CN117106781B - Modified nucleic acids and products and uses thereof - Google Patents
Modified nucleic acids and products and uses thereof Download PDFInfo
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- CN117106781B CN117106781B CN202311332720.3A CN202311332720A CN117106781B CN 117106781 B CN117106781 B CN 117106781B CN 202311332720 A CN202311332720 A CN 202311332720A CN 117106781 B CN117106781 B CN 117106781B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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Abstract
The invention belongs to the field of small molecule medicines, and particularly relates to modified nucleic acid, a product and application thereof. The nucleic acid of the invention is siRNA, and discloses the structure of the forward chain and the reverse chain, and the specific modification mode of the nucleotide is defined, so that the PCSK9 inhibitor with better performance is obtained, the levels of hPCSK9 and LDL-C in mice can be obviously reduced, the inhibition level is still achieved in 21 days after administration, the invention has a certain treatment effect on the hyperlipidemia of the mice, the administration interval is long, and the invention can be used as an additional treatment of statin drugs in the future, thereby providing better treatment options for patients with hyperlipidemia.
Description
Technical Field
The invention belongs to the field of small molecule medicines, and particularly relates to modified nucleic acid, a product and application thereof.
Background
Hyperlipidemia is a chronic disease that severely threatens human health. Hyperlipidemia includes primary hypercholesterolemia and mixed dyslipidemia, and elevated levels of low density lipoprotein cholesterol (LDL-C) in the blood of the patient. Hyperlipidemia also increases cardiac risk, and if the concentration of LDL-C in blood increases, it will cause LDL-C deposition in arterial wall of blood vessel in heart and brain, etc., gradually form atherosclerosis plaque, block corresponding blood vessel, and finally may cause fatal and disabling serious diseases such as cerebral apoplexy and peripheral arterial disease.
Among the current drugs for the treatment of hyperlipidemia, statin drugs (oral administration) are gold standards for lowering LDL-C. Whereas non-statin drugs such as ezetimibe, PCSK9 (human proprotein convertase subtilisin/Kexin type 9) inhibitors may be used in statin intolerant patients. Statin alone may cause associated myalgia, myopathy, rhabdomyolysis and liver function changes, so treatment with statin alone may not always achieve therapeutic goals. When the statin is unable to achieve the therapeutic goal, etamic step (oral) may be used as an additional treatment. However, the effect of etamic steps on LDL-C is not great. In addition, the effect of the phenoxy acid drugs (oral administration) and the bile acid sequestering agents (oral administration) on reducing LDL-C is not as good as that of statin drugs, and the side effects are also larger than that of statin drugs.
When the statin fails to achieve the therapeutic goal or the statin is intolerant or contraindicated, the PCSK9 inhibitor may be used as the maximum additional treatment. The invention provides sequences and modifications of various PCSK9 inhibitor siRNA, can achieve therapeutic effect in vivo and in vitro, has long administration interval, and provides a second better treatment option for patients with hyperlipidemia.
Chinese patent application number CN202210762455.1 discloses: use of a PCSK9 inhibitor for the treatment of hyperlipidemia, in particular a method for treating hyperlipidemia in a patient not undergoing statin therapy is provided. Comprising administering to a patient a pharmaceutical composition comprising a PCSK9 inhibitor. In certain embodiments, the PCSK9 inhibitor is an anti-PCSK 9 antibody, such as an exemplary antibody referred to herein as mAb 316P. But they have less of a study on small molecule inhibitors.
Chinese patent application No. cn202010456825.X discloses: use of micrornas of miRNA552 cluster in the treatment of glycolipid metabolic diseases. Specifically, micrornas of miRNA552 clusters can act as FXR agonists and lxrα antagonists to regulate glycolipid metabolic disorders at cellular and animal levels, manifesting in inhibiting hyperlipidemia, hyperglycemia, liver lipid accumulation and alleviating insulin resistance. Based on this, micrornas of the miRNA552 cluster can be used to treat glycolipid metabolic diseases. Considering the action mechanism, the medicine has weak pertinence to the hyperlipidemia and is difficult to have stable treatment effect.
In the prior art, the preparation of hyperlipidemia drugs by modified siRNA is disclosed.
