUGGCUUCAACCCUG  59 CAGGGUUGAAGCCAUACACCU  404   7 GGUGUAUGGCUUCAACCCUGA  60 UCAGGGUUGAAGCCAUACACC  405   8 GUGUAUGGCUUCAACCCUGAG  61 CUCAGGGUUGAAGCCAUACAC  406   9 GUAUGGCUUCAACCCUGAGGG  62 CCCUCAGGGUUGAAGCCAUAC  408  10 AUGGCUUCAACCCUGAGGGCA  63 UGCCCUCAGGGUUGAAGCCAU  410  11 UGGCUUCAACCCUGAGGGCAA  64 UUGCCCUCAGGGUUGAAGCCA  411  12 CUGAACAUCAAGAGGGGCAUC  65 GAUGCCCCUCUUGAUGUUCAG  562  13 UGAACAUCAAGAGGGGCAUCA  66 UGAUGCCCCUCUUGAUGUUCA  563  14 GAUACCGUGUAUGGAAACUGC  67 GCAGUUUCCAUACACGGUAUC  640  15 UACCGUGUAUGGAAACUGCUC  68 GAGCAGUUUCCAUACACGGUA  642  16 GUCCAGCCCCAUCACUUUACA  69 UGUAAAGUGAUGGGGCUGGAC 1221  17 CAGCCCCAUCACUUUACAAGC  70 GCUUGUAAAGUGAUGGGGCUG 1224  18 AGCCCCAUCACUUUACAAGCC  71 GGCUUGUAAAGUGAUGGGGCU 1225  19 GCCCCAUCACUUUACAAGCCU  72 AGGCUUGUAAAGUGAUGGGGC 1226  20 CUUUACAAGCCUUGGUUCAGU  73 ACUGAACCAAGGCUUGUAAAG 1235  21 ACAAGCCUUGGUUCAGUGUGG  74 CCACACUGAACCAAGGCUUGU 1239  22 AAGCCUUGGUUCAGUGUGGAC  75 GUCCACACUGAACCAAGGCUU 1241  23 UCACAUCCUCCAGUGGCUGAA  76 UUCAGCCACUGGAGGAUGUGA 1278  24 AAAUAGAAGGGAAUCUUAUAU  77 AUAUAAGAUUCCCUUCUAUUU 2063  25 UAGAAGGGAAUCUUAUAUUUG  78 CAAAUAUAAGAUUCCCUUCUA 2066  26 GAAGGGAAUCUUAUAUUUGAU  79 AUCAAAUAUAAGAUUCCCUUC 2068  27 GGAAUCUUAUAUUUGAUCCAA  80 UUGGAUCAAAUAUAAGAUUCC 2072  28 GAGUUUGUGACAAAUAUGGGC  81 GCCCAUAUUUGUCACAAACUC 2746  29 GUUUGUGACAAAUAUGGGCAU  82 AUGCCCAUAUUUGUCACAAAC 2748  30 GUGACAAAUAUGGGCAUCAUC  83 GAUGAUGCCCAUAUUUGUCAC 2752  31 AGAUGAACACCAACUUCUUCC  84 GGAAGAAGUUGGUGUUCAUCU 2801  32 GAUGAACACCAACUUCUUCCA  85 UGGAAGAAGUUGGUGUUCAUC 2802  33 UGAACACCAACUUCUUCCACG  86 CGUGGAAGAAGUUGGUGUUCA 2804  34 GAACACCAACUUCUUCCACGA  87 UCGUGGAAGAAGUUGGUGUUC 2805  35 ACACCAACUUCUUCCACGAGU  88 ACUCGUGGAAGAAGUUGGUGU 