CN114480382A - RNAi agents and compositions for inhibiting SURF4 gene expression - Google Patents

Abstract

The present invention is an RNAi agent and composition for inhibiting SURF4 gene expression, and relates to short antisense oligonucleotides and RNAi agents capable of inhibiting SURF4 expression. The invention also relates to compositions comprising one or more of the short antisense oligonucleotides or RNAi agents. The invention also relates to the use of the short antisense oligonucleotides, RNAi agents or compositions in reducing blood lipids in a subject, or in treating or preventing a disease associated with dyslipidemia.

Description

RNAi agents and compositions for inhibiting SURF4 gene expression

Technical Field

The present invention relates to short antisense oligonucleotides and RNAi agents for inhibiting SURF4 gene expression, and compositions comprising one or more of the short antisense oligonucleotides or RNAi agents. The invention also relates to methods and uses of the short antisense oligonucleotides, RNAi agents, or compositions for reducing blood lipid in a subject, or for treating or preventing a disease associated with dyslipidemia.

Background

SURF4 is a transmembrane cargo receptor (cargo receptor) on the endoplasmic reticulum. Transmembrane cargo receptors (cargo receptors) are generally thought to mediate the recognition of selected cargo (e.g., proteins or lipids) by the envelope complex (Dancourt, j., and Barlowe, C. (2010). The yeast homolog Erv29p of SURF4 mediates the transport of the alpha-factor, wherein ER export of these cargo is accelerated by focusing the protein to be transported into COPII vesicles (Belden, W.J., and Barlowe, C. (2001). Science 294, 1528-. However, very few transmembrane receptors have been characterized in mammals, let alone elucidating their physiological functions in vivo. In contrast, there are studies in the art that this receptor may not be sufficient to deliver a sufficient number of major secretions to function in physiological transport programs that require large amounts of secretion (Warren, G., and Mellman, I. (1999). Cell 98, 125-.

Lipids are important energy sources, structural components, and also play a role in physiological functions such as signal transduction. Lipids are distinguished from other biomolecules because lipids are hydrophobic and therefore bulk transport of lipids requires the use of specialized lipoproteins. The mechanism of how to distinguish lipid-carrying lipoproteins from general proteins and to allow them to selectively enter the secretory pathway is unclear. Dyslipidemia can lead to a variety of cardiovascular and metabolic diseases. For example, low density lipoprotein cholesterol (LDL-C) is thought to be closely associated with atherosclerosis. Arteriosclerosis can further cause diseases with high fatality rate and disability rate such as cerebral apoplexy, coronary heart disease and the like, and is also related to diseases such as hypertension, fatty liver, diabetes and the like. With the increase of living standard, the incidence of dyslipidemia-related diseases in the population tends to increase.

Although mainstream drugs for lowering Low Density Lipoprotein (LDL) including MTP (microsomal triglyceride transfer protein) inhibitors and antisense oligonucleotides (ASO) that inhibit APOB (apolipoprotein B) have been successful, there is an unmet need in the art for therapeutic agents for lowering plasma lipids (Rader, D.J. (2016.). Cell Metab 23,405- & 412).

PCSK9 inhibitors are novel hypolipidemic agents that have attracted attention in recent years. PCSK9 inhibitors of lipid lowering agents including eloreuptamab (evocolumab) from ann, Alirocumab from cenofuran, and leqvio (inclisiran), a small interfering RNA lipid lowering agent from nova, are highly expected. Binding of PCSK9 (proprotein convertase, cumysin type Kexin 9) to LDLR (low density lipoprotein receptor) promotes lysosomal degradation of LDLR, which fails to return to the cell surface for LDL binding. The PCSK9 inhibitor improves the clearance of LDL (low-cholesterol) such as bad blood fat by preventing the degradation of LDLR by PSCK9, thereby realizing the effect of reducing blood fat. However, PCSK9 inhibitors fail to achieve the desired LDL lowering effect in patients with their own LDLR deficiency, for example, in patients with severe familial hyperlipidemia resulting from LDLR inactivating mutations.

Therefore, there is still a need in the art to develop new targets and effective hypolipidemic agents acting on the targets.

Disclosure of Invention

The inventors of the present invention have revealed a transfer procedure specific to lipid carriers, which enables the efficient and precise supply of lipids in response to physiological needs, and the entrance of which is defined by molecular switch SAR1B and cargo receptor SURF 4. The present inventors have for the first time found and confirmed that inactivation of the coding gene for the cargo receptor SURF4 in the liver completely prevented pathogenic lipid abnormalities and atherosclerosis, even in cases where the lipid clearance function was deficient due to lack of LDL receptor (LDLR), and did not cause significant damage or inflammation of the liver. These findings indicate that inhibition of SURF4 gene expression can reduce the production and transport of "bad blood lipid" LDL from the source, which can produce better blood lipid lowering effect for diseases such as severe familial hyperlipidemia (e.g., caused by LDL receptor (LDLR) inactivating mutation).

Based on the above-mentioned findings, the inventors have constructed antisense oligonucleotide molecules capable of inhibiting SURF4 gene expression for the regulation of lipid abnormality (lowering of blood lipids) and the further treatment and prevention of diseases associated with lipid abnormality, thereby completing the present invention.

Thus, in a first aspect, the invention provides short antisense oligonucleotides 8-30 nucleotides in length that target the nucleotides encoding SURF 4. Preferably, the short antisense oligonucleotide comprises 15-25 nucleotides, more preferably 19-23 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

In a specific embodiment, the short antisense oligonucleotide comprises or consists of a sequence selected from the group consisting of the sequences of the antisense strands of table 1 or a sequence differing by 1 or 2 nucleotides from it.

In a second aspect, the present invention provides an RNAi agent for inhibiting expression of SURF4 gene, comprising the short antisense oligonucleotide of the first aspect, and a sense strand at least partially complementary to the short antisense oligonucleotide. Preferably, the sense strand is at least 85% complementary, at least 90% complementary, at least 95% complementary, or 100% complementary to the short antisense oligonucleotide over the length of the short antisense oligonucleotide. In particular embodiments, the RNAi agent comprises a sense strand and/or an antisense strand selected from table 1. More preferably, the RNAi agent is selected from the group consisting of the combinations of sense and antisense strands listed in table 1.

In a third aspect, the present invention provides a composition for inhibiting expression of SURF4 gene, comprising (a) the short antisense oligonucleotide of the first aspect or the RNAi agent of the second aspect, and (b) a pharmaceutically acceptable excipient.

In a fourth aspect, the invention provides the use of a short antisense oligonucleotide of the first aspect or a RNAi agent of the second aspect in the manufacture of a medicament for the treatment or prevention of a disease associated with dyslipidemia, such as cardiovascular and cerebrovascular diseases or metabolic diseases associated with dyslipidemia. In specific embodiments, the disease is selected from the group consisting of: hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, cholesterol metabolism disorder, lipid metabolism disorder, high LDL-C blood disease, atherosclerotic cardiovascular and cerebrovascular diseases such as myocardial infarction, apoplexy, and atherosclerosis.

In a fifth aspect, the invention provides the use of a short antisense oligonucleotide of the first aspect or a RNAi agent of the second aspect in the manufacture of a medicament for reducing one, two or all three of Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) and triglycerides in the blood of a subject. In a specific embodiment, the medicament is for reducing Low Density Lipoprotein (LDL) in the blood of a subject.

In a sixth aspect, the present invention provides a method of treating or preventing a disease associated with dyslipidemia, such as a cardiovascular metabolic disease, in a subject, comprising administering to the subject a therapeutically effective amount of an RNAi agent of the second aspect or a composition of the third aspect. In specific embodiments, the cardiovascular metabolic disease is selected from the group consisting of: hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, cholesterol metabolism abnormality, lipid metabolism abnormality, high LDL-C blood disease, and atherosclerotic cardiovascular disease such as atherosclerosis.

In a seventh aspect, the invention provides a method of reducing one, two or all three of Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL) and triglycerides in the blood of a subject, comprising administering to the subject a therapeutically effective amount of an RNAi agent of the second aspect or a composition of the third aspect. In particular embodiments, the method is for reducing Low Density Lipoprotein (LDL) in the blood of a subject.

In a specific embodiment of the fourth, fifth, sixth, seventh aspect, the subject is deficient in LDLR expression. In particular embodiments, the subject is a familial hyperlipidemia patient with a gene deficiency, such as a hereditary LDLR deficiency.

Drawings

Fig. 1 is a schematic diagram showing the mechanism of interaction between SURF4 and SAR1B, both of which recognize lipoproteins and mediate their packaging into transport vesicles for ER export, as revealed by the inventors.

Fig. 2 is a schematic showing the transmembrane structure and sequence of SURF 4.

Figure 3 is a mass spectrum of SURF4 peptide identified as a novel copri factor using a proximity-dependent proteomics study. The protein was biotinylated using SAR1B-BirA, then purified using streptavidin beads and visualized using avidin-HRP after separation by SDS-PAGE. Asterisks: SAR1B-BirA fusion protein. Three independent experiments are shown as representative. Right side: SURF4 peptide was identified by mass spectrometry.

Fig. 4 is a schematic and experimental evidence illustrating sarf 4 packaging into reconstituted COPII vesicles in an SAR 1B-dependent manner. Left: schematic representation of in vitro recombination assay of COPII vesicles. And (3) right: immunoblotting of vesicle-marking proteins with the indicated antibodies.

Figure 5 shows a schematic of the process of tissue-specific knock-out of Surf4 in example 2.

Fig. 6A-B show results of SURF4 knockout by CRISPR-mediated gene editing, in which three different grnas targeting SURF4 and control grnas were used to knock out SURF4 protein and mRNA in mouse liver, (a) results of immunoblotting of total liver protein from mice, (B) results of total liver mRNA extracted from mice using qPCR (a: P <0.001, two-tail Student's t test).

Fig. 7A-B show the reduction of total cholesterol (a) and total triglyceride (B) levels in mouse plasma following CRISPR-mediated gene editing knockout of SURF 4. The results show data for the CRISPR gene-edited mice and controls using three grnas against Surf4, each group n-5. Time points (x-axis) represent time post AAV injection. Data are presented as mean ± SEM. P <0.001 (two-tailed Student's t test).

Fig. 8A-B show cholesterol and triglyceride levels of different classes of lipoproteins in the control mouse and Surf4 CRISPR mouse of fig. 7A, (a) cholesterol measurements after fractionation of lipoproteins in plasma into VLDL, LDL and HDL by FPLC, and (B) triglyceride measurements after fractionation of lipoproteins in plasma into VLDL, LDL and HDL by FPLC.

Fig. 9 is a dot-line graph showing that deficiency of liver Surf4 inhibited triglyceride secretion into plasma, and WT (n-3) and Surf4 LKO (n-3) mice were used as controls.

Fig. 10 is a graph of histological staining of mouse liver sections, showing that lack of liver Surf4 results in lipid accumulation in the liver. Upper part: h & E staining; the lower part: and (5) dyeing with oil red O.

