Calcium-containing lipid nanoparticles for loading and reducing transthyretin nucleic acid

Abstract

The invention relates to a calcium-containing lipid nanoparticle for loading and reducing transthyretin nucleic acid, in particular to a calcium-containing cationic lipid nanoparticle which contains calcium ions in an inner core and is used for loading and reducing transthyretin nucleic acid. The invention further relates to a calcium-containing cationic lipid nanoparticle which contains calcium ions in an inner core and is used for loading the small interfering RNA for reducing transthyretin. The calcium-containing cationic lipid nanoparticle for loading and reducing transthyretin nucleic acid disclosed by the invention has the characteristic of efficiently reducing transthyretin mRNA (messenger Ribonucleic Acid) and protein.

Description

Technical Field

[0001] The present application relates to a calcium-containing lipid nanoparticle loaded with a transthyretin nucleic acid, in particular to a calcium-containing cationic lipid nanoparticle containing calcium ions in the inner core for loading a transthyretin nucleic acid, and further to a calcium-containing cationic lipid nanoparticle containing calcium ions in the inner core for loading a transthyretin small interfering RNA. The calcium-containing cationic lipid nanoparticle for loading a transthyretin nucleic acid disclosed in the present invention has the characteristics of efficiently reducing transthyretin mRNA and protein. Background Art

[0002] Transthyretin amyloidosis (ATTR) is a disease caused by the misfolding of transthyretin (TTR) leading to its abnormal deposition in tissues. It has a low incidence and is a rare disease. The survival period after diagnosis is generally only 2-12 years. The common clinical phenotypes are transthyretin amyloidosis polyneuropathy (ATTR-PN) and transthyretin amyloidosis cardiomyopathy (ATTR-CM), which are progressively aggravated to life-threatening. TTR is mainly produced by hepatocytes and exists in the peripheral blood as a homotetramer under normal physiological conditions. It transports thyroxine and vitamin A. Mutations in the gene encoding TTR and aging can lead to instability of the protein tetramer structure of TTR. TTR monomers pathologically aggregate in tissues such as the peripheral nervous system, heart, eyes, kidneys, and meninges to form insoluble amyloid deposits.

[0003] Currently, there is only one small molecule drug Tafamidis on the market for ATTR-CM. It is a TTR stabilizer that stabilizes the tetrameric structure of wild-type and mutant TTR by binding to the thyroxine binding site of TTR, slowing down its dissociation into monomers. The drugs approved for the treatment of ATTR-PN mainly include the antisense oligonucleotide (ASO) drug Tegsedi and two siRNA drugs developed by Alnylam, with the trade names Onpattro and Amvuttra. Onpattro is a preparation product that uses lipid nanoparticles (LNP) to deliver siRNA to knock down TTR, and Amvuttra is a product that uses N-acetylgalactosamine (GalNac)-coupled siRNA to knock down TTR. Both have high liver delivery efficiency, and the knockdown of TTR reaches about 90% through RNA interference technology. As a TTR-siRNA delivery carrier, LNP has shown its high delivery efficiency and safety, and has been approved by the FDA for the treatment of ATTR-PN. However, due to the various toxic effects found in its non-clinical studies, including liver toxicity (liver cell necrosis / vacuolization, inflammatory cell infiltration, pigmentation), spleen toxicity (atrophy, cell reduction), immune system toxicity (splenic lymphocyte depletion, lymph node necrosis), etc., its dosage is limited. Even at the dose of the marketed drug Onpattro, there are still obvious side effects compared to the placebo group in clinical trials, including infusion reactions, indigestion, dyspnea, muscle spasms, etc. How to achieve the same level of TTR reduction at a lower dose, thereby reducing the side effects of the drug, is of great significance for the treatment of ATTR disease. Summary of the invention

[0004] The present application relates to a calcium-containing lipid nanoparticle loaded with a transthyretin nucleic acid, in particular to a calcium-containing cationic lipid nanoparticle containing calcium ions in the inner core for loading a transthyretin nucleic acid, and further to a calcium-containing cationic lipid nanoparticle containing calcium ions in the inner core for loading a transthyretin small interfering RNA. The calcium-containing cationic lipid nanoparticle for loading a transthyretin nucleic acid disclosed in the present invention has the characteristics of efficiently reducing transthyretin mRNA and protein.

[0005] Aspect 1. A calcium-containing cationic lipid nanoparticle loaded with nucleic acid, characterized in that the cationic lipid nanoparticle comprises a cationic lipid, a neutral lipid, a PEGylated lipid and cholesterol and / or cholesterol ester;

[0006] The cationic lipid nanoparticles contain calcium ions, and the anions corresponding to the calcium ions are not phosphate, hydrogen phosphate and dihydrogen phosphate; and

[0007] The cationic lipid nanoparticles do not contain non-PEG group-modified negatively charged lipids;

[0008] The cationic lipid nanoparticles are loaded with nucleic acids for reducing transthyretin.

[0009] Aspect 2. The calcium-containing cationic lipid nanoparticles according to aspect 1, characterized in that: the nucleic acid used to reduce transthyretin is plasmid DNA, single-stranded DNA, double-stranded DNA, siRNA, shRNA, aiRNA, miRNA, mRNA, circular RNA, gRNA, aptamer, ribozyme, antisense oligonucleotide ASO or any combination thereof; preferably, the nucleic acid used to reduce transthyretin is siRNA or ASO;

[0010] More preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand that is at least 85% complementary thereto, the sense strand has a nucleotide sequence of at least 17 contiguous nucleotides, and one of the sense strand or the antisense strand has at least 85% of a region that can complementarily act within a partial continuous region of the TTR gene sequence in Table 1;

[0011] Further more preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand complementary thereto, the sense strand having a nucleotide sequence of at least 17 contiguous nucleotides, and the antisense strand having at least 90% of a region capable of complementarity within a partial continuous region of the TTR gene sequence in Table 1; Further more preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand complementary thereto, the sense strand having a nucleotide sequence of at least 17 contiguous nucleotides, and the antisense strand having at least 95% of a region capable of complementarity within a partial continuous region of the TTR gene sequence in Table 1;

[0012] Further more preferably, the nucleic acid for reducing transthyretin is siRNA, and its sequence is independently selected from one of the following sequences:

[0013]

[0014] The lowercase letters represent 2'-O-methyl nucleotide residues, the lowercase underlined letters represent 2'-fluoro nucleotide residues, and * represents a phosphorothioate bond.

[0015] Aspect 3. The calcium-containing cationic lipid nanoparticles as described in Aspect 1 or 2, characterized in that the cationic lipid nanoparticles contain calcium ions in a non-precipitated state.

[0016] Aspect 4. The calcium-containing cationic lipid nanoparticles according to aspect 1 or 2, characterized in that the concentration of calcium in the entire preparation is 0.01 to 150 mmol / L; preferably 0.01 to 10 mmol / L or 10 to 150 mmol / L; preferably 0.01 to 0.1 mmol / L, 0.1 to 1 mmol / L, 1 to 10 mmol / L, 10 to 100 mmol / L or 100 to 150 mmol / L; more preferably 0.01 to 0.1 mmol / L, 0.1 to 1 mmol / L, 1 to 10 mmol / L, 10 to 30 mmol / L, 30 to 50 mmol / L, 50 to 70 mmol / L, 70 to 90 mmol / L, 90 to 110 mmol / L, 110 to 130 mmol / L or 130 to 150 mmol / L;

[0017] More preferably, the concentration of calcium in the entire preparation is 0.01 to 10 mmol / L; more preferably 0.02 to 8 mmol / L; more preferably 0.05 to 5 mmol / L; more preferably 0.1 to 3 mmol / L;

[0018] More preferably, the concentration of calcium in the entire preparation is 0.1 to 1 mmol / L before concentration, preferably 0.1 to 0.5 mmol / L, more preferably 0.1 to 0.5 mmol / L; and / or the concentration of calcium in the entire preparation is 1 to 10 mmol / L after concentration, preferably 1 to 5 mmol / L, more preferably 2 to 4 mmol / L.

[0019] Aspect 5. The calcium-containing cationic lipid nanoparticles according to aspect 1 or 2, characterized in that the concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 10-30000 μM, preferably 20-10000 μM, preferably 30-3000 μM;

[0020] More preferably, the concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 30-300 μM before concentration, preferably 50-200 μM, more preferably 60-180 μM; and / or the concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 500-5000 μM, preferably 1000-4000 μM, more preferably 2000-3000 μM after concentration.

[0021] Aspect 6. The calcium-containing cationic lipid nanoparticles as described in Aspect 1 or 2, characterized in that the molar ratio of calcium to lipid in the cationic lipid nanoparticles is 1:(5-50), more preferably 1:(8-30), and more preferably 1:(10-14).

[0022] Aspect 7. The calcium-containing cationic lipid nanoparticles as described in Aspect 1 or 2, characterized in that the molar ratio of calcium in the cationic lipid nanoparticles to the total amount of cholesterol in the preparation is 1:(1-20); preferably 1:(1-15); more preferably 1:(3-12); more preferably 1:(4-7).