Disclosure of Invention
In order to solve the above problems, the present invention provides a modified nucleic acid which can be used for the preparation of a medicament for the prophylaxis or treatment of hyperlipidemia as a PCSK9 inhibitor.
In one aspect, the invention provides a modified nucleic acid.
The nucleic acid sense strand is selected from SEQ ID NO.1-10, and the antisense strand is selected from SEQ ID NO.11-20; the modification is any one of M1, M12, M4, M10 and M11 modification.
In the present invention, "M1 modification" means:
sense strand 5'-3': nucleotide 1-2, nucleotide 2 '-O-methyl substitution, thio backbone, 3-6, nucleotide 2' -O-methyl substitution, 7-9, nucleotide 2 '-fluoro, nucleotide 10-19, nucleotide 2' -O-methyl substitution;
antisense strand 5'-3': nucleotide 12 ' -O-methyl substitution, thio skeleton, nucleotide 2' -fluoro, thio skeleton, nucleotide 3-5 2' -O-methyl substitution, nucleotide 62 ' -fluoro, nucleotide 7-13 2' -O-methyl substitution, nucleotide 14 2' -fluoro, nucleotide 15 2' -O-methyl substitution, nucleotide 162 ' -fluoro, nucleotide 17-18 2' -O-methyl substitution, nucleotide 19-20 2' -O-methyl substitution, thio skeleton, nucleotide 21 2' -O-methyl substitution.
In the present invention, the M1 modifications of all sequences can be replaced by M12/M4/M10/M11 modifications. All sequences were delivered by GALNAC.
The "M12 modification" in the present invention can be referred to as follows:
sense strand 5'-3': 1-2 nucleotide 2 '-O-methyl substitution, thio, 3-4 nucleotide 2' -O-methyl substitution, 5 nucleotide 2 '-fluoro, 6 nucleotide 2' -O-methyl substitution, 7-9 nucleotide 2 '-fluoro, 10-14 nucleotide 2' -O-methyl substitution, 15 nucleotide 2 '-fluoro, 16-19 nucleotide 2' -O-methyl substitution;
antisense strand 5'-3': nucleotide 12 '-O-methyl substitution, thio, nucleotide 2' -fluoro, thio, nucleotide 3-5 '-O-methyl substitution, nucleotide 6 2' -fluoro, nucleotide 7 2 '-O-methyl substitution, nucleotide 8' -deoxy-2 '-fluoro, nucleotide 9-13 2' -O-methyl substitution, nucleotide 14 2 '-fluoro, nucleotide 15 2' -O-methyl substitution, nucleotide 162 '-fluoro, nucleotide 17-18 2' -O-methyl substitution, nucleotide 19-20 2 '-O-methyl substitution, thio, nucleotide 21-2' -O-methyl substitution.
In the present invention, "M4 modification" may be referred to as follows:
sense strand 5'-3': 1-2 nucleotide 2 '-O-methyl substitution, thio, 3 nucleotide 2' -fluoro, 4 nucleotide 2 '-O-methyl substitution, 5 nucleotide 2' -fluoro, 6 nucleotide 2 '-O-methyl substitution, 7-9 nucleotide 2' -fluoro, 10 nucleotide 2 '-O-methyl substitution, 11 nucleotide 2' -fluoro, 12 nucleotide 2 '-O-methyl substitution, 13 nucleotide 2' -fluoro, 14 nucleotide 2 '-O-methyl substitution, 15 nucleotide 2' -fluoro, 16 nucleotide 2 '-O-methyl substitution, 17 nucleotide 2' -fluoro, 18 nucleotide 2 '-O-methyl substitution, 19 nucleotide 2' -fluoro;
antisense strand 5'-3': nucleotide 12 '-O-methyl substitution, thio, nucleotide 2' -fluoro, thio, nucleotide 32 '-O-methyl substitution, nucleotide 4 2' -fluoro, nucleotide 5 '-O-methyl substitution, nucleotide 6' -fluoro, nucleotide 7 '-O-methyl substitution, nucleotide 8' -fluoro, nucleotide 9 '-O-methyl substitution, nucleotide 10' -fluoro, nucleotide 11-13 2 '-O-methyl substitution, nucleotide 14 2' -fluoro, nucleotide 15 '-O-methyl substitution, nucleotide 16 2' -fluoro, nucleotide 17 2 '-O-methyl substitution, nucleotide 18' -fluoro, nucleotide 19-20 2 '-O-methyl substitution, thio, nucleotide 21' -O-methyl substitution.