Fig. 11A-B are histograms comparing hepatic tg (a) and cholesterol (B) content of control (n-6) and liver Surf4 deficient mice (n-6). P <0.001 (two-tailed Student's t test).

Figure 12 compares plasma AST and ALT levels in three-month-old control mice (n-4) and liver Surf 4-deficient mice (n-4). n.s. not significant (two-tailed Student's t test).

Figures 13A-B compare (a) plasma AST and ALT levels, n.s. ═ insignificant (two-tailed Student's t test), in 13-month old control mice (n ═ 5) and liver Surf4 deficient mice (n ═ 5); and (B) histological staining of liver.

Figure 14 is plasma TG, cholesterol and AST, ALT levels in control mice (n-6) and Surf4 deficient mice (n-6) fed a fructose diet. P <0.001 (two-tailed Student's t test).

FIG. 15 is a schematic showing Surf4(Surf4 flox) between loxP sites and deleted alleles.

Fig. 16A-C are histograms showing the dose-dependent effect of Surf4 in lipid regulation. In the control group (n ═ 10), Surf4^flox/flox(n-6) and Surf4^flox/+(n-11) plasma total cholesterol (a), triglyceride (B) and apob (c) levels measured after AAV-mediated Cre recombinase expression in mouse liver. Data are presented as mean ± SEM. Asterisks: p<0.001；****：P<0.0001 (two-tailed Student's t test).

Figures 17A-B are bar graphs showing that Surf4 reduced blood lipids in a dose-dependent manner under conditions of 12 hours of re-receiving high fructose diet after 24 hours of fasting. In control group (n ═ 11), Surf4^flox/flox(n-6) and Surf4^flox/+(n-6) mouse liver expression of AAV Cre recombinase, total plasma cholesterol (a) and triglyceride (B) levels measured. Data are presented as mean ± SEM. Asterisks: p<0.01； ****：P<0.0001 (two-tailed Student's t test).

FIGS. 18A-B show hepatic cholesterol (A) and total triglyceride (B) levels in mice of FIGS. 17A-B. n.s. not significant. Asterisks: p <0.001 (two-tailed Student's t test).

Fig. 19A-B are photographs showing the localization of SURF4 within a cell. (A) Huh7 intracellular SURF4 was localised at the ER in steady state, Huh7 cells were fixed and co-stained with anti-SURF 4 antibody and either anti-SEC 31A antibody (COPII marker protein) or anti-GM 130 (golgi marker protein) and examined by confocal microscopy at a scale bar of 5 μm; (B) SURF4 mAb was verified in immunostaining, hepatocytes isolated from WT or SURF4 KO murine livers were fixed with methanol and stained with anti-SURF 4 antibody, followed by confocal microscopy at a scale bar of 5 μm.

Fig. 20 is a photograph of cells demonstrating the cycling characteristics of SURF 4. Mutant or continuously activated form of Arf1 by the copri sorting motif of SURF4^Q71IThe expression of (a) makes SURF4 stagnate on the golgi. Upper and middle panels: huh7 cells expressing FLAG-tagged wild-type SURF4 (on), or coexpressing a continuously activated form of Arf1^Q71I(iii) (in); the lower small picture: hurh 7 cells expressing SURF4-AAA mutants. Prior to confocal microscopy, cells were fixed and stained with the indicated antibodies at a scale bar of 5 μm.

Fig. 21 is a schematic diagram of a model depicting the bidirectional shuttling of SURF4 between the ER and golgi.

Fig. 22 is a schematic depicting the design of CRISPR resistant SURF4 cDNA. Upper part: mouse Surf4 cDNA sequence (SEQ ID NO:5) and corresponding protein sequence (SEQ ID NO: 6); the following: the human SURF4 cDNA sequence (SEQ ID NO:7) and the corresponding protein sequence (SEQ ID NO:8) were used for anaplerotic experiments. The PAM region and gRNA targeting sequences are indicated in dark and light grey, respectively. Mutations in the human SURF4 cDNA eliminated the PAM motif, but did not alter protein coding.

Fig. 23A-B are bar graphs showing the results of anaplerosis experiments demonstrating that defective Surf4 in recovery failed to anaplerosis lipoprotein secretion. Total cholesterol (a) and triglycerides (B) were measured using plasma samples from mice receiving gRNA plus EGFP cDNA targeting Surf4(Surf4 LKO + EGFP), gRNA plus CRISPR resistant Surf4 cDNA targeting Surf4(Surf4 LKO + Surf4 WT) or Surf4-AAA mutant cDNA (Surf4 LKO + Surf4 AAA) (n ═ 5 per group). Data are presented as mean ± SEM. Asterisks: ***: p <0.001 (two-tailed Student's t test).

FIG. 24 is the immunoblot results demonstrating that lack of Sar1b increased the Surf4-APOB interaction. FLAG-tagged Surf4 was introduced into the liver of WT or Sar1b lko (ko) mice by AAV, liver samples were lysed and anti-FLAG IP was performed, and the presence of proteins in total liver lysates (Inputs) or immune complexes (FLAG IP) was detected by immunoblotting after SDS-PAGE. Representative results from three independent experiments are shown.

FIG. 25 is a bar graph showing the synergistic effect of Sar1b and Surf4 single dose insufficiency in lowering blood lipids. Total plasma cholesterol levels measured in control (CTL, n-12), Sar1b L-hets (n-9), Surf 4L-hets (n-13) and duplicate L-hets (n-9) mice. Data are presented as mean ± SEM. **: p < 0.01; ****: p <0.0001, compared to CTL. Statistical methods are one-way ANOVA test and Tukey posthoc test.

FIG. 26 is the results of H & E and oil Red O staining of liver samples obtained from the same mice as in FIG. 25.

Fig. 27A-D are histograms showing the fit of SAR1B and SURF4 in lipid transport. (a-B) liver expression Sar1B, Surf4, or a combination of both (n-7 per group) plasma cholesterol (a) and plasma tg (B) in mice that did not affect daily diet, n.s. -not significant (by one-way ANOVA test and Tukey posthoc test); (C) liver expression of Surf4 increased plasma cholesterol in fructose diet mice, control mice (GFP) or mice with liver expressing Sar1b, Surf4 or a combination of both (n-7 per group) were fed a fructose diet two weeks prior to lipid measurement: p <0.01, compared to GFP group (by one-way ANOVA test and Tukey posthoc test); (D) liver expression Sar1b, Surf4, or a combination of both (n-7 per group) did not affect plasma TG in mice on a fructose diet for two weeks, n.s. -not significant (by one-way ANOVA test and Tukey posthoc test).

Figures 28A-B are histograms showing that liver inactivation of Surf4 reduces plasma cholesterol (a) and triglycerides (B) in mice (each group n-8) that received PCSK9 AAV and a high cholesterol diet. Data are presented as mean ± SEM. *: p<0.05；****：P<10^-4(two-tailed Student's t test).

Fig. 29 is a bar graph of plasma AST and ALT levels after 3 months of feeding the mice in fig. 28 with a high cholesterol diet, with similar data for the three mice.

FIG. 30 shows liver inactivation of Surf4 upon receiving PCSK9 AAV and high cholesterolAtherosclerosis was prevented in the diet mice, which were shown to be derived from WT (n-8), Surf4^+/f(n-8) and Surf4^f/fRepresentative images of atherosclerotic plaques in (n-8) mice.

Fig. 31 is a graph of WT (n-8), Surf4 from fig. 30^+/f(n-8) and Surf4^f/f(n-8) bars of atherosclerotic plaques in mice were quantified.

Figures 32A-D show the effect of liver inactivation by Surf4 on cholesterol and triglyceride levels in different lipoprotein fractions. Plasma cholesterol levels (a) and their area under the curve (B) in different lipoprotein populations (VLDL, LDL, HDL) isolated by FPLC in control (CTL, circle), liver Surf4 KO (Surf4 LKO, triangle) and liver Surf heterozygous (Surf4 LHets, square) mice, and plasma triglyceride levels (C) and their area under the curve (D). The numbers in B and D indicate the percent reduction in liver Surf4 heterozygous mice compared to controls. ApoB-L: AopB-containing lipoprotein, VLDL and LDL were combined.

FIGS. 33A-D show the results of RNAi agents S4-1, S4-2, S4-3 in vitro cell experiments. (A) SURF4 mRNA levels; (B) immunoblotting results for SURF4 protein; (C) immunoblotting results for APOB protein; (D) level of APOB in the medium.

Detailed Description

Definition of

The practice of some of the methods disclosed herein, unless otherwise indicated, employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art.

The term "about" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 or a standard deviation of greater than 1, according to practice in the art. Alternatively, "about" may represent a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may represent an order of magnitude, preferably within 5-fold, more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise specified, it should be assumed that the term "about" means within an acceptable error range for the particular value.

The term "nucleotide" generally refers to an alkali-sugar-phosphate combination. Nucleotides may comprise analogs or derivatives of nucleotides, as well as synthetic nucleotides. The nucleotides may be labeled for detection by known techniques. Detectable labels may include, for example, radioisotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels.

The term "expression" refers to one or more processes of transcription of a polynucleotide from a DNA template (e.g., into mRNA or other RNA transcript) and/or subsequent translation of the transcribed mRNA into a peptide, polypeptide, or protein. The transcripts and encoded polypeptides may be collectively referred to as "gene products". If the polynucleotide is derived from genomic DNA, expression may comprise splicing of the mRNA in a eukaryotic cell. "Up-regulation" or "down-regulation" of expression generally refers to an increase or decrease in the level of expression of a polynucleotide (e.g., RNA, e.g., mRNA) and/or polypeptide sequence relative to its level of expression in the wild-type state.

The term "modulation" in relation to expression or activity refers to altering the level of expression or activity. Modulation may occur at the transcriptional level and/or the translational level.

The terms "subject," "individual," and "patient" are used interchangeably herein to refer to a vertebrate, preferably a mammal, e.g., a human. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

The term "treatment" refers herein to a method for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and/or a prophylactic benefit. For example, treatment may comprise administering a therapeutically effective amount of a short antisense oligonucleotide, RNAi agent, or composition to SURF4 of the present invention. Therapeutic benefit means that there is any treatment-related improvement in the disease or condition being treated.

The term "preventing" as used herein refers to administering a short antisense oligonucleotide, RNAi agent, or composition of the invention directed to SURF4 to a subject at risk of developing a particular disease or condition, or to a subject presenting with one or more physiological signs of a disease, prior to development of the particular disease, thereby producing a prophylactic benefit, even though the disease or condition may not have yet been manifested.

The term "effective amount" or "therapeutically effective amount" refers to an amount of a composition, e.g., a composition comprising an antisense oligonucleotide or RNAi agent of the invention, that is sufficient to produce a desired activity or effect, e.g., a blood lipid lowering effect, when administered to a subject in need thereof.

