CN117363614A - SiRNA for inhibiting expression of angiotensinogen and application thereof - Google Patents

SiRNA for inhibiting expression of angiotensinogen and application thereof Download PDF

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CN117363614A
CN117363614A CN202310460609.6A CN202310460609A CN117363614A CN 117363614 A CN117363614 A CN 117363614A CN 202310460609 A CN202310460609 A CN 202310460609A CN 117363614 A CN117363614 A CN 117363614A
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seq
sirna
hypertension
nucleotide
sense strand
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陈平
刘兆贵
马利帅
丁彦伟
陈璞
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Naptide Qingdao Biomedical Co ltd
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Abstract

The invention relates to siRNA for inhibiting expression of angiotensinogen and application thereof. The siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity complementary to the sense strand, wherein the sense strand is selected from the group consisting of nucleotide sequences that differ by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID NO. 1-SEQ ID NO. 52, and the antisense strand is selected from the group consisting of nucleotide sequences that differ by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID NO. 53-SEQ ID NO. 104. The siRNA of the invention forms RISC silencing complex and is complementarily paired with the AGT mRNA sequence, thereby degrading mRNA, inhibiting the expression of AGT, specifically reducing the synthesis of AGT by hepatic cells, reducing blood pressure in vivo and effectively preventing or treating hypertension.

Description

SiRNA for inhibiting expression of angiotensinogen and application thereof
Technical Field
The invention belongs to the technical field of biological medicines, in particular to siRNA for inhibiting expression of angiotensinogen and application thereof, and more particularly relates to siRNA, siRNA conjugate, pharmaceutical composition and application thereof.
Background
Hypertension is a long-term disease in which arterial blood pressure continues to rise. Hypertension is classified as primary (primary) hypertension or secondary hypertension. Essential hypertension is hypertension due to non-specific lifestyle and genetic factors, and about 90 to 95% of cases are essential hypertension. Hypertension affects the systemic blood vessels and is a major risk factor for a variety of diseases, disorders and conditions, such as retinopathy, chronic kidney disease, stroke, myocardial infarction, heart failure, aneurysms (e.g., aortic aneurysms), peripheral arterial disease, cardiac injury (e.g., heart distention or hypertrophy), and other cardiovascular-related diseases, disorders and/or conditions. Furthermore, hypertension has been shown to be an important risk factor for cardiovascular morbidity and mortality, accounting for or constituting 62% of all strokes and 49% of all heart disease cases.
The refractory hypertension (Treatment resistant hypertension, TRH) is a type of hypertension which is not up to standard for more than 1 month by applying a reasonable and tolerable sufficient quantity of 3 or more than 3 antihypertensive drugs (including diuretics) on the basis of improving life style, or can be effectively controlled by taking 4 or more than 4 antihypertensive drugs. The prevalence of TRH disease in hypertensive patients is approximately 5% to 30%. It is a difficulty in hypertension treatment, and TRH is becoming an increasingly common clinical problem as the population ages and diseases such as obesity, sleep apnea-hypopnea syndrome, chronic kidney disease, etc. increase. Compared with patients with the blood pressure easily reaching the standard, the risk of myocardial infarction, apoplexy and death of patients with intractable hypertension is increased by 20-60%.
Therefore, there is an urgent need to develop a medicament effective in treating hypertension.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides an siRNA for inhibiting the expression of angiotensinogen and application thereof, and the siRNA can inhibit the expression of the angiotensinogen and effectively treat related diseases caused by the excessive angiotensinogen.
The present invention has been completed based on the following findings by the inventors:
renin (AGT), an alpha-2-globulin synthesized in the liver, is a precursor of angiotensin, also known as renin substrate. The renin-angiotensin system (RAS) is a hormonal system that regulates blood pressure, fluid and electrolyte balance, and systemic vascular resistance. When renal blood flow decreases, glomerular cells in the kidney convert the precursor renin (already present in the blood) to renin and secrete it directly into the circulation. Plasma renin converts liver released angiotensinogen to angiotensin I. Angiotensin Converting Enzyme (ACE) on the surface of vascular endothelial cells converts angiotensin I to angiotensin II, principally pulmonary angiotensin II. Angiotensin ii is an effective vasoconstrictor peptide that narrows the blood vessel, resulting in an increase in blood pressure. Angiotensin ii also stimulates the secretion of aldosterone by the adrenal cortex. Aldosterone causes the tubules to increase sodium reabsorption, resulting in water reabsorption into the blood, while simultaneously leading to potassium excretion (maintenance of electrolyte balance). This increases the volume of extracellular fluid in the body, which also increases blood pressure. If the RAS is abnormally active, the blood pressure may be too high.