In the present invention, "M10 modification" may be referred to as follows:
sense strand 5'-3': 1-2 nucleotide 2 '-O-methyl substitution, thio, 3-4 nucleotide 2' -O-methyl substitution, 5 nucleotide 2 '-fluoro, 6 nucleotide 2' -O-methyl substitution, 7-8 nucleotide 2 '-fluoro, 9 nucleotide non-modified, 10-19 nucleotide 2' -O-methyl substitution;
antisense strand 5'-3': nucleotide 12 ' -O-methyl substitution, nucleotide 2' -fluoro, thio, nucleotide 32 ' -O-methyl substitution, nucleotide 4 2' -fluoro, nucleotide 5' -O-methyl substitution, nucleotide 6' -fluoro, nucleotide 7-13 2' -O-methyl substitution, nucleotide 14 2' -fluoro, nucleotide 15 2' -O-methyl substitution, nucleotide 16 ' -fluoro, nucleotide 17-18 2' -O-methyl substitution, nucleotide 19-20 2' -O-methyl substitution, thio, nucleotide 21 2' -O-methyl substitution.
In the present invention, "M11 modification" may be referred to as follows:
sense strand 5'-3': nucleotide 1 (LNA), thio, nucleotide 2 '-O-methyl substitution, thio, nucleotide 3-4 2' -O-methyl substitution, nucleotide 52 '-fluoro, nucleotide 6' -O-methyl substitution, nucleotide 7-9 2 '-fluoro, nucleotide 10-19 2' -O-methyl substitution;
antisense strand 5'-3': nucleotide 12 ' -O-methyl substitution, thio, nucleotide 2' -fluoro, thio, nucleotide 32 ' -O-methyl substitution, nucleotide 4 2' -fluoro, nucleotide 5' -O-methyl substitution, nucleotide 6' -fluoro, nucleotide 7-13 2' -O-methyl substitution, nucleotide 14 2' -fluoro, nucleotide 15 2' -O-methyl substitution, nucleotide 16 ' -fluoro, nucleotide 17-18 2' -O-methyl substitution, nucleotide 19-20 2' -O-methyl substitution, thio, nucleotide 21 2' -O-methyl substitution.
When the invention relates to nucleotide modifications (as in the general formulae), the person skilled in the art will be aware of:
m represents that the nucleotide contains 2' -O-methyl substitution modification;
f represents that the nucleotide contains 2' -fluoro modification;
ms represents that the nucleotide contains a 2' -O-methylthio modification;
fs indicates that the nucleotide contains 2' -fluoro, thio-modification.
In some embodiments, the positive strand of nucleic acid has the general formula: X1-X2-Am-Gm-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14-X15-X16-Um-X18-Um;
wherein X1 and X2 are 2' -O-methylthionucleotides;
x7, X8 and X9 are 2' -fluoro nucleotides;
x5, X6, X10-X16, X18 are 2' -O-methyl substituted nucleotides;
the inverted chain has the general formula:
Ams-X2’-Am-X4’-X5’-X6’-X7’-X8’-X9’-X10’-X11’-X12’-X13’-X14’-X15’-Cf-Um-X18’-X19’-X20’-X21’；
wherein X2 'is 2' -fluoro, a thio nucleotide;
x6', X14' are 2' -fluoronucleotides;
x19', X20' are 2' -O-methylthionucleotides;
x4', X5', X7'-X13', X15', X18', X21 'are 2' -O-methyl substituted nucleotides.
Preferably, the nucleic acid is of the formula:
x1 and X2 are each independently selected from Cms, gms, ums, ams;
x7, X8, X9 are each independently selected from Cf, gf, uf, af;
x5, X6, X10-16, X18 are each independently selected from Cm, gm, um, am;
x2' is selected from Afs and Ufs;
x6', X14' are each independently selected from Cf, gf, uf, af;
x19', X20' are each independently selected from Cms, gms, ums, ams.