Lipoprotein

Lipoproteins are complexes comprising proteins and lipids. Lipoproteins existing in human blood are classified according to lipid content and ultracentrifuge density and mainly include chylomicron, Very Low Density Lipoprotein (VLDL), Low Density Lipoprotein (LDL) and High Density Lipoprotein (HDL). Among these, LDL is responsible for the transport of cholesterol from the liver to human cells, and is a major risk factor for atherosclerosis, also known as "bad cholesterol lipoprotein" or "bad blood lipids".

The RNAi agents of the invention are capable of lowering blood lipids, particularly LDL, VLDL and triglycerides in plasma.

SURF4

Human SURF4 is a 269 amino acid protein with about 99% identity to its mouse homolog. Human SURF4 has six predicted transmembrane domains and a short cytoplasmic tail containing a trilysine sorting motif for COPI interaction and ER recovery (Jackson, L.P. et al (2012) Dev Cell 23,1255-1262), as shown in FIG. 2.

The mechanism by which newly synthesized proteins and lipids enter the secretory pathway through COPII-coated vesicles, assemble on the Endoplasmic Reticulum (ER) via GTPase SAR1, but how to distinguish lipid-bearing lipoproteins from common protein cargo in the endoplasmic reticulum and selectively secrete these lipoproteins has not been clear prior to the present invention.

The inventors of the present invention demonstrated that this process is quantitatively controlled by GTPase SAR1B and SURF 4. Specific experiments and results are provided in the detailed description section below.

First, the inventors surprisingly found that liver-specific knockdown of Sar1b effectively and selectively blocks lipid secretion, but does not affect transport of other major secretory proteins. Based on this finding, the inventors demonstrated that the partner SURF4 of SAR1B is associated with changes in plasma lipid levels. SURF4 is a highly potent COPII cargo receptor with multiple transmembrane domains. Genome-wide association analysis (GWAS) and functional characterization studies also found that plasma LDL-cholesterol in humans is closely related to SURF4, and that acute inactivation of SURF4 resulted in almost complete disappearance of plasma lipids and lipoproteins in mice, and thus protected mice from atherosclerosis induced by PCSK9 and dietary challenges. Active repetitive cycling enables SUFR4 to act as a limiting, dose-dependent factor that supports robust transport of lipid carriers. These findings indicate that SURF4 in combination with SAR1B can operate a specialized, dose-sensitive transport procedure for lipid entry into circulation, and also indicate applications for the treatment of dyslipidemia, atherosclerosis and related cardiometabolic diseases.

The function of cargo receptors such as SURF4 in lipid transport has never been confirmed in the art, nor has it been demonstrated to what extent SURF4 can inhibit blood lipids, and whether SURF4 is dependent on the assistance of other participants such as LDL receptors and the like for its modulating effects on blood lipids. Those skilled in the art will appreciate that these aspects provide the invention.

RNAi agents and compositions

Based on the results of studies on the function of cargo receptor SURF4 in lipid transport, the inventors propose that SURF4 is inhibited by RNA interference (RNAi) agents to thereby regulate upstream of the lipid transport process, achieving the effect of lowering lipids ultimately secreted into the circulation, for the treatment or prevention of diseases and symptoms associated with lipid abnormalities, particularly elevated lipid abnormalities.

Accordingly, the present invention provides antisense oligonucleotides targeting the SURF4 gene, as well as double stranded RNAi agents targeting the SURF4 gene.

The antisense oligonucleotides and double-stranded RNAi agents of the invention are capable of inducing degradation of SURF4 mRNA by acting on SURF4 mRNA. Specific examples of double stranded RNAi agents are detailed in table 1, but those skilled in the art will appreciate that the scope of the present invention is not limited to these specific RNAi agents, but encompasses any RNAi agent that targets SURF4 mRNA and is capable of acting on SURF4 mRNA to produce an effect of reducing SURF4 mRNA levels.

Basic methods for designing antisense oligonucleotides and double-stranded RNAi agents against known sequences are known in the art. The sequence of the human SURF4 gene and the transcribed mRNA sequence are known. The 7 different transcripts of the human SURF4 gene are described in the prior art, the sequences of which can be obtained from the public database of NCBI by GenBank accession numbers (SURF4_ Homo sapiens _ transcript 1, NM _ 033161; SURF4_ Homo sapiens _ transcript 2, NM _ 001280788; SURF4_ Homo sapiens _ transcript 3, NM _ 001280789; SURF4_ Homo sapiens _ transcript 4, NM _ 001280790; SURF4_ Homo sapiens _ transcript 5, NM _ 001280791; SURF4_ Homo sapiens _ transcript 6, NM _ 001280792; SURF4_ Homo sapiens _ transcript 7, NM _ 001280793). Transcript 1(SEQ ID NO:10) is the most classical, most common transcript, and is preferably used as the basis for sequence design for antisense oligonucleotides and RNAi agents. The antisense oligonucleotides and RNAi agents of the invention can also be designed based on other transcripts.

It is known in the art that antisense oligonucleotides and RNAi agents can be endowed with certain properties, such as increased silencing efficiency for target genes, tissue/cell specificity, etc., by modification of RNA molecules (e.g., modifying nucleotides or linking to other groups), but fall within the scope of the present invention as long as the modified RNA molecules still comprise 8-30 nucleotides as defined herein and have the ability to target the SURF4 gene.

Therapeutic uses

The antisense oligonucleotides, RNAi agents and compositions directed to SURF4 of the present invention can be used for preventing or treating diseases or conditions associated with lipid metabolism, particularly cardiovascular metabolic diseases, more particularly atherosclerotic cardiovascular disease (ASCVD). The disease or condition is associated with abnormal lipids, particularly abnormal elevated lipids, such as diseases or conditions caused by abnormal elevated lipids, such as abnormal elevated triglycerides and/or cholesterol.

Preferably the cardiovascular metabolic disease is selected from the group consisting of: hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, cholesterol metabolism disorder, lipid metabolism disorder, high LDL-C blood disease, and atherosclerotic cardiovascular disease such as atherosclerosis.

The antisense oligonucleotides, RNAi agents, and compositions of the invention directed to SURF4 are particularly suitable for patients with high LDL-C blood. The antisense oligonucleotides, RNAi agents and compositions of the invention are also particularly suitable for patients with LDLR deficiencies, such as genetic deficiencies, and thus unresponsive or unresponsive responses to drugs that improve LDLR function or act on LDLR inhibitors (such as PCSK9 inhibitors), e.g., familial hyperlipidemia patients. In preferred embodiments, for patients with severe familial hyperlipidemia, circulating LDL-C in such patients can be reduced by 30%, 40%, 50%, or even more by administering the RNAi agents of the invention.

Delivery mode

The RNAi agent of the invention or a composition comprising the RNAi agent can be delivered to a subject in a variety of ways.

In preferred embodiments, the RNAi agent or composition of the invention is delivered to a subject in a tissue-specific manner, preferably in a liver-specific manner.

Tissue-specific delivery can be achieved in a variety of forms. For example, liposomes with tissue specific antibodies can be used, or non-nucleotide groups can be linked to the RNAi agent to confer tissue specific distribution and uptake. For example, US9370582B2 discloses a method of achieving liver-targeted delivery by coupling RNAi molecules to GalNAc (N-acetylgalactosamine), a highly potent ligand for asialoglycoprotein receptors expressed on hepatocytes and thus capable of conferring liver-targeting specificity to the molecules coupled thereto.

In particular embodiments, Lipid Nanoparticles (LNPs) are used to deliver the RNAi agents or compositions. Current research has found that LNP preferentially reaches liver cells in vivo, making it particularly suitable for delivery of the RNAi kit compositions of the invention. Thus, efficient and relatively specific hepatic delivery of the RNAi molecules of the invention can be achieved using LNPs.

Detailed Description

For a more complete understanding and appreciation of the invention, the invention will be described in detail below with reference to examples and the accompanying drawings, which are intended to illustrate the invention and not to limit the scope thereof. The scope of the invention is specifically defined by the appended claims.

The experimental materials and methods used in the examples are described below.

Mouse model

Animal containment and all experimental procedures were approved by the Beijing university Committee for laboratory animal care and use, which is an AAALAC approved animal institute. All mice used were incubated on a C57BL6/J background basis. Mice were maintained at 22 ℃ for a 12 hour light/dark cycle, and were free to eat normal food and water unless otherwise indicated. All mice used for the experiments were from laboratory mating (in-housemating). Animals were monitored for overall development and wellness according to AAALAC guidelines and littermates were used as controls in the experiments, unless otherwise indicated.

Sar1b liver-specific knockout mice were prepared by mixing Sar1b^fl/flMice (KOMP: 061774, RRID: MMRRC-061774-UCD) were generated by crossing an albumin promoter driven Cre recombinase transgenic mouse (JAX: 016833, RRID: IMSR-JAX: 016833).

Surf4^fl/flMice were generated by CRISPR/Cas 9-based protocols using wild-type C57BL6/J mice. Briefly, Cas9 mRNA, sgRNA, and donor plasmid were injected into fertilized eggs. Microinjection was performed by bioctygen corporation. Founder Surf4^fl/+Mice were backcrossed with C57BL6/J for two rounds. Surf4^fl/flMice were immunized with Surf4^fl/+Mouse progeny were generated. Cre-dependent spCas9 knock-in (KI) mice were purchased from Jackson Lab (RRID: IMSR _ JA)X:026556) (Platt et al (2014). Cell 159, 440-455).

Unless otherwise stated, male mice 8-16 weeks old were used for all experiments. Mice were divided into different groups or randomly assigned according to genotype. For studies with a particular diet, 8 week old mice received a high cholesterol diet for a period of 12 weeks, or 8 week old mice received a high fructose diet for a period of 2 weeks.

Cell culture

HEK293A cells (Thermo Fisher, R70507) were obtained from Thermo Fisher. 293T cells (ATCC, CRL-3216, RRID: CVCL _0063) and HepG2(ATCC, HB-8065, RRID: CVCL _0027) were obtained from the ATCC. Huh7 cells (JCRB Cell Ban, JCRB0403, RRID: CVCL _0336) were obtained from JCRB. Sex information for the cell lines may be obtained at the web site of the relevant supplier. Primary hepatocytes of mice were isolated from the liver of male mice with the indicated genotype.

All cells were cultured in Dulbecco' S Modified Eagle Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (P/S) at 37 deg.C and 5% CO₂And (5) atmosphere.

For plasmid transfection, Polyethyleneimine (PEI) was used according to the manufacturer's protocol. For gene editing in Huh7 cells, cells were infected with lentiviruses packaged with CRISPR elements with the aid of 8 μ g/ml polypropylene.

Lipid analysis

Unless otherwise stated, after fasting of the mice for 16 hours, blood was taken from the tip of the tail using a heparin capillary and centrifuged at 6000rpm at 4 ℃ for 5 minutes, thereby obtaining plasma. Triglyceride and total cholesterol levels were measured using a commercially available kit (Zhongsheng Beijing and Sigma) according to the instructions.