[0023] Aspect 8. The calcium-containing cationic lipid nanoparticles according to any of the preceding aspects, characterized in that the calcium ions are derived from calcium salts, preferably soluble calcium salts, more preferably calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, calcium gluconate, more preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, calcium gluconate, more preferably calcium acetate;

[0024] Preferably, the calcium ions come from a calcium salt solution with a concentration of 50 mmol / L to 1000 mmol / L; preferably, the calcium ions come from a calcium salt solution with a concentration of 50 to 150 mmol / L, a calcium salt solution with a concentration of 150 to 300 mmol / L, a calcium salt solution with a concentration of 300 to 500 mmol / L, or a calcium salt solution with a concentration of 500 to 800 mmol / L;

[0025] More preferably, the calcium ions come from a calcium salt solution having a concentration of 100±50mmol / L, a calcium salt solution of 200±50mmol / L, a calcium salt solution of 300±50mmol / L, a calcium salt solution of 400±50mmol / L, a calcium salt solution of 500±50mmol / L, a calcium salt solution of 600±50mmol / L, a calcium salt solution of 700±50mmol / L, a calcium salt solution of 800±50mmol / L or a calcium salt solution of 900±50mmol / L;

[0026] More preferably, the calcium ions come from a calcium salt solution with a concentration of 200±20 mmol / L.

[0027] Aspect 9. The calcium-containing cationic lipid nanoparticles as described in any of the preceding aspects are characterized in that the molar number of phosphate groups of nucleic acids: the molar number of positive charges of cationic lipids is 1:(0.5~20), preferably 1:(1~10), more preferably 1:(1.5~6), more preferably 1:(1.5~3) or 1:(3~6), more preferably 1:3.

[0028] Aspect 10. The calcium-containing cationic lipid nanoparticles as described in any of the preceding aspects are characterized in that the nucleic acid:lipid mass ratio is 1:(1-100), preferably 1:(5-90), more preferably 1:(10-70), further preferably 1:(10-30), and further preferably 1:(15-20).

[0029] Aspect 11. The calcium-containing cationic lipid nanoparticle according to any of the preceding aspects, characterized in that: the amount of the loaded nucleic acid is 5 μg / ml to 15 mg / ml;

[0030] The amount of nucleic acid loaded is preferably 5 μg / ml to 10 μg / ml, 10 μg / ml to 20 μg / ml, 20 μg / ml to 30 μg / ml, 30 μg / ml to 70 μg / ml, 70 μg / ml to 150 μg / ml, 150 μg / ml to 300 μg / ml, 300 μg / ml to 400 μg / ml, 400 μg / ml to 800 μg / ml, 800 μg / ml to 1 mg / ml, 1 mg / ml 1-1.5 mg / ml, 1.5 mg / ml-2 mg / ml, 2 mg / ml-4 mg / ml, 4 mg / ml-6 mg / ml, 6 mg / ml-8 mg / ml, 8 mg / ml-10 mg / ml or 10-15 mg / ml; more preferably, the amount of the loaded nucleic acid is 10 μg / ml-100 μg / ml (preferably 30 μg / ml-70 μg / ml), and / or optionally reduced to the desired concentration as required after preparation;

[0031] More preferably, the amount of nucleic acid loaded is preferably 30 μg / ml to 70 μg / ml, and / or optionally after preparation, it is reduced to the required concentration of 4 mg / ml to 6 mg / ml, 6 mg / ml to 8 mg / ml, 8 mg / ml to 10 mg / ml or 10 to 15 mg / ml as needed. Aspect 12. The calcium-containing cationic lipid nanoparticles according to any of the above aspects, characterized in that the lipids constituting the cationic lipid nanoparticles include a combination of one or more of the following groups: cationic lipids, cholesterol and / or cholesterol esters, neutral lipids, PEGylated lipids;

[0032] Preferably, the lipids constituting the cationic lipid nanoparticles include the following lipids:

[0033] (1) Cationic lipids: the cationic lipids are selected from ionizable cationic lipids;

[0034] (2) cholesterol lipids: the cholesterol lipids are selected from cholesterol and / or cholesterol esters;

[0035] (3) neutral lipids: the neutral lipids are selected from phospholipids, fatty acid glycerides or glycolipids or any combination thereof; and optionally (4) PEGylated lipids;

[0036] More preferably, the lipids constituting the cationic lipid nanoparticles include:

[0037] (1) Cationic lipids: the cationic lipids are selected from ionizable cationic lipids;

[0038] (2) Cholesterol lipid: the cholesterol lipid is selected from cholesterol;

[0039] (3) neutral lipids: the neutral lipids are selected from phospholipids; and

[0040] Optional (4) PEGylated lipid.

[0041] Aspect 13. The calcium-containing cationic lipid nanoparticles according to any of the preceding aspects, characterized in that the lipids constituting the cationic lipid nanoparticles include a molar ratio of 10%-50% of cationic lipids and / or 25%-75% of cholesterol lipids;

[0042] Preferably, the cationic lipid nanoparticles include 20%-40% cationic lipids and / or 40%-60% cholesterol lipids in a molar ratio;

[0043] More preferably, the cationic lipid nanoparticles comprise cationic lipids and / or cholesterol lipids in a molar ratio of 33.3%±5% and / or 51.3%±5%.

[0044] Aspect 14. The calcium-containing cationic lipid nanoparticles according to any one of the preceding aspects, characterized in that the lipids constituting the cationic lipid nanoparticles include a combination of the following groups in molar ratios:

[0045] (1) Cationic lipids: 10%-50%,

[0046] (2) Cholesterol lipids: 25%-75%,

[0047] (3) Neutral lipids: 1%-30%,

[0048] (4) PEGylated lipids: 0.5%-5%;

[0049] More preferably, the lipids constituting the cationic lipid nanoparticles include a combination of the following groups in molar ratios:

[0050] (1) Cationic lipids: 20%-40%,

[0051] (2) Cholesterol lipids: 40%-60%,

[0052] (3) Neutral lipids: 10%-15%,

[0053] (4) PEGylated lipids: 1%-3%;

[0054] More preferably, the lipids constituting the cationic lipid nanoparticles include a combination of the following groups in molar ratios:

[0055] (1) Cationic lipids: 33.3% ± 5%,

[0056] (2) Cholesterol lipids: 51.3%±5%,

[0057] (3) Neutral lipids: 13.3% ± 5%,

[0058] (4) PEGylated lipid: 2%±1%.

[0059] Aspect 15. The calcium-containing cationic lipid nanoparticles according to any of the preceding aspects, characterized in that the cationic lipid is selected from ionizable cationic lipids; preferably, the ionizable cationic lipid is selected from DSDMA, DLinDMA, DLenDMA, DODMA, A6, OF-02, A18-Iso5-2DC18, 98N 12 -5, 9A1P9, C12-200, cKK-E12, 7C1, G0-C14, L319, 304O 13 , OF-Deg-Lin, 306-O12B, 306O i10, FTT5, SM102, ALC-0315, A9, Lipid 2, 2(8,8)4CCH3, CL1, LP01, DLin-MC3-DMA or any of the foregoing cationic lipid analogs and combinations; more preferably, the ionizable cationic lipid is selected from DLin-MC3-DMA; and / or the neutral phospholipid is selected from egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylcholine, Preferably, the neutral phospholipid is selected from one or more of distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, distearoylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, dioleoylphosphatidylinositol, 9A1P9, 10A1P10; preferably, the neutral phospholipid is selected from one or more of distearoylphosphatidylcholine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin and phosphatidylethanolamine; more preferably, the neutral phospholipid is selected from distearoylphosphatidylcholine; and / or the PEGylated lipid is selected from methoxy polyethylene glycol-distearoylphosphatidylethanolamine (mPEG-DSPE) , methoxy polyethylene glycol-dioleoylphosphatidylethanolamine (mPEG-DOPE), methoxy polyethylene glycol-dipalmitoylphosphatidylethanolamine (mPEG-DPPE), polyethylene glycol-dilauroylglycerol (PEG-DAG), polyethylene glycol-dimyristoylglycerol (PEG-DMG), polyethylene glycol-dipalmitoylglycerol (PEG-DPG), polyethylene glycol-distearoylglycerol (PEG-DSG), polyethylene glycol-dioleoylglycerol (PEG-DOG), polyethylene glycol-dilinoleoylglycerol (PEG-DLinG), polyethylene glycol-di Lauroyl propylamide (PEG-DAA), polyethylene glycol-dimyristoyl propylamide (PEG-DMA), polyethylene glycol-bispalmitoyl propylamide (PEG-DPA), polyethylene glycol-bisoleoyl propylamide (PEG-DOA), polyethylene glycol-bislinoleoyl propylamide (PEG-DLinA), polyethylene glycol-ceramide (PEG-ceramide), stearoyl polyethylene glycol ester, vitamin E polyethylene glycol succinate (TPGS) and any combination thereof; preferably, the PEGylated lipid is selected from polyethylene glycol-dimyristoyl glycerol (PEG-DMG);

[0060] wherein PEG is a PEG group having a degree of polymerization selected from 3 to 100; preferably, PEG is a PEG group having a degree of polymerization selected from 3 to 50 or 50 to 100; more preferably, PEG is a PEG group having a degree of polymerization selected from 3 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100; more preferably, PEG is a PEG group having a degree of polymerization selected from about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100.