Currently, the primary control of hypertension is by treatment with blood pressure lowering drugs. Such as angiotensin converting enzyme inhibitors, cytarabine and renin inhibitors, which can interrupt different steps in the RAS system to improve blood pressure, are one of the main methods of controlling the deleterious effects of hypertension, heart failure, renal failure and diabetes. Despite the large number of anti-compression drugs available for the treatment of hypertension, more than two-thirds of subjects cannot be controlled with one anti-compression drug, but need two or more anti-compression drugs selected from different drug classes. Furthermore, there are some adverse effects of existing drugs, which further reduce the number of subjects with controlled blood pressure, as compliance and side effects increase with increasing medication.
Based on this, in one aspect of the present invention, the present invention proposes an siRNA. According to an embodiment of the present invention, the siRNA comprises a sense strand and an antisense strand, the antisense strand comprising a region of complementarity that is complementarily paired with the sense strand, wherein the sense strand is selected from the group consisting of nucleotide sequences that differ by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID NO: 1-SEQ ID NO:52, SEQ ID NO: 105-110, and the antisense strand is selected from the group consisting of nucleotide sequences that differ by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID NO: 53-SEQ ID NO:104, SEQ ID NO: 111-SEQ ID NO:116. The inventor obtains the above-mentioned suitable small interfering RNA (siRNA) through experimental design, can specifically reduce the synthesis of AGT by hepatic cells, meanwhile avoid off-target effect, and the siRNA forms a silencing complex (RNA-induced silencing complex, RISC) to complementarily pair with the sequence of mRNA of a target gene (AGT), so that the mRNA of the AGT is degraded, the expression of the AGT is inhibited, the blood pressure in the body is reduced, and then the related diseases caused by the excessive angiotensinogen such as hypertension are effectively prevented or treated.
In the present invention, the "difference of not more than 2 nucleotides" means that there may be a difference of 1 or 2 nucleotides from the target nucleotide sequence, including, but not limited to, deletion of nucleotides, insertion of nucleotides (which may be inserted to the 3 '-end, the 5' -end or between any two nucleotides of the nucleotide sequence), substitution of nucleotides, and the like.
According to an embodiment of the present invention, the above siRNA may further include at least one of the following additional technical features:
according to an embodiment of the invention, the sense strand comprises, in addition to at least one of SEQ ID NO:1 to SEQ ID NO:52 and SEQ ID NO:105 to SEQ ID NO:110 shown in Table 1, a continuous nucleotide sequence differing from the sense strand shown in Table 1 by 1, 2 nucleotides.
According to an embodiment of the invention, the antisense strand comprises, in addition to at least one of SEQ ID NO:53 to SEQ ID NO:104 and SEQ ID NO:111 to SEQ ID NO:116 shown in Table 1, a continuous nucleotide sequence differing from the antisense strand shown in Table 1 by 1, 2 nucleotides.
Table 1: nucleotide sequence of siRNA
According to an embodiment of the invention, the sense strand comprises one of the following nucleotide sequences, or a nucleotide sequence differing therefrom by no more than 2 nucleotides: SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109 and 4SEQ ID NO. 110. And/or, the antisense strand comprises one of the following nucleotide sequences, or a nucleotide sequence differing therefrom by no more than 2 nucleotides: SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 91, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115 and 4SEQ ID NO 116.
According to an embodiment of the invention, the length of the complementarity region is at least 19bp.
According to an embodiment of the invention, the length of the complementarity region is 19-23 bp.
According to an embodiment of the present invention, one or more overhanging bases are added to the 3' -end of the sense strand and/or antisense strand of the siRNA.
According to an embodiment of the invention, the overhanging bases are dA, dT, dG and/or dU.
According to an embodiment of the invention, the siRNA comprises at least one modified nucleotide.
According to an embodiment of the invention, the modification comprises at least one selected from the group consisting of phosphorylation, methoxylation, thio, cholesterol, vitamins, folic acid, cholic acid and PEG.
According to an embodiment of the invention, the modified nucleotide is selected from at least one of the following: a 5 '-phosphorothioate diester modified nucleotide, a 5' -methylated cytosine nucleotide, a 2 '-O-methyl modified nucleotide, a 2' -O-2-methoxyethyl modified nucleotide, a 2 '-fluoro modified nucleotide, a 3' -nitrogen substituted modified nucleotide, a 2 '-deoxy-2' -fluoro modified nucleotide, a 2 '-deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2' -amino modified nucleotide, a morpholino nucleotide, a polypeptide nucleotide, an phosphoramidate, and a nucleotide comprising a non-natural base.