For Fast Protein Liquid Chromatography (FPLC) analysis, plasma samples of the same genotype were pooled and then FPLC was performed using a Superose 6 chromatography column. The sample was eluted at a flow rate of 0.5ml/min and 500. mu.l of fractions per tube were collected for cholesterol and triglyceride measurements.

For lipidomic analysis, serum lipids were extracted by the Bligh-Dyer lipid extraction method, adding a lipid internal standard mixture (splasttm lipedomix) during extraction. LC-MS analysis of the extracted lipids was performed as described in previous reports. Lipid separation was performed on an AQUITY UPLC system (Waters, Milford, MA) equipped with a CSH C18 column with an internal diameter of 100X 2.1mm, 1.7 μm (Waters, Milford, MA). The gradient elution was carried out at a flow rate of 0.4ml/min and the column was thermostatted at 55 ℃. Mobile phase a consisted of water-ACN (40:60, v/v) supplemented with 10mM ammonium formate and 0.1% formic acid, and mobile phase B consisted of ACN-IPA (10:90, v/v) supplemented with 10mM ammonium formate and 0.1% formic acid. The initial conditions started at 40% B and a linear gradient from 40% to 43% B was initiated immediately within 2 minutes (curve 6). The percentage of mobile phase B increased to 50% in the next 0.1 minutes. In the next 9.9 minutes, the gradient further increased to 54% B and the amount of mobile phase B increased to 70% in 0.1 minutes. In the final part of the gradient, mobile phase B increased to 99% in 5.9 minutes. The eluent composition returned to the original state in 0.1 minute and the column was equilibrated at the original conditions for 1.9 minutes before the next injection, so that the total run time was 20 minutes. The sample was injected in 2. mu.l and the autosampler temperature was set to 4 ℃. For all experiments, the same chromatographic conditions were used.

The UPLC system was used in conjunction with a equipped SYNAPT G2-S qTOF HDMS mass spectrometer (SYNAPT G2-S HDMS, Waters Corporation) for analysis. Electrospray (ESI source) positive ionization and negative ionization modes were used. Data were acquired by MassLynx 4.1 software in a full scan mode over a range of m/z from 50 to 1,200 Da. The unfragmented ion data and the fragmented ion data are collected by an alternating acquisition function operating with alternating collision energy. Data alignment and ion chromatogram extraction were analyzed by prognesis QI version. Lipid identification was performed using the LIPID MAPS database. R3.5.1 are used to generate heat maps.

VLDL secretion assay in mice

Mice were fasted for 16 hours and given an intravenous injection of tyloxapol at a dose of 500mg/kg body weight. Blood samples were collected at the indicated time points and plasma was separated by centrifugation. Triglycerides were determined as described above.

Histology and oil red O staining

For histological analysis, liver tissue was fixed in 4% PFA and then paraffin embedded. Hematoxylin and eosin (H & E) staining and Sirius red (Sirius red) staining were performed on 5 μm paraffin sections. For oil Red o (oil Red o) staining, liver samples were embedded in OCT gel and then rapidly frozen. Frozen tissues were cryosectioned at 8 μm and stained with oil red O according to the manufacturer's instructions. Liver macrophages were visualized by immunohistochemical staining for F4/80.

Quantification of hepatic triglyceride and cholesterol

The flash frozen liver samples were weighed and homogenized in PBS and the lipids extracted by the Bligh-Dyer method. Briefly, the homogenate was mixed with chloroform-methanol (2:1) by vortexing thoroughly. After centrifugation, the organic phase was collected and resuspended in a 15% aqueous triton X-100 solution in a rotary evaporator. Triglyceride and cholesterol were measured as described above.

Quantitative proteomics

The protein concentration of the sample was adjusted to 3mg/mL using 100mM TEAB. The enriched protein was denatured in 6M Urea/TEAB and treated with 10mM dithiothreitol (DTT, J) at 35 deg.C&K Scientific) for 35 minutes and blocked with 20mM iodoacetamide (Sigma-Aldrich) with stirring in the dark for 30 minutes at 35 ℃. The reaction mixture was diluted to 2M urea/TEAB and digested with 0.6. mu.g trypsin overnight at 37 ℃ with stirring. The digested sample was subjected to reductive dimethylation. Briefly, 4. mu.L of 4% (v/v) CH₂O or¹³CD₂O (SigmaAldrich) was added separately to the digested samples and incubated with 4. mu.L of 0.6M NaBH at room temperature₃CN was reduced for 1 hour. The reaction was quenched with 16. mu.L of 1% (v/v) ammonia solution and 8. mu.L of formic acid. The light and heavy sample pairs were combined, desalted using a C18 spin column and dried by vacuum centrifugation. The dried sample was finally resuspended in 15. mu.L of buffer A (95% water, 5% acetonitrile, 0.1% formic acid) for LC-MS/MS analysis.

Immunochromatography, immunoblotting and Blue Native PAGE

Immunoprecipitation experiments at 4 ℃ with buffer A (50mM Tris, pH 7.5)150mM sodium chloride, 1% Nonidet P-40, and 10% glycerol, supplemented with protease inhibitor tablets (Roche)) protein extracted from mouse plasma, cultured cells, or mouse liver. The cell extracts were centrifuged at 13000g for 15 minutes at 4 ℃ and the supernatants were collected and 60ul of cell lysate was taken. The remaining lysate was incubated with 15 μ l M2 or anti-HA agarose beads (Sigma-Aldrich) for 3h at 4 ℃ and then washed 4 to 8 times with buffer A (for mass spectrometry). The immunocomplexes were then solubilized with 1.5x SDS/PAGE sample buffer and separated with a 3-15% Tris-acetate SDS/PAGE gel. After SDS/PAGE, the proteins in the gel were transferred to nitrocellulose and analyzed with IB. Rabbit polyclonal antibody and mouse monoclonal antibody against the C-terminal epitope (SEQ ID NO:9) of SURF4 were prepared by Proteitech group (www.ptglab.com)。

For Blue Native-polyacrylamide gel electrophoresis (PAGE), HEK293A cells were transfected with FLAG-SURF4 and proteins were extracted with buffer A and centrifuged at 13000 g. The supernatant was dissolved and then separated using 4% -16% Blue Native-PAGE before IB.

Identification of proteins by LC-MS/MS

To identify the proteins, silver stained protein from each sample was excised from the gel and destained with 100mM ammonium bicarbonate in 50% acetonitrile. Following dithiothreitol reduction and iodoacetamide alkylation, the protein was digested with porcine trypsin (sequencing grade modification; Promega, Madison, Wis.) overnight at 37 ℃ (Olsen JV et al, 2004). The resulting trypsin-treated peptides were extracted from the gel strips with acetonitrile containing 0.1% Formic Acid (FA). The samples were dried in a vacuum centrifugal concentrator at 30 ℃ and then resuspended in 10ul of 0.1% FA.

Using the Easy-nLC 1200 system, 5ul of sample was loaded onto a capture column (C18, Acclaim PepMap) at a rate of 0.3ul/min in 0.1% FA^TM10075 um x 2cm NanoViper Thermo) and passed through a fritless analytical column (C18, Acclaim PepMap)^TM75um x15cm nanoViper RSLC Thermo), with a 75-min gradient of 4% to 30% LC-MS buffer B (LC-MS buffer a contains 0.1% formic acid; LC-MS buffer B contained 0.1% formic acid and 80% acetonitrile) at a flow rate of 300 nl/min.

Peptides were directly injected into Thermo Orbitrap Fusion Lumos using a nano electrospray ion source with an electrospray voltage of 2.2 kV. Full scan mass spectra were obtained on an Orbitrap mass analyser (m/z range: 300-1500 Da) with a resolution set at 70,000(FWHM) at m/z 200 Da. The full scan target is 1e6, and the maximum fill time is 50 milliseconds. All data were collected in profile mode (profile mode) using positive polarity. MS/MS spectral data were also obtained in Orbitrap, with a resolution of 15,000(FWHM), m/z 200Da, High Collisional Dissociation (HCD) MS/MS fragmentation. The isolation width was 1.6 m/z. MS data were compared to the protocol discover 2.2 software via the Mouse Review Swiss-Port database.

In vitro Budding Assay using semi-intact cells (Budding Assay)

Semi-whole cells were freshly prepared by permeabilizing Huh7 cells with 40ug/ml digoxin in B88(20mM Hepes, pH 7.2, 250mM sorbitol, 150mM potassium acetate and 2mM magnesium acetate). The final volume of each reaction was 100. mu.L, containing an ATP regeneration system (1mM ATP, 40mM creatine phosphate and 0.2mg/mL creatine phosphokinase), GTP (0.2mM), murine liver cytosol containing 100. mu.g protein, and semi-whole cells containing 50. mu.g protein. The reaction mixture was incubated in a siliconized 1.5mL microcentrifuge tube at 30 ℃ for 60 minutes and then cooled on ice to terminate the reaction. After centrifugation at 14,000g for 10 minutes to remove the moderate speed precipitate (donor membrane), 75 μ L of supernatant was taken and centrifuged in a Beckman TLA100 rotor at 55,000RPM for 30 minutes at 4 ℃. The pellet (vesicle fraction) was used for immunoblot analysis.

Cell fixation, immunostaining and confocal microscopy

Cells cultured on glass coverslips were washed with pre-warmed (37 ℃) PBS and then fixed with methanol for an additional 5 minutes. Cells were rehydrated in PBS and then blocked with 2% BSA for 30 min. Cells were incubated with primary antibodies diluted in blocking buffer overnight at 4 ℃ and then with secondary antibodies for 2 hours at room temperature. For lipid droplet staining, murine primary hepatocytes cultured on glass coverslips were fixed with 4% PFA and incubated with BODIPY 493/503 for 30 minutes. Cells were fixed with anti-quenching (anti-fade) medium and images were acquired by zeiss rotating disk microscope in a double blind fashion.

LocusZoom database correlation analysis

Genome-wide association data analysis of SURF4 gene with lipid metabolism Using LocusZoom: (http:// csg.sph.umich.edu/locuszoom/) The process is carried out.

Production of AAV DNA vectors

An AAV DNA vector for CRISPR mediated liver acute KO (pX 602-AAV-Cre-sgRNA) comprises a liver specific TBG promoter, Myc-tagged Cre recombinase (for recombination flanked by loxP stop codons), and a human U6 promoter for non-coding sgRNA transcription. Using pX602-AAV-TBG: NLS-SaCas 9-NLS-HAOLLAS-bGHpA; u6 BsaI-sgRNA (PX602, Addgene, 61593-) generated vector pX 602-Cre-sgRNA. Briefly, Myc-tagged Cre recombinase was cloned between AgeI and EcoRI restriction enzyme sites (Platt et al, 2014). AAV-TBG-CRE, AAV-TBG-FLAG-hSURF4 and AAV-TBG-FLAG-hSURF4 AAA were generated from AAV-TBG-GFP (Addgene, 105535) by replacing the GFP sequence with the corresponding cDNA. For complementation experiments, the PAM sequence in hSURF4(CGG) was mutated to CTT as shown in fig. 22 and then cloned into pAAV-TBG-hSURF4 by standard mutagenesis procedures.

sgRNA design and target-directed sequence cloning

sgRNA uses the Benching platform (https://benchling.com/) Designed to maximize editing efficiency and minimize off-target effects. The sequence of sgRNA is shown in SEQ ID Nos: 1-4. For cloning of the guide sequences, synthetic oligonucleotides were inserted either into the pX602-AAV-Cre-sgRNA backbone for genome editing in the mouse liver, or into the pLenti-CRISPR V2 Cas 9D 10A for genome editing in cell lines according to the previously disclosed methods (Platt et al, 2014).