[0061] Aspect 16. The calcium-containing cationic lipid nanoparticles according to any of the preceding aspects, characterized in that: the particle size of the calcium-containing cationic lipid nanoparticles is 25 to 1000 nm; preferably, the particle size of the lipid nanoparticles is 25 to 50 nm, 50 to 200 nm, 200 to 500 nm or 500 to 1000 nm; more preferably, the particle size of the lipid nanoparticles is 25 to 75 nm, 75 to 150 nm, 150 to 225 nm, 225 to 350 nm, 350 nm to 500 nm, 500 to 800 nm or 800 to 1000 nm; more preferably Preferably, the particle size of the liposome nanoparticles is 40±10nm, 50±10nm, 60±10nm, 70±10nm, 80±10nm, 90±10nm, 100±10nm, 110±10nm, 120±10nm, 125±10nm, 130±10nm, 140±10nm, 150±10nm, 160±10nm, 170±10nm, 180±10nm, 190±10nm, 200±10nm, 210±10nm, 220±10nm or 250±10nm.

[0062] Aspect 17. The calcium-containing cationic lipid nanoparticles according to any one of the preceding aspects, characterized in that the calcium-containing cationic lipid nanoparticles are selected from the following nanoparticle preparations:

[0063] Cationic lipid nanoparticles containing calcium and loaded with siRNA, using cationic lipids (preferably DLin-MC3-DMA), cholesterol, neutral phospholipids (preferably DSPC), PEGylated lipids (preferably PEG2000-DMG) as lipids and calcium acetate as a calcium ion source;

[0064] Calcium-containing cationic lipid nanoparticles loaded with mRNA using cationic lipids (preferably DLin-MC3-DMA), cholesterol, neutral phospholipids (preferably DSPC), PEGylated lipids (preferably PEG2000-DMG) as lipids and calcium acetate as a calcium ion source;

[0065] DNA-loaded cationic lipid nanoparticles containing calcium, using cationic lipids (preferably DLin-MC3-DMA), cholesterol, neutral phospholipids (preferably DSPC), PEGylated lipids (preferably PEG2000-DMG) as lipids and calcium acetate as a calcium ion source;

[0066] Preferably, the calcium-containing cationic lipid nanoparticles are selected from the following nanoparticle formulations:

[0067] Cationic lipid nanoparticles containing calcium and loaded with siRNA are prepared by using cationic lipids (preferably DLin-MC3-DMA), cholesterol, neutral phospholipids (preferably DSPC), and PEGylated lipids (preferably PEG2000-DMG) as lipids and calcium acetate as a calcium ion source; wherein the molar ratio of the cationic lipids (preferably DLin-MC3-DMA), cholesterol, neutral phospholipids (preferably DSPC), and PEGylated lipids (preferably PEG2000-DMG) is cationic lipids: 33.3%±5%, cholesterol: 51.3%±5%, neutral lipids: 13.3%±5%, and PEGylated lipids: 2%±1%.

[0068] Aspect 18. The calcium-containing cationic lipid nanoparticles as described in any of the preceding aspects, characterized in that the calcium-containing cationic lipid nanoparticles have a liver-targeting effect.

[0069] Aspect 19. A calcium-containing cationic lipid nanoparticle composition, characterized in that it is prepared by mixing a calcium-containing nucleic acid solution with a lipid solution,

[0070] Preferably the pH is adjusted to neutral after mixing;

[0071] Preferably, the nucleic acid solution is a solution of siRNA in a calcium salt aqueous solution, and / or the lipid solution is a solution of a cationic lipid (preferably DLin-MC3-DMA), cholesterol, a neutral phospholipid (preferably DSPC), or a PEGylated lipid (preferably PEG2000-DMG) in ethanol.

[0072] Aspect 20. Use of the nucleic acid-loaded calcium-containing cationic lipid nanoparticles of Aspects 1-19 or the calcium-containing cationic lipid nanoparticle composition of Aspect 20 for preparing a gene drug for intravenous injection in vivo.

[0073] Aspect 21. Use of the nucleic acid-loaded calcium-containing cationic lipid nanoparticles of Aspects 1-19 or the calcium-containing cationic lipid nanoparticle composition of Aspect 20 for the preparation of a medicament for treating transthyretin-mediated amyloidosis and hereditary TTR amyloidosis (hATTR);

[0074] Preferably, the transthyretin-mediated amyloidosis is familial transthyretin amyloidosis or sporadic transthyretin amyloidosis;

[0075] More preferably, the familial transthyretin amyloidosis is familial amyloid cardiomyopathy (FAC), familial amyloid polyneuropathy (FAP) or selective central nervous system amyloidosis (CNSA); the sporadic transthyretin amyloidosis is senile systemic amyloidosis (SSA) or senile cardiac amyloidosis (SCA).

[0076] Aspect 22. A method for preparing calcium-containing cationic lipid nanoparticles loaded with nucleic acid for reducing transthyretin, characterized in that step (1): mixing cationic lipids, cholesterol lipids, neutral phospholipids, PEGylated lipids and ethanol to form a lipid solution;

[0077] Step (2): mixing an aqueous phase containing a water-soluble calcium salt with a nucleic acid for reducing transthyretin to form a nucleic acid solution;

[0078] Step (3): mixing the solutions in step (1) and step (2) to form the calcium-containing cationic lipid nanoparticles loaded with nucleic acid for reducing transthyretin; and

[0079] Optional step (4) dialysis to adjust pH.

[0080] Aspect 23. In the preparation method described in Aspect 22, the organic phase solvent in step (1) is selected from a solvent miscible with water; preferably, the organic phase solvent is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, sec-butanol, DMSO, DMF, acetic acid and any combination thereof; more preferably, the organic phase solvent is selected from ethanol, n-propanol, isopropanol; more preferably, the organic phase solvent is selected from ethanol.

[0081] Aspect 24. The preparation method described in aspect 22, wherein the lipids in step (1) comprise a combination of the following groups in molar ratios: (1) cationic lipids: 10%-50%,

[0082] (2) Cholesterol lipids: 25%-75%,

[0083] (3) Neutral lipids: 1%-30%,

[0084] (4) PEGylated lipids: 0.5%-5%;

[0085] More preferably, in step (1), the lipid comprises a combination of the following groups in molar ratios:

[0086] (1) Cationic lipids: 20%-40%,

[0087] (2) Cholesterol lipids: 40%-60%,

[0088] (3) Neutral lipids: 10%-15%,

[0089] (4) PEGylated lipids: 1%-3%;

[0090] Further preferably, in step (1), the lipids include a combination of the following groups in molar ratios:

[0091] (1) Cationic lipids: 33.3% ± 5%,

[0092] (2) Cholesterol lipids: 51.3%±5%,

[0093] (3) Neutral lipids: 13.3% ± 5%,

[0094] (4) PEGylated lipid: 2%±1%.

[0095] Aspect 25. In the preparation method described in Aspect 22, the lipid in step (1) is selected from:

[0096] The cationic lipid is selected from ionizable cationic lipids; preferably, the ionizable cationic lipid is selected from DSDMA, DLinDMA, DLenDMA, DODMA, A6, OF-02, A18-Iso5-2DC18, 98N 12 -5, 9A1P9, C12-200, cKK-E12, 7C1, G0-C14, L319, 304O 13 , OF-Deg-Lin, 306-O12B, 306O i10, FTT5, SM102, ALC-0315, A9, Lipid2, 2(8,8)4CCH3, CL1, LP01, DLin-MC3-DMA or any of the foregoing cationic lipid analogs and combinations; more preferably, the ionizable cationic lipid is selected from DLin-MC3-DMA; and / or the neutral phospholipid is selected from egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, distearoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, Phosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, distearoylphosphatidylinositol, dimyristoylphosphatidylinositol, dipalmitoylphosphatidylinositol, dioleoylphosphatidylinositol, 9A1P9, 10A1P10 or more; preferably, the neutral phospholipid is selected from one or more of distearoylphosphatidylcholine, phosphatidylcholine, egg yolk lecithin, soybean lecithin, hydrogenated soybean lecithin and phosphatidylethanolamine; more preferably, the neutral phospholipid is selected from distearoylphosphatidylcholine; and / or the PEGylated lipid is selected from methoxy polyethylene glycol-distearoylphosphatidylcholine PEG-DSPE, methoxy polyethylene glycol-dioleoylphosphatidylethanolamine (mPEG-DOPE), methoxy polyethylene glycol-dipalmitoylphosphatidylethanolamine (mPEG-DPPE), polyethylene glycol-dilauroylglycerol (PEG-DAG), polyethylene glycol-dimyristoylglycerol (PEG-DMG), polyethylene glycol-dipalmitoylglycerol (PEG-DPG), polyethylene glycol-distearoylglycerol (PEG-DSG), polyethylene glycol-dioleoylglycerol (PEG-DOG), polyethylene glycol-dilinoleoylglycerol (PEG-DLinG ), polyethylene glycol-bis-lauroyl propylamide (PEG-DAA), polyethylene glycol-bis-myristoyl propylamide (PEG-DMA), polyethylene glycol-bis-palmitoyl propylamide (PEG-DPA), polyethylene glycol-bis-oleoyl propylamide (PEG-DOA), polyethylene glycol-bis-linoleoyl propylamide (PEG-DLinA), polyethylene glycol-ceramide (PEG-ceramide), stearoyl polyethylene glycol ester, vitamin E polyethylene glycol succinate (TPGS) and any combination thereof; preferably, the PEGylated lipid is selected from polyethylene glycol-dimyristoyl glycerol (PEG-DMG);

[0097] wherein PEG is a PEG group having a degree of polymerization selected from 3 to 100; preferably, PEG is a PEG group having a degree of polymerization selected from 3 to 50 or 50 to 100; more preferably, PEG is a PEG group having a degree of polymerization selected from 3 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90 or 90 to 100; more preferably, PEG is a PEG group having a degree of polymerization selected from about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95 or about 100.