When one modified nucleotide is selected from the group consisting of a 2 '-alkyl modified nucleotide and a 5' -phosphorothioate diester modified nucleotide, the modified nucleotide has both a 2 '-alkyl modification and a 5' -phosphorothioate diester modification.
As will be appreciated by those skilled in the art, ribonucleotide molecules are composed of phosphate, ribose and bases. In this context, a "modified nucleotide" refers to the presence of a change in the group attached to a different C atom on the ribose, a change in the phosphate group, or a deletion of the base as compared to A, U, C or G nucleotides.
According to an embodiment of the invention, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides, each independently selected from at least one of a 2' -methoxy modified nucleotide, a 2' -fluoro modified nucleotide and a 5' -phosphorothioate diester modified nucleotide.
According to an embodiment of the invention, the 2' -fluoro modified nucleotide is present in the sense strand and/or the antisense strand at a position consisting of: 2 '-fluoro modified nucleotides at positions 9, 10 and 11 of the 5' -terminal nucleotide of the sense strand as a starting point; and/or, the nucleotide at the 5 '-end of the antisense strand is a 2' -fluoro modified nucleotide at the 2 nd, 6 th, 14 th and 16 th positions of the starting point.
Illustratively, the nucleotide at position 1, where the nucleotide at the 5 'end of the sense strand is the starting point, is a 2' -methoxy modified nucleotide.
Illustratively, the nucleotide at position 1 of the 5 'terminus of the antisense strand is the starting point, the nucleotides at positions 1 and 2 of the 3' terminus of the antisense strand are both a 2 '-methoxy modified nucleotide and a 5' -phosphorothioate diester modified nucleotide.
According to an embodiment of the invention, the siRNA comprises one or more of the following groups:
wherein, in the sense strand and the antisense strand, aM, gM, cM and uM are respectively 2 '-O-methyl a, 2' -O-Me G, 2'-O-Me C and 2' -O-Me U; aF. gF, cF and uF are 2 '-fluoro A, 2' -fluoro C, 2 '-fluoro G and 2' -fluoro U, respectively; * Indicating that the nucleotide has a 5' -phosphorothioate modification.
In yet another aspect of the invention, the invention provides an siRNA conjugate. According to an embodiment of the invention, the siRNA conjugate comprises: the siRNA and a ligand of the foregoing, wherein the siRNA is covalently linked to the ligand. The inventor finds through experiments that in the siRNA conjugate, the ligand is added, so that the degradation of AGT mRNA can be further improved, the synthesis of AGT by hepatic cells is specifically reduced, the blood pressure in a human body is reduced, and related diseases caused by excessive angiotensinogen, such as hypertension, can be effectively prevented or treated.
Ligand refers to a class of substances having a profile, targeting, or lifetime that can alter siRNA, e.g., a ligand that provides enhanced affinity for a selected target (e.g., a molecule, cell, or cell type, compartment (e.g., cell or organ compartment, body tissue, organ, or region)) as compared to a species in which the ligand is not present. The ligand may be a natural protein (e.g., human Serum Albumin (HSA), etc.), a carbohydrate (e.g., dextran, chitosan, etc.), or a lipid; recombinant or synthetic molecules, such as synthetic polymers, are also possible. Preferably, the ligand does not participate in the pairing of the sense strand and the antisense strand in the siRNA.
According to an embodiment of the invention, the ligand is linked to the sense strand in the siRNA.
According to an embodiment of the invention, the ligand is linked to the 5 'end or the 3' end of the sense strand in the siRNA via a phosphoester bond or a phosphorothioate bond.
According to an embodiment of the present invention, the ligand is a GalNAc targeting compound;
according to an embodiment of the present invention, the GalNAc targeting compound includes at least one selected from 1043, 1046 and 1048;
in yet another aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the siRNA as described above; or the aforementioned siRNA conjugates. The pharmaceutical composition according to the embodiment of the invention can specifically reduce the synthesis of AGT by liver cells, reduce blood pressure in a human body, and effectively prevent or treat related diseases caused by excessive angiotensinogen, such as hypertension.
It is noted that the pharmaceutical compositions herein may include one or more of the foregoing siRNAs or siRNA conjugates.