Genomic DNA extraction and SURVEYOR nuclease analysis

Genomic DNA was extracted from both cells and tissues using the TIANamp genomic DNA kit (Tiangen) following the manufacturer's protocol. The isolated genomic DNA was then used as a template for PCR for sequencing and SURVEYOR nuclease assay using high fidelity polymerase as described previously (Platt et al, 2014).

AAV production and delivery

AAV is packaged and produced in HEK293T cells. Briefly, cells were transfected with AAV transfer plasmids (7. mu.g/5 cm dish), Rep/Cap (2/8) plasmids (7. mu.g/15 cm dish), and helper plasmids (20. mu.g/15 cm dish) using Polyethyleneimine (PEI). 60 hours after transfection, cells were removed and collected. Viral purification and titer quantification were performed as described in the previous article (Platt et al, 2014). The virus was titrated by qPCR using a custom SYBR Green assay (Tiangen). AAV delivery was performed by tail vein injection. To acutely inactivate the Surf4 gene in the mouse liver by CRISPR/Cas9, 8-week-old spCas9 KI mice were administered 2x 10¹¹AAV-Cre-sgRNA having a viral genomic copy. For anaplerotic experiments, 2 × 10 with targeted sgRNA¹¹Viral genomes were copied together and 1X 10 were simultaneously added¹¹GFP-expressing or human SURF 4-expressing viral copies were injected into each spCas9 KI mouse.

Analysis of Gene expression

Tissues were homogenized in TRIzol reagent (Thermo Fisher) and RNA was extracted according to the manufacturer's protocol. A total of 1. mu.g of RNA was used to generate a 20. mu.l reaction volume of complementary cDNA by M-MLV reverse transcriptase kit (invitrogen). Quantitative PCR reactions were performed on a 7500FAST system (Applied Biosystems) using SYBR green. The gene of interest was normalized to β -actin. The RT-qPCR primers were designed according to the routine methods in the art.

Glucose Tolerance Test (GTT) and insulin resistance test (ITT)

GTT and ITT analyses were performed in WT and acute KO mice at 4 months of age (each genotype n ═ 7). For GTT, mice were fasted overnight and had free access to water. Glucose (1g/kg body weight) was injected intraperitoneally. Blood glucose was measured by tail blood sampling using a glucometer at 0, 15, 30, 45, 60 and 120 minutes after glucose injection. For ITT, mice were fasted for 4 hours, then injected intraperitoneally with insulin (0.75U/kg body weight). Blood glucose was measured at the same time point as the GTT analysis.

Observation with an electron microscope

Mice were euthanized and perfused with sodium phosphate buffer (PB, 100mM, pH7.4) and pre-fixation solution (2.5% (vol/vol) glutaraldehyde in PB, 0.8% paraformaldehyde). Liver tissue samples were cut and fixed in a pre-fixing solution for 2 hours at room temperature. The liver tissue samples were then cut into small pieces (0.2x0.3x0.5 mm)³) And fixed in the same pre-fixing solution at 4 ℃ overnight. After washing with PB, the tissue was immersed in 0.1M imidazole in PB for 30 minutes, followed by post-fixation with 2% osmium tetroxide in PB. After rinsing with high purity water, the samples were stained with 1% uranium acetate overnight at 4 ℃. Gradient dehydration was accomplished by stepwise increasing the acetone concentration and embedding in epoxy resin (60 ℃, 24 hours). The samples were sectioned using a Leica EM UC7 (approximately 60nm thick) and placed on a copper grid. In the presence of Eagle^TMImages were taken in a double-blind manner on a FEI Tecnai G220 Twin electron microscope (FEI, usa) of a 4k CCD digital camera.

Organelle isolation from mouse liver

Wild type or Surf4 depleted mouse liver was homogenized in HES buffer (20mM Hepes, pH7.4, 1mM EDTA, 250mM sucrose) and centrifuged at 3,000g for 5 min to remove nuclei or unbroken cells. 1.5ml of the supernatant was mixed with 1.5ml of 60% iodixanol in a centrifuge tube, and then 1.3ml of 20% iodixanol and 1.2ml of 10% iodixanol were added layer by layer in this order. The gradient mixture was centrifuged at 350,000g for 4 hours at 4 ℃ and separated into 25 fractions starting at the top of the gradient.

Atherosclerosis analysis

Surf4 receiving AAV-HCRApoE/hAAT-hPCSK 9D 374Y with or without AAV-TBG-Cre^fl/flAnd Surf4^fl/+Mice were fed on western diet for 3 months. Mice were anesthetized and sacrificed, then perfused sequentially with Phosphate Buffered Saline (PBS) and 4% Paraformaldehyde (PFA). Aorta was isolated and stained with oil red O and photographed with olympus stereoscopic microscope equipped with SCMOS camera. The lesion area of the thoracic aorta was quantified using Image J.

Quantitative and statistical analysis

Immunoblotting and quantification of atherosclerotic plaque area were performed by Image J. No statistical method is used to predetermine the sample size. In particular, at least triplicate replicates are included for any statistical analysis, whereas at least 6-8 mice are typically included for physiological assays, including plasma lipid measurements. Data are presented as mean ± SEM as shown in the figure. Statistical analysis was performed by GraphPad Prism 7. Statistical significance was calculated by the two-tailed Student's t test or the one-way ANOVA test with Tukey post hoc test, as shown in the figure. When P <0.05, the results were considered significant. The number indicates the corresponding statistical significance. P <0.05, P <0.01, P <0.001 and P < 0.0001. For mouse experiments, "n" corresponds to the number of mice used. For experiments using cell culture, "n" corresponds to the number of independent replicates. Experiments involving measurement of blood lipid, blood glucose and body weight of mice were performed in a blind fashion, where mice were randomly grouped according to ear tag identification numbers. Imaging, histology, and analysis of atherosclerotic plaques were also performed blindly prior to decoding the sample identity. No data points were excluded from the study. Data were assumed to be continuously normal distributed.

Example 1 characterization of SURF4 as SAR1B partner in relation to changes in blood lipids in humans

By liver-specific knock-out of Sar1b in mice using the Cre-loxP recombinase system, it was surprisingly found that Sar1b LKO mice exhibited significantly lower blood lipid levels than littermate controls under fasting conditions, and no signs of death were observed (data not shown). Then, through a secretogomics research, the liver Sar1b defect only has an effect on the secretion of proteins related to lipoprotein circulation, such as apolipoprotein B (APOB), apolipoprotein E (APOE), apolipoprotein A1(APOA1), SAA4 and SAA1, and has little effect on other common secretory proteins in plasma, such as albumin, transferrin and alpha 1 antitrypsin. In other words, the secretion of lipid carriers is separate from the secretion of general proteins, an unexpected result suggesting that there may be regulatory factors acting on SAR1B that would otherwise not direct these unconventional cargo to the cytoplasmic COPII structure across the endoplasmic reticulum membrane.

The inventors used a BioID approach to capture the dynamics of the COPII complex and its cofactors in previous studies (Nie et al, (2018.) Proceedings of the National Academy of Sciences of the United States of America 115, E3155-E3162) using a fusion protein of SAR1B and biotin ligase birA. Using a similar approach, ortho-dependent proteomics studies were performed using SAR1B-BirA and focused on transmembrane proteins present as high molecular weight oligomers as potential features of cargo receptors. The mass spectrum identified SURF4 (fig. 3). Consistent with the proximity-dependent proteomics data, SURF4 was efficiently packaged in vitro into reconstituted colpii-coated transport vesicles in an SAR 1B-dependent manner (fig. 4).

Example 2 CRISPR/Cas 9-mediated lipid-clearing Effect of liver Surf4 inactivation

To study the function of SURF4 in vivo, SURF4 of the liver was selectively inactivated using a gene editing system recently established in adult mice (Platt et al, 2014, supra). This system is achieved by knock-in alleles of "silent" spCas9, and spCas9 is initiated in a tissue-specific manner by Cre recombinase (fig. 5).

Three gRNAs (SEQ ID NOs:1-3) targeting different regions of the Surf4 gene were designed to avoid the potential "off-target" effects of any single gene editing event. AAV was used to co-deliver hepatocyte-specific Cre and three gRNAs targeting different exons of the mouse Surf4 gene, and a gRNA targeting LacZ (SEQ ID NO:4) was used as a control. High inactivation of liver Surf4 was confirmed by immunoblotting experiments (fig. 6A) and quantitative PCR experiments of Surf4 mRNA (fig. 6B) on liver lysates, whereas control liver Surf4 was not inactivated.

It was surprisingly found that inactivation of liver Surf4 in adult mice by three grnas resulted in almost complete clearance of both plasma cholesterol and triglycerides in fasting conditions within 4 weeks post AAV delivery compared to LacZ gRNA treated control mice (fig. 7A-B). The lipid-lowering effect became apparent starting at about 2 weeks after gRNA introduction and continued until 8 weeks after gRNA delivery when mice were sacrificed.

Plasma samples were fractionated by size exclusion chromatography, and as a result, it was confirmed that cholesterol was hardly present in the VLDL, LDL and HDL fractions of liver Surf 4-deficient mice as compared to the control (fig. 8A), and a similar triglyceride-clearing effect was observed in the VLDL and LDL fractions (fig. 8B). This reduction in blood lipids was attributed to a significant inhibition of hepatic total Triglyceride (TG) secretion by Surf4 deletion (fig. 9). Consistent with the secretion blocking results, oil red O staining revealed lipid accumulation in the liver of mice deficient in liver Surf4 (fig. 10). Biochemical experiments also confirmed TG and cholesterol accumulated in the liver (fig. 11). Thus, partial rather than full knock-out (e.g., half knock-down) of Surf4 may also be contemplated, thereby achieving a lipid lowering effect while still retaining a portion of the ability of Surf4 such that lipids do not accumulate in the liver, and this effect is achieved in dependence upon the "dose-dependent" nature of Surf4 as discovered by the present invention.

Despite the near zero blood lipid levels, liver Surf4 deficient mice appeared normal overall with no signs of death during the entire experiment. In addition, their plasma transaminase (AST and ALT) levels were also similar to control mice (fig. 12), and no significant signs of liver injury, fibrosis, or inflammation were observed in Surf 4-deficient livers until 13 months of age (fig. 13).