2807  36 CACCAACUUCUUCCACGAGUC  89 GACUCGUGGAAGAAGUUGGUG 2808  37 CAAAUGGACUCAUCUGCUACA  90 UGUAGCAGAUGAGUCCAUUUG 3547  38 GGACUCAUCUGCUACAGCUUA  91 UAAGCUGUAGCAGAUGAGUCC 3552  39 UCUGUGGGAUUCCAUCUGCCA  92 UGGCAGAUGGAAUCCCACAGA 4075  40 AUUCCAUCUGCCAUCUCGAGA  93 UCUCGAGAUGGCAGAUGGAAU 4083  41 ACAAUUUGAUCAGUAUAUUAA  94 UUAAUAUACUGAUCAAAUUGU 6636  42 CAAUUUGAUCAGUAUAUUAAA  95 UUUAAUAUACUGAUCAAAUUG 6637  43 UAAAUCAAGUGUCAUCACACU  96 AGUGUGAUGACACUUGAUUUA 10116  44 AUCAAGUGUCAUCACACUGAA  97 UUCAGUGUGAUGACACUUGAU 10119  45 UCAAGUGUCAUCACACUGAAU  98 AUUCAGUGUGAUGACACUUGA 10120  46 CAAGUGUCAUCACACUGAAUU  99 AAUUCAGUGUGAUGACACUUG 10121  47 GUCAUCACACUGAAUACCAAU 100 AUUGGUAUUCAGUGUGAUGAC 10126  48 UCAUCACACUGAAUACCAAUG 101 CAUUGGUAUUCAGUGUGAUGA 10127  49 CAUCACACUGAAUACCAAUGC 102 GCAUUGGUAUUCAGUGUGAUG 10128  50 UAACACUAAGAACCAGAAGAU 103 AUCUUCUGGUUCUUAGUGUUA 10959  51 AUUGGGAAGAAGAGGCAGCUU 104 AAGCUGCCUCUUCUUCCCAAU 12167  52 GAUUGAUUGACCUGUCCAUUC 105 GAAUGGACAGGUCAAUCAAUC 13478  53 UGAUUGACCUGUCCAUUCAAA 106 UUUGAAUGGACAGGUCAAUCA 13481  54 GAUUGACCUGUCCAUUCAAAA 107 UUUUGAAUGGACAGGUCAAUC 13482  55 GACCUGUCCAUUCAAAACUAC 108 GUAGUUUUGAAUGGACAGGUC 13486  56 ACCUGUCCAUUCAAAACUACC 109 GGUAGUUUUGAAUGGACAGGU 13487  57 CUGUCCAUUCAAAACUACCAC 110 GUGGUAGUUUUGAAUGGACAG 13489 111 CAGCACCUAGCUGGAAAGUUA 112 UAACUUUCCAGCUAGGUGCUG 113 CUCCAUGGAAUUUAAGUAUGA 114 UCAUACUUAAAUUCCAUGGAG 115 UUCCCUGAAGUUGAUGUGUUA 116 UAACACAUCAACUUCAGGGAA 117 GUCCAAUAAGAUCAAUAGCAA 118 UUGCUAUUGAUCUUAUUGGAC 119 AACUCUCAAACCCUAAGAUUA 120 UAAUCUUAGGGUUUGAGAGUU 121 UCGGAACAAUCCUCAGAGUUA 122 UAACUCUGAGGAUUGUUCCGA 123 AAGCAAGAACUUAAUGGAAAU 124 AUUUCCAUUAAGUUCUUGCUU 125 GGCCAUUAGGCAAAUUGAUGA 126 UCAUCAAUUUGCCUAAUGGCC