[0098] Aspect 26. In the preparation method described in Aspect 22, the water-soluble calcium salt in step (2) is selected from calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, and calcium gluconate, and is more preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, and calcium gluconate, and is more preferably calcium acetate;

[0099] Preferably, the water-soluble calcium salt concentration is a calcium salt solution of 50 mmol / L to 1000 mmol / L; preferably, the water-soluble calcium salt concentration is a calcium salt solution of 50 to 150 mmol / L, a calcium salt solution of 150 to 300 mmol / L, a calcium salt solution of 300 to 500 mmol / L, or a calcium salt solution of 500 to 800 mmol / L;

[0100] More preferably, the water-soluble calcium salt concentration is 100±50mmol / L calcium salt solution, 200±50mmol / L calcium salt solution, 300±50mmol / L calcium salt solution, 400±50mmol / L calcium salt solution, 500±50mmol / L calcium salt solution, 600±50mmol / L calcium salt solution, 700±50mmol / L calcium salt solution, 800±50mmol / L calcium salt solution or 900±50mmol / L calcium salt solution;

[0101] More preferably, the calcium salt solution has a water-soluble calcium salt concentration of 200±20 mmol / L.

[0102] Aspect 27. In the preparation method described in Aspect 22, the calcium salt solution used in step (2) is a weak acidic solution, preferably a weak acidic solution with a pH value of 4-6.8, and more preferably a weak acidic solution with a pH value of 5±0.5.

[0103] Aspect 28. In the preparation method described in Aspect 22, the mixing method in step (3) is

[0104] The solution obtained in step (1) and the solution obtained in step (2) are mixed by flowing through the same pipeline; preferably, the mixer in the same pipeline is a three-way pipe, a microfluidic chip, or any variant thereof; more preferably, the mixer in the same pipeline is a Y-shaped pipe, a T-shaped pipe, a microfluidic chip, or any variant thereof;

[0105] More preferably, mixing is regulated by flow rate ratios.

[0106] Aspect 29. In the preparation method described in Aspect 22, in step (3), the flow rate ratio of the solution obtained in step (1) to the solution obtained in step (2) is 1:(3-6), preferably 1:(4-5), preferably 1:4.5.

[0107] Aspect 30. In the preparation method described in Aspect 22, step (4) is to adjust the pH value to neutral by dialysis.

[0108] The organic phase containing lipids is mixed with the aqueous phase containing calcium and TTR siRNA to form calcium-containing cationic lipid nanoparticles loaded with TTR siRNA. Based on the research, the inventor surprisingly found that when the inner core of the cationic lipid nanoparticle contains soluble calcium salt, the use of lipid nanoparticles to load nucleic acid drugs has the characteristics of high transfection efficiency, stable encapsulation, uniform and controllable particle size, and specific targeting of liver organs. Through experiments to detect the effect of nucleic acid onset, it was found that the interfering RNA loaded by the calcium-containing cationic lipid nanoparticles of the present invention can significantly reduce the content of related mRNA, and the loaded TTR siRNA can significantly reduce the amount of related TTR mRNA and protein.

[0109] Unless otherwise indicated, the following terms have the meanings ascribed to them.

[0110] Unless the context requires otherwise, the word "comprise" and variations such as "include" and "comprising" in this specification and claims, and the open and inclusive meaning are to be interpreted as meaning "including, but not limited to".

[0111] The term "precipitated calcium ions" refers to calcium salts whose pure substances are in a poorly soluble state under normal conditions, such as calcium phosphate, calcium carbonate, calcium fluoride, etc. The term "non-precipitated calcium ions" refers to calcium ions whose pure substances are in a non-poorly soluble state under normal conditions, that is, calcium ions in a soluble, easily soluble, or slightly soluble state, including but not limited to calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium hydrogen sulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, calcium gluconate, etc.

[0112] The term "DNA" refers to a non-replicating gene or a replicating gene vector used as an expression vector.

[0113] The term "interfering RNA" or "RNAi" or "interfering RNA sequence" refers to a single-stranded RNA (e.g., a mature miRNA) or a double-stranded RNA (i.e., a duplex RNA such as siRNA, aiRNA, or pre-miRNA) that can reduce or inhibit the expression of a target gene or sequence when the interfering RNA and the target gene or sequence are in the same cell (e.g., by mediating degradation and inhibiting the translation of an mRNA complementary to the interfering RNA sequence). Interfering RNA therefore refers to a single-stranded RNA complementary to a target mRNA sequence or a double-stranded RNA formed by two complementary strands or by a single self-complementary strand. Interfering RNA may have substantial or complete identity to a target gene or sequence, or may include a mismatch region (i.e., a mismatch motif). The sequence of an interfering RNA may correspond to a full-length target gene or a subsequence thereof.

[0114] Interfering RNA includes "small interfering RNA" or "siRNA," for example, about 15-60, 15-50, or 15-40 (duplex) nucleotides in length, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotides in length, and preferably about 20-24, 21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementary strand of a double-stranded siRNA is about 100 nt long). In some embodiments, the siRNA duplex may include a 3' overhang of about 1 to about 4 nucleotides or about 2 to about 3 nucleotides and a 5' phosphate terminus. Examples of siRNA include, but are not limited to, a double-stranded polynucleotide molecule assembled from two separate strand molecules, one of which is a sense strand and the other is a complementary antisense strand; a double-stranded polynucleotide molecule assembled from single-stranded molecules, wherein the sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule containing a hairpin secondary structure having self-complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule containing a stem having two or more loop structures and self-complementary sense and antisense regions, wherein the circular polynucleotide can be processed in vivo or in vitro to generate an active double-stranded siRNA molecule.

[0115] Preferably, the siRNA is chemically synthesized. siRNA can also be produced by cleaving longer dsRNAs (eg, dsRNAs greater than about 25 nucleotides in length) using E. coli RNase III or Dicer. These enzymes process dsRNA into biologically active siRNA (see, e.g., Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegari et al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al., Ambion Tech Notes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res., 31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); and Robertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, the dsRNA length is at least 50 nucleotides - about 100, 200, 300, 400, or 500 nucleotides. The dsRNA length can be up to 1000, 1500, 2000, 5000 nucleotides or longer. The dsRNA can encode a complete gene transcript or a partial gene transcript. In some instances, the siRNA can be encoded by a plasmid (e.g., transcribed as a sequence that automatically folds into a duplex with a hairpin loop).

[0116] The term "substantially identical" or "substantial identity" in the context of two or more nucleic acids refers to two or more sequences or subsequences that are identical or have a specific percentage of identical nucleotides (i.e., at least about 60%, preferably at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity) when compared and aligned for maximum correspondence over a comparison window, or a specified region, by using one of the following sequence comparison algorithms or by manual alignment and visual measurement. This definition, when indicated in the context, also similarly refers to the complement of the sequence. Preferably, the substantial identity exists over a region of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

[0117] The term "nucleic acid" as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in single-stranded or double-stranded form and includes DNA and RNA. DNA can take the following forms: for example, antisense molecules, plasmid DNA, pre-concentrated DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA can take the following forms: siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or bonds, which are synthetic, naturally occurring, and non-naturally occurring, and which have binding properties similar to reference nucleic acids. Examples of the analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides and peptide-nucleic acids (PNAs). Unless specifically limited, the term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid. Unless otherwise indicated, a specific nucleic acid sequence also inherently includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the explicitly indicated sequence. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed basic and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem., 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8: 91-98 (1994)). "Nucleotides" comprise the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. The nucleotides are linked together by the phosphate groups. "Bases" include purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0118] The term "gene" refers to a nucleic acid (eg, DNA or RNA) sequence that contains a partial or full-length coding sequence necessary for producing a polypeptide or precursor polypeptide.

[0119] The term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derived lipids" such as steroids.

[0120] When " lipid granule " is used herein, refer to can be used for delivering active agent or therapeutic agent, such as nucleic acid (for example, interfering RNA), to the lipid preparation of target target site.In the lipid granule formed by the combination lipid of cationic lipid, non-cationic lipid and prevention particle aggregation of the typical of the present invention, described active agent or therapeutic agent can be encapsulated in lipid, thus protecting reagent from enzymatic degradation.