Further preferred, the nucleic acid is of the formula:
x1 and X2 are selected from any one of the following combinations:
(1) X1 is Cms, X2 is Ums;
(2) X1 is Gms and X2 is Ums;
(3) X1 is Cms, X2 is Ams;
x7, X8, X9 are selected from any combination of the following:
(1) X7 is Cf, X8 is Uf, X9 is Gf;
(2) X7 is Uf, X8 is Uf, X9 is Uf;
(3) X7 is Af, X8 is Gf, X9 is Cf;
x6', X14' are selected from any combination of:
(1) X6 'is Cf, X14' is Gf;
(2) X6 'is Af and X14' is Uf;
(3) X6 'is Uf, X14' is Uf;
x19', X20' are selected from any combination of:
(1) X19' is Gms; x20' is Ams;
(2) X19' is Cms; x20' is Ams;
(3) X19' is Gms; x20' is Gms.
Still further, the nucleic acid:
the sense strand is selected from SEQ ID NO.21-30, and the antisense strand is selected from SEQ ID NO.31-40.
In some embodiments, the nucleic acid is selected from any one or more of the following:
(1) Sense strand SEQ ID NO.21, antisense strand SEQ ID NO.31;
(2) Sense strand SEQ ID NO.22, antisense strand SEQ ID NO.32;
(3) Sense strand SEQ ID NO.23, antisense strand SEQ ID NO.33;
(4) Sense strand SEQ ID NO.24, antisense strand SEQ ID NO.34;
(5) Sense strand SEQ ID NO.25, antisense strand SEQ ID NO.35;
(6) Sense strand SEQ ID NO.26, antisense strand SEQ ID NO.36;
(7) Sense strand SEQ ID NO.27, antisense strand SEQ ID NO.37;
(8) Sense strand SEQ ID NO.28, antisense strand SEQ ID NO.38;
(9) Sense strand SEQ ID NO.29, antisense strand SEQ ID NO.39;
(10) Sense strand SEQ ID NO.30, antisense strand SEQ ID NO.40.
In another aspect, the invention provides the use of the aforementioned nucleic acid for the preparation of a PCSK9 inhibitor.
The general formula of the invention provides PCSK9 inhibitors comprising the nucleic acid as described above.
In still another aspect, the invention provides the use of the aforementioned nucleic acid or PCSK9 inhibitor for the preparation of a medicament for the prevention or treatment of hyperlipidemia and the prevention of hyperlipidemia complications.
In particular, the hyperlipidemia includes, but is not limited to, primary hypercholesterolemia or mixed dyslipidemia.
In particular, the hyperlipidemia complications include, but are not limited to, one or more of heart disease, atherosclerosis, cerebral apoplexy, thrombosis, hypertension, peripheral arterial disease, cerebral hemorrhage.
Preferably, the medicament is in the form of injection.
Preferably, the administration of the drug comprises intraperitoneal injection, intravenous injection or subcutaneous injection.
The invention also provides medicaments comprising the aforementioned nucleic acids or PCSK9 inhibitors.
Pharmaceutically acceptable auxiliary materials such as cosolvent, stabilizer, excipient and the like can be also included in the medicament.
In view of the fact that the nucleic acid or PCSK9 inhibitor of the present invention may be used in combination with existing drugs, drugs that have been disclosed in the art for treating hyperlipidemia may also be optionally included in the drug of the present invention, including but not limited to one or more of statins, ezetimibe, phenoxy acids, bile acid sequestrants, ANGPTL3 inhibitors (as disclosed in prior art PCTUS 2017020221). Preferably in combination with statin drugs.
The invention has the beneficial effects that:
the siRNA sequence and the modification combination screened by the research can obviously reduce the levels of hPCSK9 and LDL-C in mice, has the inhibition level in 35 days after administration, has long administration interval, can be used as a substitute or an additional treatment of statin drugs in the future, and provides better treatment options for patients with hyperlipidemia.
Drawings
FIG. 1 siRNA sequence single concentration screening results.
FIG. 2 shows the results of three-concentration verification of siRNA sequences.
FIG. 3 is a diagram of siRNA IC 50 And (5) detecting a value.