TABLE-US-00009 TABLE 2 A library of 40 duplex siRNAs was synthesized by Horizon Discovery. The table shows the sequences of both strands of RNA for each siRNA. The following DNA sequence (dTdCdAdCdCdTdCdAdTdCdCdCdGdCdGdAdAdGdC) was appended to the 3′ end of either the sense strand (siRNAs C1 to C20, thereafter referred to as sense siRNAs) or the antisense strand (siRNAs C21 to C40, thereafter referred to as antisense siRNAs). siRNA ID Sense Antisense Sense sequence (5′-3′) Antisense sequence (5′-3′) C1 C21 UAGAAGGGAAUCUUAUAUUUG CAAAUAUAAGAUUCCCUUCUA (SEQ ID NO: 25) (SEQ ID NO: 78) C2 C22 CACCAACUUCUUCCACGAGUC GACUCGUGGAAGAAGUUGGUG (SEQ ID NO: 36) (SEQ ID NO: 89) C3 C23 GGUGUAUGGCUUCAACCCUGA UCAGGGUUGAAGCCAUACACC (SEQ ID NO: 7) (SEQ ID NO: 60) C4 C24 GACCUGUCCAUUCAAAACUAC GUAGUUUUGAAUGGACAGGUC (SEQ ID NO: 55) (SEQ ID NO: 108) C5 C25 UACCGUGUAUGGAAACUGCUC GAGCAGUUUCCAUACACGGUA (SEQ ID NO: 15) (SEQ ID NO: 68) C6 C26 GCCCCAUCACUUUACAAGCCU AGGCUUGUAAAGUGAUGGGGC (SEQ ID NO: 19) (SEQ ID NO: 72) C7 C27 GAUUGAUUGACCUGUCCAUUC GAAUGGACAGGUCAAUCAAUC (SEQ ID NO: 52) (SEQ ID NO: 105) C8 C28 GAGGUGUAUGGCUUCAACCCU AGGGUUGAAGCCAUACACCUC (SEQ ID NO: 5) (SEQ ID NO: 58) C9 C29 UCUGUGGGAUUCCAUCUGCCA UGGCAGAUGGAAUCCCACAGA (SEQ ID NO: 39) (SEQ ID NO: 92) C10 C30 GUCAUCACACUGAAUACCAAU AUUGGUAUUCAGUGUGAUGAC (SEQ ID NO: 47) (SEQ ID NO: 100) C11 C31 GUGACAAAUAUGGGCAUCAUC GAUGAUGCCCAUAUUUGUCAC (SEQ ID NO: 30) (SEQ ID NO: 83) C12 C32 ACCUGUCCAUUCAAAACUACC GGUAGUUUUGAAUGGACAGGU (SEQ ID NO: 56) (SEQ ID NO: 109) C13 C33 CAGCACCUAGCUGGAAAGUUA UAACUUUCCAGCUAGGUGCUG (SEQ ID NO: 111) (SEQ ID NO: 112) C14 C34 CUCCAUGGAAUUUAAGUAUGA UCAUACUUAAAUUCCAUGGAG (SEQ ID NO: 113) (SEQ ID NO: 114) C15 C35 UUCCCUGAAGUUGAUGUGUUA UAACACAUCAACUUCAGGGAA (SEQ ID NO: 115) (SEQ ID NO: 116) C16 C36 GUCCAAUAAGAUCAAUAGCAA UUGCUAUUGAUCUUAUUGGAC (SEQ ID NO: 117) (SEQ ID NO: 118) C17 C37 AACUCUCAAACCCUAAGAUUA UAAUCUUAGGGUUUGAGAGUU (SEQ ID NO: 119) (SEQ ID NO: 120) C18 C38 UCGGAACAAUCCUCAGAGUUA UAACUCUGAGGAUUGUUCCGA (SEQ ID NO: 121) (SEQ ID NO: 122) C19 C39 AAGCAAGAACUUAAUGGAAAU AUUUCCAUUAAGUUCUUGCUU (SEQ ID NO: 123) (SEQ ID NO: 124) C20 C40 GGCCAUUAGGCAAAUUGAUGA UCAUCAAUUUGCCUAAUGGCC (SEQ ID NO: 125) (SEQ ID NO: 126)