[0121] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, sphingolipid family, diacylglycerol and β-acyloxy acid are also included in the group referred to as amphipathic lipids. In addition, the above-mentioned amphipathic lipids can be mixed with other lipids, and the other lipids include triglycerides and sterols.

[0122] The term "neutral lipid", when present in lipid particles, can be any of many lipid species present in the form of uncharged or neutral zwitterions at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cholesterol, cephalin and cerebrosides. The selection of neutral lipids for particles described herein is usually guided by considerations, such as liposome size and the stability of liposomes in the bloodstream. Preferably, the neutral lipid component is a lipid with two acyl groups (i.e., diacylphosphatidylcholine and diacylphosphatidylethanolamine). Liposomes with acyl chain groups of multiple different chain lengths and saturation are available or can be separated or synthesized by known technology. In one group of embodiments, lipids containing saturated fatty acids with carbon chain lengths within the range of C10 to C20 are preferred. In another group of embodiments, lipids with mono- or di-unsaturated fatty acids with carbon chain lengths within the range of C10 to C20 are used. In addition, lipids with mixtures of saturated and unsaturated fatty acid chains can be used. Preferably, neutral lipids used for the present invention are DOPE, DSPC, POPC, DPPC or any relevant phosphatidylcholine. Neutral lipids used for the present invention can also be made of sphingomyelin, dihydrosphingomyelin or have other head groups, for example the phospholipids of serine and inositol form.

[0123] The term "negatively charged lipid" refers to a lipid containing a free phosphohydroxyl group in the molecule. Due to the presence of the phosphohydroxyl group, this type of lipid can ionize positively charged hydrogen ions in solution and become negatively charged, including but not limited to acidic phospholipids such as phosphatidylserine.

[0124] The term "negatively charged lipids with a PEG group" refers to negatively charged lipids modified with PEG," including but not limited to distearoylphosphatidylethanolamine-methoxypolyethylene glycol 2000 (DSPE-MPEG2000).

[0125] The term "non-PEG group-modified negatively charged lipid" refers to a negatively charged lipid that is not modified with PEG, such as phosphatidylserine, phosphatidic acid, and phosphatidylglycerol.

[0126] The term "non-cationic lipid" refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0127] The term "anionic lipid" refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0128] The term "cationic lipid" refers to any of a number of lipid species carrying a net positive charge at a selected pH value, such as a physiological pH value or a pH value used in a preparation process. It has been unexpectedly found that cationic lipids comprising alkyl chains with multiple unsaturated sites, for example, at least 2 or 3 unsaturated sites, are particularly effective for forming lipid particles with increased membrane fluidity. Many cationic lipids and related analogs that are also effective for use in the present invention have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Patent Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96 / 10390, the disclosure of which is incorporated herein by reference for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cationic lipid comprises a protonatable tertiary amine (e.g., pH titrable) head group, a C18 alkyl chain, an ether bond between the head group and the alkyl chain, and 0 to 3 double bonds. Such lipids include, for example, DSDMA, DLinDMA, DLenDMA, DODMA, A6, OF-02, A18-Iso5-2DC18,98N 12-5, 9A1P9, C12-200, cKK-E12, 7C1, G0-C14, L319, 304O 13 , OF-Deg-Lin, 306-O12B, 306O i10 , FTT5, SM102, ALC-0315, A9, Lipid 2, 2(8,8)4CCH3, CL1, LP01, MC3 or an analog of any of the foregoing cationic lipids.

[0129] The term "analogs of cationic lipids" refers to cationic lipids with similar structures produced by changing the number of carbon atoms in any carbon chain of a cationic lipid.

[0130] In some examples, the cationic lipid is selected from the cationic lipid of formula (Ia):

[0131]

[0132] Where R 1a and R 2a Each time it occurs, it is independently C 10 -C 30 Alkyl, C 10 -C 30 Alkenyl or C 10 -C 30 Alkynyl; preferably, wherein R 1a and R 2a include at least one unsaturated site; more preferably, wherein R 1a and R 2a include at least two unsaturated sites; more preferably, wherein R 1a and R 2a All include at least two double bonds;

[0133] R 3a NH2-C 1-8 Alkyl-, NH(C 1-8 Alkyl)-C 1-8 Alkyl- or NH(C 1-8 Alkyl)2-C 1-8 Alkyl-; preferably, R 3ais NH2-(CH2)-, NH2-(CH2)2-, NH2-(CH2)3-, NH2-(CH2)4-, NH2-(CH2)5-, NH2-(CH2)6-, NH2-(CH2)7-, NH2 -(CH2)8-, NH(CH3)-(CH2)-, NH(CH3)-(CH2)2-, NH(CH3)-(CH2)3-, NH(CH3)-(CH2)4-, NH(CH3)-(CH2)5-, NH(CH3)-(CH2)6-, NH(CH3)-(CH2)7-, NH(CH3)-(CH2)8-, N(CH3)2-(CH2)-, N(CH3)2-(CH2)2-, N(CH3)2- (CH2)3-, N(CH3)2-(CH2)4-, N(CH3)2-(CH2)5-, N(CH3)2-(CH2)6-, N(CH3)2-(CH2)7- or N(CH3)2-(CH2)8-;

[0134] E is O, N(Q)C(O), C(O)N(Q), (Q)NC(O)O, OC(O)N(Q), SS or O=N, and Q is hydrogen or methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl; preferably, E is O, NHC(O), N(CH3)C(O), C(O)NH, C(O)N(CH3), NHC(O)O, N(CH3)C(O)O, C(O)ONH or C(O)ON(CH3).

[0135] In some examples, the cationic lipid is selected from the cationic lipid of formula (Ib):

[0136]

[0137] R 1b Choose from C 5-30 Alkyl, C 5-20 Alkenyl, -R b* Y c R b ”, -Y b R b ” and -R b ”M b 'R b 'Composition of groups;

[0138] R 2b and R 3b Independently selected from H, C 1-14 Alkyl, C 2-14 Alkenyl, -R b* Y b R b ”, -Y b Rb ” and -R b *OR b ", or R 2b and R 3b Together with the atoms to which they are attached, they form a heterocyclic or carbocyclic ring;

[0139] R 4b Choose from C 3-6 Carbocyclic ring, -(CH2) 1-5 Q, -(CH2) 1-5 CHQ b R b 、-CHQ b R b 、-CQ b (R b )2 and unsubstituted C 1-6 A group consisting of alkyl groups, wherein Q b Selected from carbocyclic, heterocyclic, -OR b 、-O(CH2) 1-5 N(R b )2. -C(O)OR b 、-OC(O)R b , -CX3, -CX2H, -CXH2, -CN, -N(R b )2、-C(O)N(R b )2、-N(R b )C(O)R b 、-N(R b )S(O)2R b 、-N(R b )C(O)N(R b )2、-N(R b )C(S)N(R b )2、-N(R b )R 8b 、-O(CH2) 1-5 OR b 、-N(R b )C(=NR 9b )N(R b )2、-N(R b )C(=CHR 9b )N(R b )2、-OC(O)N(R b )2、-N(R b )C(O)OR b 、-N(OR b )C(O)R b 、-N(OR b )S(O)2R b 、-N(ORb )C(O)OR b 、-N(OR b )C(O)N(R b )2、-N(OR b )C(S)N(R b )2、-N(OR b )C(=NR 9b )N(R b )2、-N(OR b )C(=CHR 9b )N(R b )2, -C(=NR 9b )N(R b )2, -C(=NR 9b )R b 、-C(O)N(R b )OR b and -C(R b )N(R b )2C(O)OR b ; Each R 5b Independently selected from C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; each R 6b Independently selected from C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; M and M b ' is independently selected from -C(O)O-, -OC(O)-, -C(O)N(R b ')-、-N(R b ')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR b ')O-, -S(O)2-, -SS-, aryl groups and heteroaryl groups; R 7b Choose from C 1-3 Alkyl, C 2-3 A group consisting of an alkenyl group and H; R 8b Choose from C 3-6 A group consisting of carbocyclic and heterocyclic rings; R 9b Selected from H, CN, NO2, C 1-6 Alkyl, -OR b 、-S(O)2R b 、-S(O)2N(R b 2. C 2-6 Alkenyl, C 3-6 A group consisting of carbocyclic and heterocyclic rings; each R b Independently selected from C 1-3 Alkyl, C 2-3A group consisting of an alkenyl group and H; each R b 'Independently choose from C 1-18 Alkyl, C 2-18 Alkenyl, -R b *YR b ”, -YR b " and H; each R b "Independently selected from C 3-14 Alkyl and C 3-14 A group consisting of alkenyl; each R b *Independently selected from C 1-12 Alkyl and C 2-12 alkenyl group; each Y b Independently C 3-6 Carbon ring; each X b Independently selected from the group consisting of F, Cl, Br and I.