Surf4 deficient mice still maintained depleted plasma cholesterol and TG levels when challenged with a lipofructose diet, although moderate increases in plasma AST and ALT were observed compared to control mice (fig. 14). Liver Surf4 deficient mice exhibited similar performance in terms of body weight, glucose tolerance, and insulin response compared to control LacZ gRNA treated mice.

The results of these in vivo experiments demonstrated that the knock-out of Surf4 was able to significantly reduce blood lipids to a level of almost complete clearance, and no signs of death or serious side effects were observed.

Example 3 dose-dependent Effect of SURF4

To further confirm the function of SURF4, the inventors constructed SURF4 alleles flanked by lox sites (fig. 15) and used AAV-mediated Cre delivery to the liver for inactivation of this gene in mice. And pair ofMice (Surf 4)^+/+TBG-Cre) in comparison, complete deletion of the Surf4 allele in the liver cleared plasma cholesterol, triglyceride, or ApoB levels, consistent with the results observed using CRISPR-mediated Surf4 inactivation in example 2 (fig. 16A-C). It is important that mice with liver Surf4 in heterozygous state also exhibited a significant reduction in blood lipids of 20-25% compared to control mice (data at the far right of each panel in fig. 16A-C).

Liver Surf4 deficiency caused about 80% reduction in plasma cholesterol and total triglycerides compared to controls when measured under fasting post-feeding (re-fed) conditions, suggesting that lipid uptake from the small intestine may contribute to circulating lipids in this experimental setting (fig. 17). Liver Surf4 heterozygosity still reduced blood lipids under refeed conditions compared to control mice (data at the far right of the two panels in fig. 17), but did not result in significant accumulation of lipids in the liver (fig. 18).

This dose effect suggests that SURF4 is a limiting factor in lipid regulation, further supporting the results observed in human genetic studies, that quantitative changes in SURF4 levels affect blood lipid levels. This quantity-dependent effect can be exploited to partially, but not completely, suppress SURF4 levels in a subject, thereby achieving a reduction in blood lipids.

Example 4 shuttling ability of SURF4 between ER and Golgi

As mentioned previously, previous studies generally held that cargo receptors are not capable of achieving a transport process that is strong enough to transport abundant cargo, such as lipoproteins (Warren, g. and Mellman, I. (1999), supra).

The inventors found by confocal microscopy that endogenous SURF4 exhibited a punctate, concentrated localization at steady state to be substantially localized to the ER in Huh7 cells and mouse primary hepatocytes (fig. 19A-B). SURF4 plaques exhibited significant co-localization with the COPII subunit SEC31A (fig. 19A), focusing on what is referred to as the ER exit site, i.e., where COPII vesicles are produced. In contrast, SURF4 in steady state exhibited little golgi localization (fig. 19A), which is the endpoint of the COPII vesicle.

This phenomenon allows the inventors to consider whether SURF4 as a cargo recipient has the potential to actively circulate back to the ER for the next round of lipoprotein export. By expressing constitutively active ARF1^Q71IMutant (fig. 20, middle three panels), or by mutating the trilysine COPI sorting motif (KKK to AAA) at the tail of SURF4 (fig. 20, lower three panels), blocking the recovery of cargo receptors from golgi, which would allow SURF4 to essentially relocate from ER to golgi. On the other hand, SURF4 trapped at the golgi apparatus also differed in localization to ER proteins such as SEC31A (fig. 21).

To directly test the necessity of SURF4 recovery in lipid transport, a back-up experiment was performed using Cas9 resistant SURF4 cDNA (fig. 22). Reintroduction of wild-type Surf4 to a large extent reversed plasma triglyceride and cholesterol clearance due to CRISPR-mediated inactivation of hepatic Surf4 (fig. 23, Surf4-WT), further demonstrating the specificity of the gene editing effect shown in fig. 6-8. However, the recovery-deficient mutant Surf4-AAA, although able to be transported to the golgi (FIG. 20, bottom three panels), still failed to achieve anaplerotic effects (FIG. 23, Surf 4-AAA). These data illustrate that SURF4 performs efficient bidirectional shuttling between the ER and golgi, thereby maintaining high volume transport of lipid cargo, while also serving as a limiting factor in this process.

EXAMPLE 5 synergistic Effect with Sar1b

To investigate the physiological relationship of SAR1B and SURF4, the following experiments were performed.

FLAG-Surf4 was first introduced into the liver of control or Sar1b LKO mice by AAV. Sar1b deficiency caused APOB accumulation in the liver and also caused an increase in the interaction between APOB and SURF4 (as determined by anti-FLAG immunoprecipitation) (fig. 24), thus demonstrating the continued cooperation of SURF4 and Sar1B in recognizing lipoproteins and mediating lipoprotein packaging into transport vesicles for ER export (fig. 1).

Synergistic effects of Sar1b and Surf4 single-deficient (haploinsufficiency) in lipid lowering were also observed (FIG. 25). Liver Sar1b and Surf4 heterozygous caused approximately 15% and 25% reductions in plasma cholesterol compared to WT littermates, respectively. Liver double-heterozygosis of Sar1b and Surf4 resulted in a synergistic approximately 55% reduction in plasma cholesterol. Meanwhile, little change was observed in the liver by H & E staining in the double heterozygous mice compared to the littermate control, although mild steatosis was observed (fig. 26).

In contrast, although ectopic expression of Sar1b and/or Surf4 in the liver did not increase blood lipids in daily diet (chow diet) mice, feeding a raw fructosyl diet resulted in a small increase in plasma cholesterol in either Surf4 or Surf4/Sar1b mice compared to controls (GFP overexpressing mice) (figure 27). These data demonstrate a robust and flexible in vivo lipid-specific export procedure, and the fit of SAR1B/SURF4 controls the entry to the ER in a quantitative, dose-dependent manner.

Example 6 lipid lowering effects of SURF4 independent of LDL receptor function

The quantitative nature of ER export of the lipid cargo motivates the inventors to investigate the possibility of targeting SURF4 to prevent pathological dyslipidemia and atherosclerosis, in particular by acute inactivation of SURF4 in mice receiving PSCK9 AAV to degrade LDL receptors.

Feeding these wild-type mice expressing PCSK9 on a Western Diet (WD) resulted in an approximately 10-fold increase in plasma cholesterol concentration over normal, consistent with that previously reported (Goettsch, C. et al (2016. Atheroscleosis 251, 109-. Despite pathological hyperlipidemia in these control mice, Cre-loxP-mediated homozygous inactivation of the liver Surf4 allele still resulted in almost complete clearance of plasma cholesterol and triglycerides (fig. 28A-B). These data demonstrate that SURF4 selectively transports lipids into the circulation, independent of LDLR-mediated lipid uptake. Notably, the single-fold deletion of Surf4 also reduced blood lipid levels in the presence of dyslipidemia compared to control mice (fig. 28A-B, data far right).

In contrast, Surf4 mutant mice did not observe induction of plasma AST or ALT compared to control mice (fig. 29). Atherosclerosis caused by dyslipidemia at the pathological level was observed by evaluation of the arterial area in control mice by prior oil-red-O staining. However, formation of atherosclerosis was completely prevented in mice with liver Surf4 removed (fig. 30 and 31).

It is noteworthy that liver Surf4 heterozygosity also produced a significant protective effect against atherosclerosis (lesion area 2.32 ± 0.51%, 11.95 ± 1.28% in the control, n ═ 8, P <0.0001, fig. 30 and 31, last column), probably because circulating cholesterol associated with ApoB-containing lipoproteins that cause atherosclerosis was reduced by nearly 50% in dyslipidemic conditions (fig. 32A-D).

The above results demonstrate that the lipid lowering effect by inhibition of Surf4 acts independently of LDL receptors, since Surf4 is located further upstream in lipid export, which is particularly beneficial for certain populations with congenital defects in LDL receptors, since these populations do not benefit from PCSK9 inhibitors.

Example 7 preparation of RNAi molecules for inhibition of SURF4

With human SURF4 mRNA as a target, potential functional siRNA sequences with small off-target effect are selected by a siDESIGN Center based on the sequence of transcript 1 of human SURF4 (SEQ ID NO:10), and candidate SURF4 RNAi agents (SEQ ID Nos:11-214) shown in the following list 1 are formed. RNA duplexes of the sequences in table 1 below were synthesized by the optisco biotechnology company as candidate SURF4 RNAi agents. RNAi control reagents (which do not target mRNA in any mammalian cell) provided by the phyloco biosciences company were used as controls in all RNAi evaluation experiments.

TABLE 1 SURF4 RNAi Agents

Example 8 characterization of the Effect of SURF4 RNAi

In vitro testing

In vitro evaluation of SURF4 RNAi was performed in the human liver cell line Huh 7. Cells were seeded in 6-well plates and candidate SURF4 RNAi agents and control RNAi agents in table 1 were transfected with the transfection agent LipoFectamine RNAiMax (Thermo Fisher), respectively. 48 hours after transfection, the effect of different SURF4 RNAi agents on reduction of SURF4 was evaluated by comparing the endogenous SURF4 mRNA levels of cells transfected with different SURF RNAi agents and cells transfected with control RNAi agents using RT-PCR, and comparing the endogenous SURF4 protein levels of cells transfected with different SURF RNAi agents and cells transfected with control RNAi agents using immunoblotting. Supernatants from cells transfected with different SURF RNAi agents and cells transfected with control RNAi agents were collected and compared for how much APOB protein was in the supernatant to assess the effect of SURF RNAi agents on reducing lipid secretion at the cellular level.

The results for SURF4 RNAi agents S4-1, S4-2 and S4-3 are shown in FIG. 33. From the results in fig. 33, it can be seen that all three SURF4 RNAi agents significantly reduced the level of SURF4 mRNA (fig. 33A-B), and reduced the level of APOB in the culture medium (fig. 33C-D). These results indicate that RNAi agents targeting SURF4 reduced expression of SURF4, reduced lipid secretion, and thereby achieved a blood lipid lowering effect.

Animal experiments

AAV-mediated human Surf4 cDNA was recruited in the liver of liver-specific Surf4 knockout mice for animal level testing of Surf4 RNAi agents. Mice expressing human SURF4 were randomly grouped into 5 or more groups, and candidate SURF4 RNAi agents in Table 1 encapsulated by lipid nanoparticles were injected through tail vein, and control RNAi agents were injected into control groups. At weeks 1,2 and 4, sera were collected and measured for LDL-C, HDL-C. After the fourth week, mice injected with different RNAi agents were harvested from the liver and SURF4 mRNA and protein levels were measured in the mouse liver. The effect of SURF4 RNAi agents in reducing SURF4 expression levels and reducing blood lipid was evaluated in combination with the expression level of liver SURF4 and LDL-C, HDL-C levels in serum. Meanwhile, the obtained liver is stained by H/E, oil red O, sirius red and the like, and the health condition of the liver injected with different SURF4 RNAi reagents is evaluated.

The effect and safety of the SURF4 RNAi agent can be further verified through animal experiments. The beneficial effects of anticipatory inhibition of SURF4 expression would outweigh the potential burden on the liver due to lipid accumulation.