EXAMPLE 1

[0167] A pilot in vivo mouse experiment was performed to assess activity of GaINAc-conjugated Crook anti-mouse ApoB siRNA compared to control siRNA constructs. Conjugated (GaINAc) and unconjugated (without GaINAc) versions of ApoB Crook siRNA (sequence C10 in Table 2; Covance) were administered to adult male wild-type (WT) C57BL/6 mice by sub-cutaneous (SC) and intravenous (IV) routes, respectively described previously in Material & Methods section.

[0168] Blood plasma ApoB was measured by ELISA (described earlier) at time 0 (prior to administration of siRNA construct) and at 96 hours following siRNA construct administration, as indicated in the four Treatment groups (5 mice per group) as detailed above under Dosing Details.

[0169] Plasma ApoB levels (micrograms/ml) from 5 mice in each treatment group, were used to calculate a mean ApoB value+/− standard error of the mean (SEM). Change in plasma ApoB level after 96 hours following SC administration of GaINAc-conjugated Crook siRNA was compared to levels in mice receiving either control (i) vehicle saline, or (ii) unconjugated siRNA with Crook. Statistical analysis was applied using the two-tailed paired T test algorithm.

[0170] With reference to FIG. 1 (a), plasma ApoB levels (micrograms/ml) of mice 96 hours following treatment with GaINAc-conjugated ApoB Crook siRNA were compared with the control treatment group administered with saline. Statistical analysis was applied using the two-tailed paired T test algorithm. Results show a substantive reduction in mean plasma ApoB levels in mice treated with GaINAc-conjugated Crook siRNA, compared to control. However, it just fails significance (p=0.11), most likely due to small sample size and variation in ApoB levels between control animals.

[0171] With reference to FIG. 1 (b), plasma ApoB levels (micrograms/ml) measured 96 hours following administration of GaINAc-conjugated ApoB Crook siRNA were compared to the control group, treated with siRNA construct unconjugated (without GaINAc) ApoB Crook siRNA. Statistical analysis was applied using the two-tailed paired T test algorithm.

[0172] Results show a highly significant reduction in plasma ApoB levels in this GaINAc-conjugated Crook siRNA treatment group when compared to control unconjugated siRNA with Crook (P=0.00435832).

[0173] Importantly, the selected ApoB Crook siRNA sequence (C10 in Table 2; Covance) used in this pilot in vivo experiment was performed prior to the in vitro ApoB Crook siRNA screen. Our subsequent in vitro data (Example 2) shows that there are other siRNA sequences with greater ApoB mRNA knockdown (KD) efficiency (Table 3) eg. C23 gives 89% KD at 25 nM when compared to C10 (74%).

EXAMPLE 2

[0174] With reference to FIG. 2 (a-d), an arrayed RNAi screen in HepG2 cells was performed to evaluate a custom library of 40 “crook” siRNAs targeting the human ApoB gene (Table 2). All siRNAs in this library possess a DNA extension (or “crook”) appended to the sense RNA strand (sense siRNA) or to the antisense RNA strand (antisense siRNA).

[0175] Prior to performing the screen, suitable conditions for HepG2 reverse transfection were identified. First, siRNAs targeting essential genes were used to evaluate a number of transfection conditions before the selected condition was taken forward to knockdown expression of ApoB using an ON-TARGETplus siRNA and assess the molecular detection tools for analysis of ApoB gene and protein expression. Homogeneous Time-Resolved Fluorescence (HTRF) and Duplex Real-Time quantitative PCR (RT-qPCR) assays were developed to quantify ApoB expression change at the protein and mRNA levels, respectively. In the Screening phase, the 40 custom crook siRNAs were assessed over a five-point dose range. 72 h post transfection, ApoB expression was evaluated by Duplex RT-qPCR. With a few notable exceptions, the knockdown of ApoB expression was similar between the sense and antisense siRNAs sharing the same RNA sequence, when the data is normalised to its relevant negative control (NEG sense for sense siRNAs and NEG antisense for the antisense siRNAs). However, it should be noted that a decrease in ApoB expression was observed for the NEG sense control and so the knockdown of ApoB for siRNAs C1-C20 may be underestimated.

[0176] HepG2 cells were reverse transfected with a library of 40 custom crook siRNAs (20 sense siRNAs and 20 antisense siRNAs) alongside the siRNA controls using conditions identified in the assay development phase. 72 h post transfection, ApoB mRNA levels in transfected cells were quantified by duplex RT-qPCR, normalizing the ApoB mRNA levels to the levels of the housekeeping reference gene GAPDH mRNA (FIG. 2 (a-d)).

[0177] As the assessment of the 40 custom crook siRNA molecules covered a number of assay plates, in order to be able to perform an assay QC step, each plate contained a number of controls. These included the ON-TARGETplus (OT+) siRNAs targeting ApoB and a matched non-targeting control assessed at 25 nM as well as the Negative controls for the sense and antisense siRNAs (NEG sense and NEG antisense, respectively) and the Argonaute control ApoB siRNA (POS ApoB).