[0140] In some examples, the cationic lipid is selected from the cationic lipid of formula (Ic):

[0141]

[0142] Among them, G 1c and G 2c Each is independently unsubstituted -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9- or -(CH2) 10 -;

[0143] G 3c is unsubstituted -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, -(CH2)6-, -(CH2)7-, -(CH2)8-, -(CH2)9-, -(CH2) 10 -,-(CH2) 11 -or-(CH2) 12 -;

[0144] R 1c and R 2c Each independently is C 6-24 Alkyl or C 6-24 alkenyl;

[0145] R 3c OR 5c 、CN、-C(=O)OR 4c 、-OC(=O)R 4c or –NR 5c C(=O)R 4c ;

[0146] R 4c C1-12 a hydrocarbon group; and

[0147] R 5c It is H, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl.

[0148] In some examples, the cationic lipid is selected from a cationic lipid of formula (Ida) or formula (Idb):

[0149]

[0150] L 1d and L 2d Each independently represents -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -NR ad C(=O)-, -C(=O)NR ad -、-NR ad C(=O)NR ad -、-OC(=O)NR ad -、-NR ad C(=O)O- or bond;

[0151] G 3d is -(CH2)-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5- or -(CH2)6-;

[0152] R ad H or C 1-12 Hydrocarbon;

[0153] R 1ad and R 1bd is independently at each occurrence: (a) H or C 1-12 or (b) R 1ad H or C 1-12 Hydrocarbon, and R 1bd Together with the carbon atom to which it is attached and the adjacent R 1bd Together with the carbon atom to which it is attached, it forms a carbon-carbon double bond;

[0154] R 2ad and R 2bd is independently at each occurrence: (a) H or C 1-12 Hydrocarbyl; or (b) R 2ad H or C 1-12 Hydrocarbon, and R 2bd Together with the carbon atom to which it is attached and the adjacent R 2bd Together with the carbon atom to which it is attached, it forms a carbon-carbon double bond;

[0155] R 3ad and R 3bdIndependently at each occurrence: (a) is H or C 1-12 Hydrocarbyl; or (b) R 3ad H or C 1-12 Hydrocarbon, and R 3bd Together with the carbon atom to which it is attached and the adjacent R 3bd Together with the carbon atom to which it is attached, it forms a carbon-carbon double bond;

[0156] R 4ad and R 4bd Independently at each occurrence: (a) is H or C 1-12 Hydrocarbyl; or (b) R 4ad H or C 1-12 Hydrocarbon, and R 4bd Together with the carbon atom to which it is attached and the adjacent R 4bd Together with the carbon atom to which it is attached, it forms a carbon-carbon double bond;

[0157] R 5d and R 6d are each independently H or methyl;

[0158] R 7d -(C=O)OR bd , -O(C=O)R bd 、-C(=O)R bd 、-OR bd 、-C(=O)SR bd 、-NR ad R bd 、-NR ad C(=O)R bd 、-C(=O)NR ad R bd 、-NR ad C(=O)NR ad R bd 、-OC(=O)NR ad R bd 、-NR ad C(=O)OR bd Optionally substituted C 6-16 Hydrocarbyl, where R ad H or C 1-12 Hydrocarbon; R bd C 1-15 Hydrocarbon;

[0159] R 8d and R 9d Each independently is C 1-12 Hydrocarbyl; or R 8d and R 9d , together with the nitrogen atom to which they are attached, form a 5-, 6- or 7-membered heterocyclic ring.

[0160] The term "DLin-MC3-DMA" refers to the substance with CAS number 1224606-06-7. The structure of the substance is

[0161]

[0162] The term "DSDMA" refers to the substance with CAS number 871258-14-9, which has the structure

[0163]

[0164] The term "DLenDMA" refers to the substance with CAS number 874291-25-5, which has the structure

[0165]

[0166] The term "DODMA" refers to the substance with CAS number 104162-47-2, which has the structure

[0167]

[0168] The term "A6" refers to a structure of substance.

[0169] The term "OF-02" refers to a of substance.

[0170] The term "A18-Iso5-2DC18" refers to a of substance.

[0171] The term "98N 12 -5" refers to the structure of substance.

[0172] The term "9A1P9" refers to a of substance.

[0173] The term "C12-200" refers to of substance.

[0174] The term "cKK-E12" refers to a of substance.

[0175] The term "G0-C14" refers to a structure of substance.

[0176] The term "L319" refers to a of substance.

[0177] The term "SM-102" refers to CAS No. 2089251-47-6, structure

[0178] of substance.

[0179] The term "ALC-0315" refers to ALC-0315 with CAS number 2036272-55-4 and structure

[0180] of substance.

[0181] The term "A9" refers to a structure of substance.

[0182] The term "Lipid 2,2(8,8)4CCH3" refers to a of substance.

[0183] The term "CL1" refers to a of substance.

[0184] The term "LP01" refers to a of substance.

[0185] The term "MC3" or "DLin-MC3-DMA" refers to a of substance.

[0186] The term "PEG2000-DMG" refers to 1,2-dimyristoyl-rac-glycero-3-methoxypolyethyleneglycol-2000, whose Chinese name is dimyristoylglycerol-polyethylene glycol 2000. Its molecular formula is C 122 H 242 O 50 , molecular weight 2509.2, structural formula:

[0187] n=45.

[0188] In addition to the cationic lipids explicitly described above, other cationic lipids that carry a net positive charge at approximately physiological pH may also be included in the lipid particles of the present invention. Such cationic lipids include, but are not limited to, N, N-dioleyl-N, N-dimethylammonium chloride ("DODAC"); N-(2,3-dioleyloxy)propyl-N, N-triethylammonium chloride ("DOTMA"); N, N-distearoyl-N, N-diethylammonium bromide ("DDAB"); N-(2,3-dioleyloxy)propyl)-N, N, N-triethylammonium chloride ("DOTAP"); 1,2-dioleyloxy-3-trimethylaminopropane hydrochloride ("DOTAP.Cl"); 3-(N-(N', N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-C hol″), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (“DOSPA”), dioctadecylaminoglycylcarboxyspermine (“DOGS”), 1,2-dioleyl-sn-3-phosphoethanolamine (“DOPE”), 1,2-dioleoyl-3-dimethylpropylamine (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (“DMRIE”). In addition, a number of commercial preparations of cationic lipids may be used, such as, for example, LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and LIPOFECTAMINE (including DOSPA and DOPE, available from GIBCO / BRL). In particular embodiments, the cationic lipid is an amino lipid.

[0189] The abbreviation "Chol" refers to cholesterol.

[0190] The ratio "N / P" refers to the number of moles of positive charge of all cationic lipids contained in the composition: the number of moles of phosphate groups.

[0191] The term "CaLNP" refers to the calcium-containing cationic lipid nanoparticles described in any of the above aspects.

[0192] The term "mammal" refers to any mammalian species such as humans, mice, rats, dogs, cats, hamsters, guinea pigs, rabbits, livestock, and the like.

[0193] The human TTR gene is shown below:

[0194] Table 1: Human TTR gene (NCBI Reference Sequence: NM_000371.4)

[0195]

[0196]

[0197] The nucleic acid (preferably, siRNA) acting on the TTR gene in the present application comprises a sense strand and an antisense strand that is at least 85% complementary thereto. The sense strand of the nucleic acid has a nucleotide sequence of at least 17 adjacent nucleotides, and wherein the sense strand sequence has a region that is at least 85% complementary to the antisense strand over the 17 adjacent nucleotides. One of the sense strand or antisense strand of the nucleic acid (preferably, the antisense strand) has at least 85% of a region that can complementarily act within a partial continuous region of the TTR gene sequence in Table 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0198] Figure 1 . Serum TTR levels of animals after administration of different preparations (n=5)

[0199] Figure 2. Liver TTR mRNA levels in animals after administration of different preparations (n=5)

[0200] Figure 3. TTR mRNA levels in HepG2 cells after administration of different preparations DETAILED DESCRIPTION

[0201] The specific embodiments of the present invention will be described in detail below. It should be noted that the embodiments described herein are only for illustration and are not intended to limit the present invention. In the following description, in order to provide a thorough understanding of the present invention, a large number of specific details are set forth, however, it is apparent to those skilled in the art that these specific details need not be adopted to implement the present invention.

[0202] The detection methods used in the present invention are all conventional or common detection methods in the art. For the use of kits in the detection methods, the operation is performed according to the instructions in the commercial kits. If it is necessary to use instruments and equipment for detection, the general operation method is to follow the conventional operation method in the art for operation and detection. Unless otherwise specified, the following detection methods are performed according to the description of the present invention.

[0203] Abbreviations

[0204]

[0205]

[0206] Experimental Materials:

[0207] The following siRNA sequences were synthesized by Suzhou Ouli Biotech Co., Ltd.

[0208]

[0209] Lower case letters represent 2'-O-methyl nucleotide residues

[0210] Lowercase underlined letters represent 2'-fluoro nucleotide residues

[0211] * represents phosphorothioate bond

[0212]

[0213] Example 1

[0214] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 25:10:38.5:1.5 to form a solution with a total lipid concentration of 8.8 mg / ml. siRNA (sequence 1) was dissolved in 200 mM calcium acetate solution (PH5.0) to form a solution with a concentration of 111 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with Tris solution (PH7.0) for 4 hours. Store at 2-8°C for later use.