FIG. 4 shows the silencing effect of siRNA on PCSK9 on Hep3B and HeLa cells.
Fig. 5 shows plasma hPCSK9 levels in mice during the administration of example 3.
FIG. 6 is a map of psiCHECK2 plasmid.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
The siRNA of the present invention may be obtained by any method in the prior art, such as artificial synthesis based on sequence, unless otherwise specified.
EXAMPLE 1 screening and validation of modified nucleotides
1. PCSK9 siRNA sequence screening
1. PCSK9 siRNA sequence design
The siRNA sequence was designed against the hPCSK9 3' UTR region sequence (Gene bank ID: 255738). 193 siRNA sequences were designed in total, all sequences were modified with M1 and subjected to the next screening.
2. PCSK9 siRNA sequence screening
(1) Construction of PCSK9 plasmid.
For the PCSK9 3' UTR region, 4 psiCHECK2-PCSK9-Renilla plasmids were constructed in total.
psiCHECK2 plasmid was purchased from Promega under the accession number C8021 and the plasmid map is shown in fig. 6;
renilla is a fluorescent label commonly used in the art, at positions 694-1629 on the psiCHECK2 plasmid;
the PCSK9 has an insertion site between 1636 and 1680 on the psiCHECK2 plasmid; since the complete sequence of the PCSK9 3' utr region is long and cannot be inserted into the plasmid completely at one time, it is split into four insertions, as shown in table 1:
TABLE 1
(2) Plasmid single concentration selection
1) Conventionally cultured 293T cells were digested with pancreatin to adjust the cell concentration to 5X 10 5 individual/mL;
2) Cell transfection:
a) Transfection reagent dilution
Diluting the transfection reagent with opti-MEM, and adding the diluted transfection reagent into a 96-well plate by a row gun, wherein the diluted transfection reagent plate is used for diluting the transfection reagent;
b) siRNA dilution
siRNA was diluted to a final concentration of 10nM with DEPC water and added to 96-well plates (transfection reagent plate+rna plate);
c) Plasmid dilution
Taking corresponding plasmids according to the arrangement condition of siRNA on the pore plate, and diluting with opti-MEM to ensure that each pore contains 60ng of plasmids;
d) 70 mu L of plasmid is sucked by a row gun and added into a corresponding transfection reagent and RNA plate, and the mixture is kept stand for 30min.
And (3) sucking 70 [ mu ] L of the transfection reagent compound, adding the transfection reagent compound into the cell subplate, adding 70 [ mu ] L of the cell suspension in the step (1) into each hole, and placing the cell suspension into an incubator.
3) Cell lysis assay:
a) Taking out the cell plate from the incubator, and sucking and discarding 40 mu L of primary cell culture solution;
b) Adding 50 mu L Dual-Glo Luciferanse Reagent into each hole; standing at room temperature for 30min, transferring 50 mu L of lysate to a whiteboard, and reading a Firefly fluorescence value by using a multifunctional microplate reader;
c) Adding 50 mu L Dual-Glo cube and Glo cube Reagent into each hole of the white board; renilla luciferase was detected using a microplate reader.
4) Statistical analysis:
a) Statistical analysis adopts GraphPad Prism statistical analysis software;
b) Linear regression: multiple linear regression was used.
The screening results are shown in FIG. 1. 10 sequences were selected out in total by single concentration screening.
103, 105, 108, 145, 160, 161, 162, 165, 190, 193, respectively, the specific sequences of which are shown in table 2:
TABLE 2
(3) Verification of plasmid three concentrations
1) Conventionally cultured 293T cells were digested with pancreatin to adjust the cell concentration to 5X 10 5 individual/mL;
2) Cell transfection:
a) Diluting the transfection reagent with opti-MEM, and adding the diluted transfection reagent into a 96-well plate by a row gun, wherein the diluted transfection reagent plate is used for diluting the transfection reagent;
b) siRNA dilution
The siRNA was diluted to final concentrations of 0.1nM, 1nM, 10nM with DEPC water and added to 96-well plates (transfection reagent plate+RNA plate);
c) Plasmid dilution
Taking corresponding plasmids according to the arrangement condition of siRNA on the pore plate, and diluting with opti-MEM to ensure that each pore contains 60ng of plasmids;
d) Sucking 70uL of plasmid by a row gun, adding the plasmid into a corresponding transfection reagent and RNA plate, and standing for 30min; and (3) adding 70uL of transfection reagent compound into the cell subplate, adding 70uL of the cell suspension in the step (1) into each hole, and placing into an incubator.