The above experiments reveal and confirm the role of SURF4 in lipid transport, and experiments verify that SURF4 inhibition can effectively reduce blood lipids, including cholesterol and triglycerides, and that RNAi agents against SURF4 can effectively reduce mRNA levels of SURF4, thereby achieving lipid lowering function.

Sequence listing

<110> Beijing university

<120> RNAi agents and compositions for inhibiting SURF4 gene expression

<130> PR12863PKU33CN

<160> 214

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

acagaacgac ctgatgggca 20

<210> 2

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gcatccgcat gtggttccag 20

<210> 3

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cagcaggttg aggaacacga 20

<210> 4

<211> 20

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tgcgaatacg cccacgcgat 20

<210> 5

<211> 45

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gacaccagca aacatgctct tcccttctga gcgggattct gccaa 45

<210> 6

<211> 15

<212> PRT

<213> Mus musculus

<400> 6

Val Gly Ala Phe Met Ser Lys Gly Glu Ser Arg Ser Glu Ala Leu

1 5 10 15

<210> 7

<211> 45

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gacgcccgca aacatgctct tcccttcaga tcttgattct gctag 45

<210> 8

<211> 15

<212> PRT

<213> Homo sapiens

<400> 8

Val Gly Ala Phe Met Ser Lys Gly Glu Ser Arg Ser Glu Ala Leu

1 5 10 15

<210> 9

<211> 22

<212> PRT

<213> Homo sapiens

<400> 9

Gly Leu Leu Leu Val Val Ala Leu Gly Pro Gly Gly Val Ser Met Asp

1 5 10 15

Glu Lys Lys Lys Glu Trp

20

<210> 10

<211> 2929

<212> DNA/RNA

<213> Homo sapiens

<400> 10

cttcctgtgg aggccgcagc gggtgcgggc gccgacgggc gagagccagc gagcgagcga 60

gcgagccgag ccgagcctcc cgccgtcgcc atgggccaga acgacctgat gggcacggcc 120

gaggacttcg ccgaccagtt cctccgtgtc acaaagcagt acctgcccca cgtggcgcgc 180

ctctgtctga tcagcacctt cctggaggac ggcatccgta tgtggttcca gtggagcgag 240

cagcgcgact acatcgacac cacctggaac tgcggctacc tgctggcctc gtccttcgtc 300

ttcctcaact tgctgggaca gctgactggc tgcgtcctgg tgttgagcag gaacttcgtg 360

cagtacgcct gcttcgggct ctttggaatc atagctctgc agacgattgc ctacagcatt 420

ttatgggact tgaagttttt gatgaggaac ctggccctgg gaggaggcct gttgctgctc 480

ctagcagaat cccgttctga agggaagagc atgtttgcgg gcgtccccac catgcgtgag 540

agctccccca aacagtacat gcagctcgga ggcagggtct tgctggttct gatgttcatg 600

accctccttc actttgacgc cagcttcttt tctattgtcc agaacatcgt gggcacagct 660

ctgatgattt tagtggccat tggttttaaa accaagctgg ctgctttgac tcttgttgtg 720

tggctctttg ccatcaacgt atatttcaac gccttctgga ccattccagt ctacaagccc 780

atgcatgact tcctgaaata cgacttcttc cagaccatgt cggtgattgg gggcttgctc 840

ctggtggtgg ccctgggccc tgggggtgtc tccatggatg agaagaagaa ggagtggtaa 900

cagtcacaga tccctacctg cctggctaag acccgtggcc gtcaaggact ggttcggggt 960

ggattcaaca aaactgccag cttttatgta tcctcttccc ttcccctccc ttggtaaagg 1020

cacagatgtt ttgagaactt tatttgcaga gacacctgag aatcgatggc tcagtctgct 1080

ctggagccac agtctggcgt ctgacccttc agtgcaggcc agcctggcag ctggaagcct 1140

cccccacgcc gaggctttgg agtgaacagc ccgcttggct gtggcatctc agtcctattt 1200

ttgagttttt ttgtgggggt acaggagggg gccttcaagc tgtactgtga gcagacgcat 1260

tggtattatc attcaaagca gtctccctct tatttgtaag tttacatttt tagcggaaac 1320

tactaaatta ttttgggtgg ttcagccaaa cctcaaaaca gttaatctcc ctggtttaaa 1380

atcacaccag tggctttgat gttgtttctg ccccgcattg tattttatag gaatagtgaa 1440

aacatttagg gacacccaaa gaatgatgca gtattaaagg ggtggtagaa gctgctgttt 1500

atgataaaag tcatcggtca gaaaatcagc ttggattggt gccaagtgtt ttattgggta 1560

acaccctggg agttttagta gcttgaggca aggtggaggg gcaagaagtc cttggggaag 1620

ctgctggtct gggtgctgct ggcctccaag ctggcagtgg gaagggctag tgagaccaca 1680

caggggtagc cccagcagca gcaccctgca agccagcctg gccagctgct cagaccagct 1740

tgcagagccg cagccgctgt gggcaggggg tgtggcagga gctcccagca ctggagaccc 1800

acggactcaa cccagttacc tcacatgggg ccttttctga gcaaggtctc gaaagcgcag 1860

gccgccctgg ctgagcagca ccgccctttc ccagctgcac tcgccctgtg gacagccccg 1920

acacaccact ttcctgaggc tgtcgctcac tcagattgtc cgtttgctat gccgaatgca 1980

gccaaaattc ctttttacaa tttgtgatgc cttaccgatt tgatcttaat cctgtattta 2040

aagttttcta acactgcctt atactgtgtt tctctttttg ggggagctta actgcttgtt 2100

gctccctgtc gtctgcacca tagtaaatgc cacaagggta gtcgaacacc tctctggccc 2160

ctagacctat ctggggacag gctggctcag cctgtctcca gggctgctgc ggcccagccc 2220

cgagcctgcc tccctcttgg cctctcatcc attggctctg cagggcaggg gtgaggcagg 2280

tttctgctca taagtgcttt tggaagtcac ctaccttttt aacacagccg aactagtccc 2340

aacgcgtttg caaatattcc cctggtagcc tacttcctta cccccgaata ttggtaagat 2400

cgagcaatgg cttcaggaca tgggttctct tctcctgtga tcattcaagt gctcactgca 2460

tgaagactgg cttgtctcag tgtttcaacc tcaccagggc tgtctcttgg tccacacctc 2520

gctccctgtt agtgccgtat gacagccccc atcaaatgac cttggccaag tcacggtttc 2580

tctgtggtca aggttggttg gctgattggt ggaaagtagg gtggaccaaa ggaggccacg 2640

tgagcagtca gcaccagttc tgcaccagca gcgcctccgt cctagtgggt gttcctgttt 2700

ctcctggccc tgggtgggct agggcctgat tcgggaagat gcctttgcag ggaggggagg 2760

ataagtggga tctaccaatt gattctggca aaacaatttc taagattttt ttgctttatg 2820

tgggaaacag atctaaatct cattttatgc tgtattttat atcttagttg tgtttgaaaa 2880

cgttttgatt tttggaaaca catcaaaata aataatggcg tttgttgta 2929

<210> 11

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ucauagcucu gcagacgau 19

<210> 12

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

aucgucugca gagcuauga 19

<210> 13

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

ccaccuggaa cugcggcua 19

<210> 14

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

uagccgcagu uccaggugg 19

<210> 15

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

uggccucguc cuucgucuu 19

<210> 16

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

aagacgaagg acgaggcca 19

<210> 17

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

agaagaagaa ggaguggua 19

<210> 18

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

uaccacuccu ucuucuucu 19

<210> 19

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gcaggaacuu cgugcagua 19

<210> 20

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

uacugcacga aguuccugc 19

<210> 21

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ccauggauga gaagaagaa 19

<210> 22

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

uucuucuucu cauccaugg 19

<210> 23

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ugcaugacuu ccugaaaua 19

<210> 24

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

uauuucagga agucaugca 19

<210> 25

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gaugagaaga agaaggagu 19

<210> 26

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

acuccuucuu cuucucauc 19

<210> 27

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

ggcgagagcc agcgagcga 19

<210> 28

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

ucgcucgcug gcucucgcc 19

<210> 29

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

ccagcgagcg agcgagcga 19

<210> 30

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

ucgcucgcuc gcucgcugg 19

<210> 31

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ccgccgucgc caugggcca 19

<210> 32

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

uggcccaugg cgacggcgg 19

<210> 33

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

ucgccauggg ccagaacga 19

<210> 34

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

ucguucuggc ccauggcga 19

<210> 35

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

cagaacgacc ugaugggca 19

<210> 36

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

ugcccaucag gucguucug 19

<210> 37

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

agaacgaccu gaugggcac 19

<210> 38

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

gugcccauca ggucguucu 19

<210> 39

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ugggcacggc cgaggacuu 19

<210> 40

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

aaguccucgg ccgugccca 19

<210> 41

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 41

ccgaggacuu cgccgacca 19

<210> 42

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 42

uggucggcga aguccucgg 19

<210> 43

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 43

ggagcgagca gcgcgacua 19

<210> 44

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 44

uagucgcgcu gcucgcucc 19

<210> 45

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 45

gcgagcagcg cgacuacau 19

<210> 46

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 46

auguagucgc gcugcucgc 19

<210> 47

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 47

cagcgcgacu acaucgaca 19

<210> 48

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 48

ugucgaugua gucgcgcug 19

<210> 49

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 49

gcgacuacau cgacaccac 19

<210> 50

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 50

guggugucga uguagucgc 19

<210> 51

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 51

ccaccuggaa cugcggcua 19

<210> 52

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 52

uagccgcagu uccaggugg 19

<210> 53

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 53

cguccuucgu cuuccucaa 19

<210> 54

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 54

uugaggaaga cgaaggacg 19

<210> 55

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 55

uccucaacuu gcugggaca 19

<210> 56

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 56

ugucccagca aguugagga 19

<210> 57

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 57

gcuucgggcu cuuuggaau 19

<210> 58

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 58

auuccaaaga gcccgaagc 19

<210> 59

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 59

cgggcucuuu ggaaucaua 19

<210> 60

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 60

uaugauucca aagagcccg 19

<210> 61

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 61

ucauagcucu gcagacgau 19

<210> 62

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 62

aucgucugca gagcuauga 19

<210> 63

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 63

agacgauugc cuacagcau 19

<210> 64

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 64

augcuguagg caaucgucu 19

<210> 65

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 65

acgauugccu acagcauuu 19

<210> 66

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 66

aaaugcugua ggcaaucgu 19

<210> 67

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 67

gaaccuggcc cugggagga 19

<210> 68

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 68

uccucccagg gccagguuc 19

<210> 69

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 69

cguucugaag ggaagagca 19

<210> 70

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 70

ugcucuuccc uucagaacg 19

<210> 71

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 71

guucugaagg gaagagcau 19

<210> 72

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 72

augcucuucc cuucagaac 19

<210> 73

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 73

ucugaaggga agagcaugu 19

<210> 74

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 74

acaugcucuu cccuucaga 19

<210> 75

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 75

gggaagagca uguuugcgg 19

<210> 76

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 76

ccgcaaacau gcucuuccc 19

<210> 77

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 77

ccaccaugcg ugagagcuc 19

<210> 78

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 78

gagcucucac gcauggugg 19

<210> 79

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 79

ccaaacagua caugcagcu 19

<210> 80

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 80

agcugcaugu acuguuugg 19

<210> 81

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 81

gggucuugcu gguucugau 19

<210> 82

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 82

aucagaacca gcaagaccc 19

<210> 83

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 83

uccagaacau cgugggcac 19

<210> 84

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 84

gugcccacga uguucugga 19

<210> 85

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 85

ccagaacauc gugggcaca 19

<210> 86

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 86

ugugcccacg auguucugg 19

<210> 87

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 87

gggcacagcu cugaugauu 19

<210> 88

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 88

aaucaucaga gcugugccc 19

<210> 89

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 89

ggcacagcuc ugaugauuu 19

<210> 90

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 90

aaaucaucag agcugugcc 19

<210> 91

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 91

gcacagcucu gaugauuuu 19

<210> 92

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 92

aaaaucauca gagcugugc 19

<210> 93

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 93

gcucuuugcc aucaacgua 19

<210> 94

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 94

uacguugaug gcaaagagc 19

<210> 95

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 95

ucuuugccau caacguaua 19

<210> 96

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 96

uauacguuga uggcaaaga 19

<210> 97

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 97

acguauauuu caacgccuu 19

<210> 98

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 98