[0215] Example 2

[0216] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 25:10:38.5:1.5 to form a solution with a total lipid concentration of 8.8 mg / ml. siRNA (sequence 1) was dissolved in 400 mM calcium acetate solution (PH5.0) to form a solution with a concentration of 111 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with Tris solution (PH7.0) for 4 hours. Store at 2-8°C for later use.

[0217] Example 3

[0218] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 25:10:38.5:1.5 to form a solution with a total lipid concentration of 8.8 mg / ml. The siRNA (sequence 2) solution was dissolved in 200 mM calcium acetate solution (PH5.0) to form a solution with a concentration of 111 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with Tris solution (PH7.0) for 24 hours. Store at 2-8°C for later use.

[0219] Example 4

[0220] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 25:10:38.5:1.5 to form a solution with a total lipid concentration of 8.8 mg / ml. siRNA (sequence 3) was dissolved in 200 mM calcium acetate solution (PH5.0) to form a solution with a concentration of 111 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with Tris solution (PH7.0) for 24 hours. Store at 2-8°C for later use.

[0221] Example 5

[0222] The preparation in Example 3 was concentrated by centrifugation at 9000 rpm using a 100 kDa ultrafiltration tube to a siRNA concentration of about 1 mg / ml.

[0223] Comparative Example 1

[0224] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to form a solution with a total lipid concentration of 12 mg / ml. siRNA (sequence 1) was dissolved in 25 mM acetic acid-sodium acetate solution (PH4.0) to form a solution with a concentration of 222 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with PBS solution (PH7.0) for 4 hours. Store at 2-8°C for later use.

[0225] Comparative Example 2

[0226] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to form a solution with a total lipid concentration of 12 mg / ml. siRNA (sequence 2) was dissolved in 25 mM acetic acid-sodium acetate solution (PH4.0) to form a solution with a concentration of 222 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with PBS solution (PH7.0) for 4 hours. Store at 2-8°C for later use.

[0227] Comparative Example 3

[0228] Dlin-MC3-DMA, DSPC, cholesterol and PEG2000-DMG were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to form a solution with a total lipid concentration of 12 mg / ml. siRNA (sequence 3) was dissolved in 25 mM acetic acid-sodium acetate solution (PH4.0) to form a solution with a concentration of 222 μg / ml. The lipid solution and siRNA solution (N / P=3) were mixed using a microfluidic device at a flow rate ratio of 1:4.5 and a total flow rate of 22 ml / min. The mixed solution was dialyzed with PBS solution (PH7.0) for 4 hours. Store at 2-8°C for later use.

[0229] Comparative Example 4

[0230] siRNA (sequence 4) was dissolved in RNase-free water to a 1 mg / ml solution.

[0231] Test example

[0232] Experimental Example 1. Characterization of Nano-siRNA Preparations:

[0233] Content encapsulation efficiency: The content encapsulation efficiency was determined using the fluorescent dye method and the qubit instrument.

[0234] siRNA control: dilute siRNA with 1% Triton to about 1-10 μg / ml of siRNA, take 10 μL, add 190 μL of fluorescent dye, mix well, and measure in a fluorometer.

[0235] Total content (X): dilute the preparation with 1% Triton to about 1-10 μg / ml of siRNA, take 10 μL, add 190 μL of fluorescent dye, mix well, and measure in a fluorometer.

[0236] Free content (Y): Take 10 μL of the preparation, add 190 μL of fluorescent dye, mix well, and measure in a fluorimeter.

[0237] Encapsulation rate (%) = (XY) / Y*100%

[0238] Particle size and potential: measured by dynamic light scattering

[0239] The preparation was diluted 100 times with water, and the particle size and polydispersity index of the nanoparticles were measured using a Malvern Zetasizer Nano ZS laser particle size analyzer. The laser beam wavelength of the instrument was set to 633 nm, the angle between the incident light and the scattered light beam was 90°, and the measurement temperature was set to 25°C.

[0240] Ca 2+ Concentration detection method

[0241] Calcium ion (Ca) was measured using the Calcium Ion (Ca) Assay Kit (Arsenic Azo III Method) (Beijing Leadman Biochemical Co., Ltd.)

[0242] Blank solution: water

[0243] Reference solution: Take the Ca 2+ Add 900ul of water to 100ul of ion standard solution (2.46mmol / L) and mix well.

[0244] Free Ca 2+ Test solution: Preparation

[0245] Total Ca 2+ Test solution: Take 100ul of the preparation and add 20ul of 1% Triton.

[0246] Determination method: Take blank solution, reference solution, free Ca 2+ Test solution, total Ca 2+ Add 900ul of calcium ion (Ca) assay reagent to 100ul of the test solution, mix well, and place in a UV-visible spectrophotometer for detection at a wavelength of 600nm and an optical path of 1cm. Record the absorbance values ​​as A 空白 , A 对照 , A 游离Ca供试 , A 总Ca供试 .

[0247] C 游离Ca供试 =(A 游离Ca供试 -A 空白 ) / (A 对照 -A 空白 )*C 对照

[0248] C 总Ca供试 =(A 总Ca供试 -A 空白 ) / (A 对照 -A 空白 )*C 对照

[0249] ΔC=C 总Ca供试 -C 游离Ca供试

[0250] Cholesterol concentration detection method

[0251] The free cholesterol (FC) content was determined using the free cholesterol (FC) content detection kit (Beijing Box Biotechnology Co., Ltd., catalog number AKFA001C).

[0252] Blank solution: anhydrous ethanol

[0253] Reference solution: Dissolve cholesterol in anhydrous ethanol to 1 μmol / ml.

[0254] Test solution: Take 100 μl of the preparation and add 20 μl of 1% triton and mix well.

[0255] Determination method: Take 50μl of blank solution, reference solution and test solution respectively, add 950μl of FC working solution, mix well, develop color at 37℃ for 15min, and then detect in UV-visible spectrophotometer with detection wavelength of 500nm and optical path length of 1cm. Record the absorbance values ​​as A blank, A control and A test respectively.

[0256] C test = (A test - A blank) / (A control - A blank) * C control

[0257] Formulation characterization results:

[0258]

[0259] ΔCa 2+ The concentration represents the calcium ion concentration in the inner core of the lipid nanoparticles.

[0260] Test Example 2 Animal Efficacy Study

[0261] Animal administration method

[0262] Fifty 6-8 week old Balb / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were randomly divided into 10 groups (5 mice in each group, all male) according to body weight, and PBS, 0.1 mg / kg of the preparation of control example 1, 0.03 mg / kg of the preparation of control example 1, 0.01 mg / kg of the preparation of control example 1, 0.1 mg / kg of the preparation of example 1, 0.03 mg / kg of the preparation of example 1, 0.01 mg / kg of the preparation of example 1, 0.1 mg / kg of the preparation of example 2, 0.03 mg / kg of the preparation of example 2, and 0.01 mg / kg of the preparation of example 2 were injected into the tail vein. The average weight of the animals was 20 g, and the preparations were diluted with 5% glucose solution, and the administration volume was 100 μL / mouse. The drug was administered once, and blood was collected 48 hours after administration, serum was separated, and TTR was determined. The mice were euthanized, the animals were dissected, the livers were collected, and mRNA was extracted and determined.

[0263] Detection of TTR expression level in mouse serum

[0264] Mouse transthyretin (TTR) ELISA kit (Wuhan Fine Biotechnology Co., Ltd., catalog number EM1435) was used to detect TTR protein according to the instructions.

[0265] Detection of TTR mRNA expression level in mouse liver tissue

[0266] Liver tissue was homogenized with a tissue homogenizer, and total RNA was extracted using an RNA extraction kit (Biyuntian, catalog number R0027) according to the instructions. The expression of TTR mRNA was detected using a one-step RT-qPCR (SYBR Green method) kit (Biyuntian, catalog number D7269M) according to its instructions. The PCR primers used to amplify mouse TTR and GAPDH as an internal reference gene are shown in the following table

[0267]

[0268] The results of the mouse animal experiment are as attached to the instruction manual. Figure 1 , Figure 2 It can be seen that after administration in mice, the preparations of Examples 1 and 2 have better TTR mRNA and protein reduction efficiency than LNP used in marketed hATTR therapeutic drugs at the same dose. To achieve the same TTR reduction level, the siRNA dose used in Example 1 or 2 is only about 1 / 10 of that in Control Example 1.

[0269] Instructions attached Figure 1 : Serum TTR levels of animals after administration of different preparations (n=5)

[0270] Instructions attached Figure 2: Liver TTR mRNA levels in animals after administration of different preparations (n=5)

[0271] Experimental Example 3. Cellular efficacy study

[0272] Cell culture and drug administration

[0273] The human liver cancer cell line HepG2 was cultured in a special culture medium for HepG2 cells (Wuhan Punosai, catalog number CM-0103), and 1% (v / v) double antibody was added thereto, and placed in a 5% CO2, 37°C constant temperature incubator. When the cells grew to about 80-90%, they were digested and passaged with 0.25% trypsin, and the passage was performed every 2-3 days on average. The cells were passaged at a ratio of 1:3, and the cells in the logarithmic growth phase were used for experiments.