3) Cell lysis assay:
a) Taking out the cell plate from the incubator, and sucking and discarding 40 mu L of primary cell culture solution;
b) 50uL Dual-Glo Luciferanse Reagent was added to each well; standing at room temperature for 30min, transferring 50 mu L of lysate to a whiteboard, and reading a Firefly fluorescence value by using a multifunctional microplate reader;
c) 50 mu L Dual-Glo cube and Glo cube Reagent are added into each hole of the white board. Renilla luciferase was detected using a microplate reader.
4) Statistical analysis:
a) Statistical analysis adopts GraphPad Prism statistical analysis software;
b) Linear regression: multiple linear regression was used.
The results of the three-concentration assay are shown in FIG. 2.
（4）IC 50 Testing
1) Conventionally cultured 293T cells were digested with pancreatin to adjust the cell concentration to 5X 10 5 individual/mL;
2) Cell transfection:
a) Diluting the transfection reagent with opti-MEM, and adding the diluted transfection reagent into a 96-well plate by a row gun, wherein the diluted transfection reagent plate is used for diluting the transfection reagent;
b) siRNA dilution
The siRNA was diluted with DEPC water to final concentrations of 0.006nM, 0.0024nM, 0.0098nM, 0.0391nM, 0.1563nM, 0.6250nM, 2.5nM, 10nM, and added to 96-well plates (transfection reagent plate+RNA plate);
c) Plasmid dilution
Taking corresponding plasmids according to the arrangement condition of siRNA on the pore plate, and diluting with opti-MEM to ensure that each pore contains 60ng of plasmids;
d) Sucking 50uL of plasmid by a row gun, adding the plasmid into a corresponding transfection reagent and RNA plate, and standing for 30min; adding 70uL of transfection reagent compound into the cell subplate, adding 70uL of the cell suspension in the step a) into each hole, and placing into an incubator.
3) Cell lysis assay:
a) Taking out the cell plate from the incubator, and sucking and discarding 20 mu L of primary cell culture solution; b) 50 [ mu ] L Dual-Glo Luciferanse Reagent was added to each well. Standing at room temperature for 30min, transferring 50uL of lysate to a whiteboard, and reading a Firefly fluorescence value by using a multifunctional microplate reader; c) 50 mu L Dual-Glo cube and Glo cube Reagent are added into each hole of the white board. Renilla luciferase was detected using a microplate reader.
4) Statistical analysis:
a) Statistical analysis adopts GraphPad Prism statistical analysis software;
b) Linear regression: multiple linear regression was used.
The results are shown in FIG. 3, which shows that the IC of 10 PCSK9 siRNA sequences 50 The values are equivalent, and the next in-vivo and in-vitro efficacy experiments are carried out.
EXAMPLE 2PCSK9 siRNA in vitro Activity assay
In this example, the complete medium is: DMEM medium (available from Transgen Biotech under the trade designation FI 101-01).
1. Cell plating
HeLa cells (obtained from cell bank of the national academy of sciences, cat# SCSP-504)/Hep 3B cells (obtained from cell bank of the national academy of sciences, cat# SCSP-5045) cultured in 10 cm culture dishes were subjected to conventional pancreatin digestion, and the whole culture medium was resuspended in 6X 10 4 Individual cells/mL, 0.5 mL/well were seeded in 24-well plates and used for subsequent experiments after overnight incubation.
2. Cell transfection
A total of 10 sequences GPYGP:103 105, 108, 145, 160, 161, 162, 165, 190, 193 additionally NC2, NC5, NC-cy3, MOCK (model control), blast (negative control), complete medium (BLANK control) as control.
Wherein capital letter U, C, G, A, T is a basic nucleotide, and its suffix is lowercase m (2 '-O-methyl substitution modification), f (2' fluoro modification), s (thio backbone) is modification, and double letter means that both modifications are included.