aaggcguuga aauauacgu 19

<210> 99

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 99

ggaccauucc agucuacaa 19

<210> 100

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 100

uuguagacug gaauggucc 19

<210> 101

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 101

ucuacaagcc caugcauga 19

<210> 102

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 102

ucaugcaugg gcuuguaga 19

<210> 103

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 103

caugcaugac uuccugaaa 19

<210> 104

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 104

uuucaggaag ucaugcaug 19

<210> 105

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 105

aaauacgacu ucuuccaga 19

<210> 106

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 106

ucuggaagaa gucguauuu 19

<210> 107

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 107

acgacuucuu ccagaccau 19

<210> 108

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

auggucugga agaagucgu 19

<210> 109

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

uuccagacca ugucgguga 19

<210> 110

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

ucaccgacau ggucuggaa 19

<210> 111

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

uggaugagaa gaagaagga 19

<210> 112

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

uccuucuucu ucucaucca 19

<210> 113

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

augagaagaa gaaggagug 19

<210> 114

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

cacuccuucu ucuucucau 19

<210> 115

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

gaagaagaag gagugguaa 19

<210> 116

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

uuaccacucc uucuucuuc 19

<210> 117

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

caacaaaacu gccagcuuu 19

<210> 118

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

aaagcuggca guuuuguug 19

<210> 119

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

ugguaaaggc acagauguu 19

<210> 120

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

aacaucugug ccuuuacca 19

<210> 121

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

gguaaaggca cagauguuu 19

<210> 122

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

aaacaucugu gccuuuacc 19

<210> 123

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

gcacagaugu uuugagaac 19

<210> 124

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

guucucaaaa caucugugc 19

<210> 125

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

acagauguuu ugagaacuu 19

<210> 126

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

aaguucucaa aacaucugu 19

<210> 127

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

gagcagacgc auugguauu 19

<210> 128

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

aauaccaaug cgucugcuc 19

<210> 129

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

acgcauuggu auuaucauu 19

<210> 130

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

aaugauaaua ccaaugcgu 19

<210> 131

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

agcggaaacu acuaaauua 19

<210> 132

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

uaauuuagua guuuccgcu 19

<210> 133

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

gcggaaacua cuaaauuau 19

<210> 134

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

auaauuuagu aguuuccgc 19

<210> 135

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

caguuaaucu cccugguuu 19

<210> 136

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

aaaccaggga gauuaacug 19

<210> 137

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

aguuaaucuc ccugguuua 19

<210> 138

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

uaaaccaggg agauuaacu 19

<210> 139

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

gcauuguauu uuauaggaa 19

<210> 140

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

uuccuauaaa auacaaugc 19

<210> 141

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

uuuuauagga auagugaaa 19

<210> 142

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

uuucacuauu ccuauaaaa 19

<210> 143

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

ggaauaguga aaacauuua 19

<210> 144

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

uaaauguuuu cacuauucc 19

<210> 145

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

gugaaaacau uuagggaca 19

<210> 146

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

ugucccuaaa uguuuucac 19

<210> 147

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

acauuuaggg acacccaaa 19

<210> 148

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

uuuggguguc ccuaaaugu 19

<210> 149

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

agggacaccc aaagaauga 19

<210> 150

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

ucauucuuug ggugucccu 19

<210> 151

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

cccaaagaau gaugcagua 19

<210> 152

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

uacugcauca uucuuuggg 19

<210> 153

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

ccaaagaaug augcaguau 19

<210> 154

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

auacugcauc auucuuugg 19

<210> 155

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

caaagaauga ugcaguauu 19

<210> 156

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

aauacugcau cauucuuug 19

<210> 157

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

aaagaaugau gcaguauua 19

<210> 158

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

uaauacugca ucauucuuu 19

<210> 159

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

agaaugaugc aguauuaaa 19

<210> 160

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

uuuaauacug caucauucu 19

<210> 161

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

guauuaaagg ggugguaga 19

<210> 162

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

ucuaccaccc cuuuaauac 19

<210> 163

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

ugguagaagc ugcuguuua 19

<210> 164

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

uaaacagcag cuucuacca 19

<210> 165

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

gguagaagcu gcuguuuau 19

<210> 166

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

auaaacagca gcuucuacc 19

<210> 167

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

caaguguuuu auuggguaa 19

<210> 168

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

uuacccaaua aaacacuug 19

<210> 169

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

gguaacaccc ugggaguuu 19

<210> 170

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

aaacucccag gguguuacc 19

<210> 171

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

gaggcaaggu ggaggggca 19

<210> 172

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

ugccccucca ccuugccuc 19

<210> 173

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

gugggaaggg cuagugaga 19

<210> 174

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

ucucacuagc ccuucccac 19

<210> 175

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

gggaagggcu agugagacc 19

<210> 176

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

ggucucacua gcccuuccc 19

<210> 177

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

gcucagacca gcuugcaga 19

<210> 178

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

ucugcaagcu ggucugagc 19

<210> 179

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

ugaucuuaau ccuguauuu 19

<210> 180

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

aaauacagga uuaagauca 19

<210> 181

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 181

cgucugcacc auaguaaau 19

<210> 182

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 182

auuuacuaug gugcagacg 19

<210> 183

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 183

aguaaaugcc acaagggua 19

<210> 184

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 184

uacccuugug gcauuuacu 19

<210> 185

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 185

guaaaugcca caaggguag 19

<210> 186

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 186

cuacccuugu ggcauuuac 19

<210> 187

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 187

ccacaagggu agucgaaca 19

<210> 188

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 188

uguucgacua cccuugugg 19

<210> 189

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 189

cauaagugcu uuuggaagu 19

<210> 190

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 190

acuuccaaaa gcacuuaug 19

<210> 191

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 191

gagcaauggc uucaggaca 19

<210> 192

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 192

uguccugaag ccauugcuc 19

<210> 193

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 193

agcaauggcu ucaggacau 19

<210> 194

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 194

auguccugaa gccauugcu 19

<210> 195

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 195

caucaaauga ccuuggcca 19

<210> 196

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 196

uggccaaggu cauuugaug 19

<210> 197

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 197

aucaaaugac cuuggccaa 19

<210> 198

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 198

uuggccaagg ucauuugau 19

<210> 199

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 199

ggucaagguu gguuggcug 19

<210> 200

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 200

cagccaacca accuugacc 19

<210> 201

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 201

gcugauuggu ggaaaguag 19

<210> 202

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 202

cuacuuucca ccaaucagc 19

<210> 203

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 203

ggaaaguagg guggaccaa 19

<210> 204

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 204

uugguccacc cuacuuucc 19

<210> 205

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 205

accaauugau ucuggcaaa 19

<210> 206

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 206

uuugccagaa ucaauuggu 19

<210> 207

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 207

ccaauugauu cuggcaaaa 19

<210> 208

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 208

uuuugccaga aucaauugg 19

<210> 209

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 209

uggaaacaca ucaaaauaa 19

<210> 210

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 210

uuauuuugau guguuucca 19

<210> 211

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 211

caaaauaaau aauggcguu 19

<210> 212

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 212

aacgccauua uuuauuuug 19

<210> 213

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 213

aauaauggcg uuuguugua 19

<210> 214

<211> 19

<212> DNA/RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 214

uacaacaaac gccauuauu 19

Claims (10)

1. Short antisense oligonucleotides 8-30 nucleotides in length that target the nucleotides encoding SURF 4.

2. The short antisense oligonucleotide of claim 1, comprising 15 to 25 nucleotides, preferably 19 to 23.

3. The short antisense oligonucleotide of claim 1, comprising or consisting of a sequence selected from the group consisting of the sequences of the antisense strands of Table 1 or a sequence differing by 1 or 2 nucleotides from it.

4. An RNAi agent for inhibiting expression of SURF4 gene, comprising the short antisense oligonucleotide of any one of claims 1-3, and a sense strand at least partially complementary to the short antisense oligonucleotide.

5. The RNAi agent of claim 4, wherein the sense strand is at least 85% complementary to the short antisense oligonucleotide over the length of the short antisense oligonucleotide.

6. The RNAi agent of claim 4, comprising a sense strand and/or an antisense strand selected from table 1.

7. A composition comprising (a) the short antisense oligonucleotide of any one of claims 1-3 or the RNAi agent of any one of claims 4-6, and (b) a pharmaceutically acceptable excipient.

8. Use of the short antisense oligonucleotide of any one of claims 1 to 3 or the RNAi agent of any one of claims 4 to 6 for the preparation of a medicament for the treatment or prevention of a disease associated with dyslipidemia, such as a cardiovascular disease or a metabolic disease associated with dyslipidemia, in a subject, preferably the disease is selected from the group consisting of: hyperlipidemia such as familial hyperlipidemia, hypertriglyceridemia, cholesterol metabolism disorder, lipid metabolism disorder, high LDL-C blood disease, atherosclerotic cardiovascular and cerebrovascular diseases such as myocardial infarction, apoplexy, and atherosclerosis.

9. Use of the short antisense oligonucleotide of any one of claims 1-3 or the RNAi agent of any one of claims 4-6 in the manufacture of a medicament for reducing one, two, or all three of Low Density Lipoprotein (LDL), Very Low Density Lipoprotein (VLDL), and triglycerides in the blood of a subject.

10. Use according to claim 8 or 9, wherein the subject is a familial hyperlipidemia patient with a gene deficiency, such as a deficiency in LDLR expression.

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