[0178] With reference to FIG. 2 (a-d), a dose dependent decrease in ApoB expression with increasing siRNA concentration was observed for the majority of the siRNA tested, however the level of knockdown observed differed between the siRNAs, as anticipated. Knockdown of ApoB by the targeting siRNAs was greater than for the NEG controls for all siRNAs tested, with the exception of siRNAs C28 and C29, where limited knockdown was observed.

[0179] With reference to Table 3, when sense and antisense siRNAs targeting the same RNA sequence are compared the knockdown was similar. Four siRNA pairs appeared to show a differential in knockdown efficiency between the sense and antisense siRNA. These were C3-C23, C8-C28, C9-C29, and C13-C33. For all of these, except C3-C23, the sense siRNA appeared to be more efficient than the antisense siRNA, however for C8-C28 and C9-C29, the antisense siRNA did not appear to knockdown ApoB expression.

[0180] Overall based on the ApoB mRNA level following treatment with 25 nM siRNA, the following siRNAs display the best knock-down efficiency: the sense crook siRNAs C3 and C13 and the antisense crook siRNAs C23, C24, C30 and C36. C13 and C23 are the only two siRNAs showing a knock-down efficiency greater than 85% at this dose; see Table 3.

TABLE-US-00010 TABLE 3 In vitro activity of ApoB Crook siRNAs (C1-C40) in HepG2 cells. Each siRNA is ranked according to ApoB mRNA knockdown (KD) performance, with highest KD at the top of the table. siRNA Crook position: Construct ApoB mRNA Knockdown Sense (S) NM_000384 Code 6.25 nM 25 nM Anti-sense (A) Start position C23 0.18 (82%) 0.11 (89%) A 423 C13 0.20 (80%) 0.15 (85%) S 6964 C36 0.18 (82%) 0.20 (80%) A 9006 C24 0.24 (76%) 0.20 (80%) A 13693 C30 0.25 (75%) 0.20 (80%) A 10168 C2 0.25 (75%) 0.22 (78%) S 2835 C22 0.27 (73%) 0.22 (78%) A 2835 C3 0.36 (64%) 0.22 (78%) S 423 C26 0.31 (69%) 0.23 (77%) A 1253 C15 0.29 (71%) 0.24 (76%) S 11500 C14 0.24 (76%) 0.25 (75%) S 10497 C17 0.29 (71%) 0.25 (75%) S 8567 C35 0.26 (74%) 0.36 (64%) A 11500 C10 0.32 (68%) 0.26 (74%) S 10168 C8 0.32 (68%) 0.26 (74%) S 430 C27 0.34 (66%) 0.26 (74%) A 13685 C16 0.30 (70%) 0.28 (72%) S 9006 C18 0.30 (70%) 0.28 (72%) S 3347 C5 0.49 (51%) 0.28 (72%) S 669 C7 0.52 (48%) 0.28 (72%) S 13685 C9 0.54 (46%) 0.30 (70%) S 4102 C4 0.26 (74%) 0.31 (69%) S 13693 C25 0.40 (60%) 0.32 (68%) A 669 C20 0.44 (56%) 0.33 (67%) S 4102 C1 0.75 (25%) 0.32 (68%) S 2093 C34 0.25 (75%) 0.33 (67%) A 10497 C33 0.31 (69%) 0.33 (67%) A 6964 C12 0.33 (67%) 0.33 (67%) S 13694 C37 0.51 (49%) 0.36 (64%) A 8567 C6 0.40 (60%) 0.37 (63%) S 1253 C21 0.62 (38%) 0.36 (64%) A 2093 C38 0.36 (64%) 0.39 (61%) A 3347 C39 0.53 (47%) 0.41 (59%) A 10450 C31 0.57 (43%) 0.43 (57%) A 2778 C19 0.67 (33%) 0.42 (58%) S 10450 C32 0.43 (57%) 0.48 (52%) A 13694 C11 0.55 (45%) 0.52 (48%) S 2778 C40 0.51 (49%) 0.58 (42%) A 4102 C28 0.60 (40%) 0.63 (37%) A 430 C29  1.25 (−25%) 0.69 (31%) A 4102

REFERENCES

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