[0274] HepG2 cells in the logarithmic growth phase were cultured at a rate of 1×10 5 / 0.5mL / well was inoculated in a 24-well plate and incubated at 37°C, 5% CO2 for 24 hours to allow the cells to adhere to the wall. After the incubation, the preparation was added to make the final concentrations of sequence 1 and sequence 2 in the well plate 50nM, 10nM, 2nM, 0.4nM and 0.08nM, respectively, and the final concentrations of sequence 3 were 1250nM, 250nM, 50nM, 10nM and 2nM, respectively. The cells treated with PBS were used as the negative control group and were placed in a cell culture incubator at 37°C and 5% CO2 for another 24 hours. The culture medium was discarded, and the cells were rinsed once with PBS and RNAeasy TM Total RNA was extracted using an animal RNA extraction kit, and then the knockdown efficiency of TTR mRNA by TTR-siRNA was detected using the fluorescence quantitative PCR method. In the fluorescence quantitative PCR method, the GAPDH gene was used as an internal reference gene.

[0275] Detection of TTR expression level in HepG2 cells

[0276] Total RNA was extracted using an RNA extraction kit (Biyuntian, catalog number R0027) according to the instructions. The expression of TTR mRNA was detected using a one-step RT-qPCR (SYBR Green method) kit (Biyuntian, catalog number D7269M) according to its instructions. The PCR primers used to amplify HepG2 cell TTR and GAPDH as an internal reference gene are shown in the following table

[0277]

[0278] The results of the cell experiment are as shown in the instruction manual. Figure 3 As shown. It can be seen that the ligand coupling sequence of Control Example 4 failed to reduce the level of TTR mRNA well within the dosage concentration range; while compared with the calcium-containing lipid nanoparticles prepared by siRNA of the same sequence and the LNP formulation used in the marketed product, the TTR mRNA levels of Examples 3 and 4 were lower at the same siRNA dose, and even if the dose was reduced by 25 times, the TTR mRNA levels of Examples 3 and 4 were still lower. The reduction in TTR mRNA levels is directly related to the reduction in TTR protein.

[0279] Instructions attached Figure 3. TTR mRNA levels in HepG2 cells after administration of different preparations.

Claims

1. A calcium-containing cationic lipid nanoparticle loaded with nucleic acid, characterized in that The cationic lipid nanoparticles comprise cationic lipids, neutral lipids, PEGylated lipids and cholesterol and / or cholesterol esters; The cationic lipid nanoparticles contain calcium ions, and the anions corresponding to the calcium ions are not phosphate, hydrogen phosphate and dihydrogen phosphate; and The cationic lipid nanoparticles do not contain non-PEG group-modified negatively charged lipids; The cationic lipid nanoparticles are loaded with nucleic acids for reducing transthyretin.

2. The calcium-containing cationic lipid nanoparticle according to claim 1, wherein: The nucleic acid used to reduce transthyretin is plasmid DNA, single-stranded DNA, double-stranded DNA, siRNA, shRNA, aiRNA, miRNA, mRNA, circular RNA, gRNA, aptamer, ribozyme, antisense oligonucleotide ASO or any combination thereof; Preferably, the nucleic acid used to reduce transthyretin is siRNA or ASO; More preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand that is at least 85% complementary thereto, the sense strand has a nucleotide sequence of at least 17 contiguous nucleotides, and one of the sense strand or the antisense strand has at least 85% of a region that can complementarily act within a partial continuous region of the TTR gene sequence in Table 1; Further more preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand complementary thereto, the sense strand having a nucleotide sequence of at least 17 contiguous nucleotides, and the antisense strand having at least 90% of a region capable of complementarity within a partial continuous region of the TTR gene sequence in Table 1; Further more preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand complementary thereto, the sense strand having a nucleotide sequence of at least 17 contiguous nucleotides, and the antisense strand having at least 95% of a region capable of complementarity within a partial continuous region of the TTR gene sequence in Table 1; Even more preferably, the nucleic acid for reducing transthyretin is siRNA, wherein the siRNA comprises a sense strand and an antisense strand complementary thereto, wherein the sense strand and the antisense strand are independently selected from a nucleotide sequence having at least 17 contiguous nucleotides with one of the following sequences: Further more preferably, the nucleic acid for reducing transthyretin is siRNA, and its sequence is independently selected from one of the following sequences: The lowercase letters represent 2'-O-methyl nucleotide residues, the lowercase underlined letters represent 2'-fluoro nucleotide residues, and * represents a phosphorothioate bond.

3. The calcium-containing cationic lipid nanoparticle according to claim 1 or 2, characterized in that The cationic lipid nanoparticles contain calcium ions in a non-precipitated state.

4. The calcium-containing cationic lipid nanoparticle according to claim 1 or 2, characterized in that The concentration of calcium in the whole preparation is 0.01-150mmol / L; preferably 0.01-10mmol / L or 10-150mmol / L; preferably 0.01-0.1mmol / L, 0.1-1mmol / L, 1-10mmol / L, 10-100mmol / L or 100-150mmol / L; more preferably 0.01-0.1mmol / L, 0.1-1mmol / L, 1-10mmol / L, 10-30mmol / L, 30-50mmol / L, 50-70mmol / L, 70-90mmol / L, 90-110mmol / L, 110-130mmol / L or 130-150mmol / L; More preferably, the concentration of calcium in the entire preparation is 0.01 to 10 mmol / L; more preferably 0.02 to 8 mmol / L; more preferably 0.05 to 5 mmol / L; more preferably 0.1 to 3 mmol / L; More preferably, the concentration of calcium in the entire preparation is 0.1 to 1 mmol / L before concentration, preferably 0.1 to 0.5 mmol / L, more preferably 0.1 to 0.5 mmol / L; and / or the concentration of calcium in the entire preparation is 1 to 10 mmol / L after concentration, preferably 1 to 5 mmol / L, more preferably 2 to 4 mmol / L.

5. The calcium-containing cationic lipid nanoparticles according to claim 1 or 2, characterized in that The concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 10-30000 μM, preferably 20-10000 μM, preferably 30-3000 μM; more preferably, the concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 30-300 μM before concentration, preferably 50-200 μM, more preferably 60-180 μM; and / or the concentration of calcium in the cationic lipid nanoparticles based on the total volume of the solution is 500-5000 μM, preferably 1000-4000 μM, more preferably 2000-3000 μM after concentration.

6. The calcium-containing cationic lipid nanoparticles according to claim 1 or 2, characterized in that The molar ratio of calcium to lipid in the cationic lipid nanoparticles is 1:(5-50), more preferably 1:(8-30), and more preferably 1:(10-14).

7. The calcium-containing cationic lipid nanoparticles according to claim 1 or 2, characterized in that The molar ratio of calcium in the cationic lipid nanoparticles to the total amount of cholesterol in the preparation is 1:(1-20); preferably 1:(1-15); more preferably 1:(3-12); more preferably 1:(4-7).

8. The calcium-containing cationic lipid nanoparticles according to any one of the preceding claims, characterized in that: The calcium ion is from a calcium salt, preferably a soluble calcium salt, more preferably calcium acetate, calcium chloride, calcium sodium EDTA, calcium gluconate, calcium dihydrogen phosphate, calcium nitrate, calcium bicarbonate, calcium hydrogen sulfate, calcium bisulfite, calcium bromide, calcium iodide, calcium citrate, calcium lactate, calcium gluconate, more preferably calcium acetate, calcium sodium EDTA, calcium gluconate, calcium citrate, calcium lactate, calcium gluconate, more preferably calcium acetate; Preferably, the calcium ions come from a calcium salt solution with a concentration of 50 mmol / L to 1000 mmol / L; preferably, the calcium ions come from a calcium salt solution with a concentration of 50 to 150 mmol / L, a calcium salt solution with a concentration of 150 to 300 mmol / L, a calcium salt solution with a concentration of 300 to 500 mmol / L, or a calcium salt solution with a concentration of 500 to 800 mmol / L; More preferably, the calcium ions come from a calcium salt solution having a concentration of 100±50mmol / L, a calcium salt solution of 200±50mmol / L, a calcium salt solution of 300±50mmol / L, a calcium salt solution of 400±50mmol / L, a calcium salt solution of 500±50mmol / L, a calcium salt solution of 600±50mmol / L, a calcium salt solution of 700±50mmol / L, a calcium salt solution of 800±50mmol / L or a calcium salt solution of 900±50mmol / L; More preferably, the calcium ions come from a calcium salt solution with a concentration of 200±20 mmol / L.

9. The calcium-containing cationic lipid nanoparticles according to any one of the preceding claims, characterized in that The molar number of phosphate groups in nucleic acids: the molar number of positive charges in cationic lipids is 1:(0.5-20), preferably 1:(1-10), more preferably 1:(1.5-6), more preferably 1:(1.5-3) or 1:(3-6), more preferably 1:

3.

10. The calcium-containing cationic lipid nanoparticles according to any of the preceding claims, characterized in that the nucleic acid:lipid mass ratio is 1:(1-100), preferably 1:(5-90), more preferably 1:(10-70), further preferably 1:(10-30), further preferably 1:(15-20).

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