NC-cy3 is fluorescent siRNA without interference effect; the MOCK group is a group which is only added with a transfection reagent and does not add any sequence; BLANK is a cell-only group. Each group had 3 duplicate wells. The specific steps are as follows:
(1) The transfection was performed with siRNA concentration of 30 nM (final 1.5. Mu.L of siRNA per well at an initial concentration of 10. Mu.M) and with a transfection reagent lipofectamine 2000 (available from ThermoFisher under the trade designation 11668-019) at a dose of 3.5. Mu.L per well. After considering the losses, the transfection complexes were prepared, i.e. the siRNA and lipofectamine 2000 transfection reagents were diluted with 50. Mu.L opti-MEM (available from ThermoFisher under the trade name 31985070) per well, respectively, and after 10min of standing, the two components were mixed and left to stand for 30min.
(2) The time to rest was reached, the transfection complex was added to the corresponding well at 100. Mu.L/well, such that a final volume of 500. Mu.L per well was achieved.
(3) After about 4 hours of transfection, the transfection complex was aspirated, 0.5. 0.5mL complete medium was added per well and placed into an incubator for further culture.
(4) In which NC-cy3 wells were transfected, washed with PBS and photographed using a fluorescent inverted microscope.
3. RNA extraction
(1) After 48 h transfection, the medium in the well plate was completely aspirated, 0.5mL Trizol Lysis Buffer (purchased from Invitrogen, cat No. 15596026) was added, the cells in the dish were thoroughly lysed by pipetting, and the completely lysed mixture was transferred to a 1.5mL centrifuge tube of RNase-free; shaking vigorously for about 10-15s to fully lyse cells, and standing at room temperature for 3min;
(2) Carefully open the cap and add 100 μl of chloroform; shaking vigorously for 15-20s, and standing at room temperature for 2min; centrifuging at 4deg.C and 10000 Xg for 10min;
(3) After centrifugation, carefully taking out the centrifuge tube onto a centrifuge tube rack, sucking the supernatant water phase into a new 2.0mL centrifuge tube, adding absolute ethyl alcohol with the volume 1.5 times that of the supernatant water phase, and mixing uniformly in a reverse way;
(4) Taking a purification column with a collecting pipe, adding 500 mu L of the mixed solution obtained in the step (3) into the purification column, and standing for 2min; centrifuging at 4deg.C and 10000 Xg for 1min, and discarding filtrate; repeating the steps of the rest mixed solution;
(5) Adding 500 μl of 80% ethanol into the purification column, centrifuging at 4deg.C 10000×g for 1min, and discarding the filtrate;
(6) Adding 500 μl of 80% ethanol into the purification column, centrifuging at 4deg.C 10000×g for 1min, and discarding the filtrate;
(7) Centrifuging the purified column at 4deg.C and 10000 Xg for 2min;
(8) After centrifugation, carefully taking out the purification column with the collection pipe (if the collection pipe contains liquid, the liquid is not splashed onto the purification column), discarding the collection pipe, putting the purification column into a new 1.5mL centrifuge tube, adding 100 mu L DEPC water into the purification column, and standing for 2min at room temperature; centrifuging at 4deg.C and 10000 Xg for 1min;
RNA was temporarily stored at 4℃for one day for PCR experiments. (e.g., long term storage should be at-80 ℃ C.).
4. qRT-PCR detection
(1) RNA reverse transcription
Step 1, genomic DNA removal: mix and template were added to the octant according to the following table, and the mixture was placed in a PCR apparatus at 45℃for 5min.
Genome removal reaction System
Step 2, reverse transcription: 5 XHiScript III qRT Supermix (available from Vazyme, cat. No. R323-01) was directly added to the reaction tube in step 1, placed in a PCR apparatus, and the reaction procedure was 38℃for 25min; cDNA was obtained after the reaction was completed at 90℃for 5 seconds.
(2) qPCR reaction system configuration
The RNA amplification system reaction solution was prepared on an ice box according to the following table:
(3) On-machine detection
And (3) subpackaging 15 mu L of each hole of the prepared PCR reaction solution into a 96-well plate, adding 5 mu L of sample, sealing by a sealing plate film, centrifuging for 1min on a 96-well plate centrifuge, and performing fluorescent quantitative PCR reaction on Step one plus.
(4) Statistical analysis
1) Statistical analysis adopts GraphPad Prism statistical analysis software; the residual activity of hPCSK9 is calculated by a QPCR relative quantitative delta Ct method, and the siRNA inhibition rate is obtained by the residual activity of 1-PCSK 9. The ΔΔct method is calculated as follows:
ΔΔct= delta CT [ (delta) CT ] treatment group mean- Δct (control group mean);
Δct (treatment group) =ct (test gene) -CT (reference gene mean);
Δct (control) =ct (test gene) -CT (reference gene mean);
2) Linear regression: adopting multiple linear regression;
the inhibition effect of siRNA in Hela and Hep3B is shown in tables 3-4:
TABLE 3 Activity in Hela
TABLE 4 Activity in Hep3B
The silencing effect of siRNA on PCSK9 is shown in figure 4. Fig. 4 shows that: siRNA 103, 105, 108, 145, 160, 161, 162, 165, 190, 193 with specifically modified sequences have potent inhibitory effect on PCSK9 expression in Hep3B, hela cells in vitro.
EXAMPLE 3PCSK9 siRNA in vivo efficacy detection
1. Mouse model
A model of hyperlipidemia was constructed by feeding B6-hPCSK9-UTR mice (from Jixiaokang, cat No. B6-hPCSK9-UTR,6-8 weeks old) with Western diet for 5-6 weeks (using Western diet feed for free feeding of mice, from Research Diets, cat No. D12079B), and blood sampling test LDL-C (blood biochemical test, hitachi 7020) and hPCSK9 levels (ELISA kit, invitrogen, cat No. EH384 RB) at the molding stage (D-3), grouping was performed with LDL-C as a main indicator, hPCSK9 as an auxiliary indicator to ensure that the mean value and SEM of each group were substantially consistent, and each group was 6 on the day of grouping was defined as D0.
2. Administration of drugs
The dosing was performed on the day of the grouping, single dosing, mice subcutaneous injection. The administration dose was 4.5mg/kg body weight.
3. Data detection and collection
Mice were tested for body weight, cholesterol, LDL-C and hPCSK9 expression levels during the drug efficacy period. Mice were tested for body weight, cholesterol, LDL-C and hPCSK9 expression levels 7 times at time points D-8, D-4, D3, D7, D14, D21, D28, D35.
The results of the in vivo activity detection of siRNA are shown in FIG. 5.
The results show that: 10 siRNAs with specific modified sequences had significant gene silencing effect on PCSK9 in humanized mice.
Claims (11)
1. A modified nucleic acid, characterized in that the sense strand is SEQ ID No.26 and the antisense strand is SEQ ID No.36, wherein the sense and antisense strands are: m represents a 2 '-O-methyl substitution modification, f represents a 2' fluoro modification, s represents a thio-skeleton modification, and double-letter represents both modifications.
2. Use of the nucleic acid of claim 1 for the preparation of a PCSK9 inhibitor.
3. A PCSK9 inhibitor comprising the nucleic acid of claim 1.
4. Use of the nucleic acid of claim 1 or the PCSK9 inhibitor of claim 3 for the manufacture of a medicament for the prevention or treatment of hyperlipidemia and the prevention of hyperlipidemia complications.
5. The use according to claim 4, wherein the hyperlipidemia comprises primary hypercholesterolemia or mixed dyslipidemia.
6. The use according to claim 5, wherein said hyperlipidemia complications comprise one or more of heart disease, atherosclerosis, cerebral stroke, thrombosis, hypertension, peripheral arterial disease, cerebral hemorrhage.
7. The use according to claim 6, wherein the medicament is in the form of an injection.
8. The use of claim 7, wherein the mode of administration of the medicament comprises intraperitoneal injection, intravenous injection or subcutaneous injection.
9. A medicament comprising the nucleic acid of claim 1 or the PCSK9 inhibitor of claim 3.
10. The medicament of claim 9, further comprising pharmaceutically acceptable excipients.
11. The medicament of claim 10, further comprising one or more of a statin, ezetimibe, a phenoxy acid, a bile acid sequestering agent.
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