According to an embodiment of the present invention, the pharmaceutical composition further comprises: pharmaceutically acceptable auxiliary materials.
In a further aspect of the invention, the invention provides the use of the aforementioned siRNA, the aforementioned siRNA conjugate or the aforementioned pharmaceutical composition for the preparation of a medicament for inhibiting the expression of angiotensinogen.
In a further aspect of the invention, the invention provides the use of the aforementioned siRNA, the aforementioned siRNA conjugate or the aforementioned pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of a related disease caused by an excessive amount of angiotensinogen.
According to an embodiment of the invention, the relevant disease caused by the excessive angiotensinogen is selected from hypertension.
According to an embodiment of the invention, the hypertension comprises borderline hypertension, primary hypertension, secondary hypertension, hypertension risk, hypertension emergency status, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, intractable hypertension, paroxysmal hypertension, renal vascular hypertension, gordbat's hypertension, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension and unstable hypertension.
In yet another aspect of the invention, the invention provides a method for preventing and/or treating diseases associated with excessive angiotensinogen. According to an embodiment of the invention, the method comprises: administering to a subject a pharmaceutically acceptable amount of the aforementioned siRNA, the aforementioned siRNA conjugate, or the aforementioned pharmaceutical composition. According to the embodiment of the invention, the method can effectively prevent and/or treat related diseases caused by the excessive angiotensinogen.
The effective amount of the siRNA, siRNA conjugate or pharmaceutical composition of the present invention may vary depending on the mode of administration, the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life etc.; the severity of the disease to be treated in the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, separate doses may be administered several times a day, e.g., as four times a day, three times a day, twice a day, once a day, or once every other day, or several times a day, may be proportionally reduced by the urgent need for the treatment of the condition.
The administration to the subject may be by any suitable route known in the art, including but not limited to: oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal and topical (including buccal and sublingual), preferably intravenous.
According to embodiments of the invention, the route of administration of the method is intravenous.
According to an embodiment of the invention, the relevant disease caused by the excessive angiotensinogen is selected from hypertension.
According to an embodiment of the invention, the hypertension comprises borderline hypertension, primary hypertension, secondary hypertension, hypertension risk, hypertension emergency status, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, intractable hypertension, paroxysmal hypertension, renal vascular hypertension, gordbat's hypertension, pulmonary arterial hypertension, portal hypertension, systemic venous hypertension, systolic hypertension and unstable hypertension.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
As used herein, the term "small interfering RNA (Small interfering RNA; siRNA)" is a double-stranded RNA of 17 to 25 nucleotides in length, comprising a sense strand and an antisense strand. siRNA mediates RNA transcript targeted cleavage of the RISC pathway by forming silencing complexes (RNA-induced silencing complex, RISC). Specifically, siRNA directs the specific degradation of mRNA sequences through known RNA interference (RNAi) processes, inhibiting translation of mRNA into protein. For example, the siRNA can modulate (e.g., inhibit) expression of AGT in the cell.
As used herein, the term "antisense strand (or guide strand)" includes a region that is substantially complementary to a target sequence, such as AGT mRNA. "sense strand (or" follower strand) "means that it contains an iRNA strand that is substantially complementary to the antisense strand. The term "substantially complementary" refers to complete complementarity or at least partial complementarity, e.g., the antisense strand is complete complementarity or at least partial complementarity to a target sequence. In the case of partial complementarity, the mismatch may be present within the interior or terminal region of the molecule, wherein the most tolerated mismatch is present within the terminal region, e.g., within 5, 4, 3, or 2 nucleotides of the 5 'end and/or 3' end of the iRNA.
It is noted that "at least partially substantially complementary" of the antisense strand to the mRNA means that the antisense strand has a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., the mRNA encoding AGT). Alternatively, if a polynucleotide is substantially non-intermittently complementary to a portion of the mRNA encoding AGT, the antisense strand is complementary to at least a portion of the AGT mRNA.
In this context, the term "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the AGT gene, including mRNA that is the RNA processing product of the primary transcript.
Herein, the term "inhibiting the expression of an AGT gene" includes any level of inhibition of AGT gene, e.g., at least partial inhibition of AGT gene expression, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%. Wherein the expression of the AGT gene, e.g.mRNA level of AGT or AGT protein level, can be evaluated based on the level of any variable related to the expression of the AGT gene. Inhibition may be assessed by a decrease in the absolute or relative level of one or more of these variables as compared to a control level. The control level may be any type of control level utilized in the art, e.g., a baseline level prior to administration, or a level determined from a similar subject, cell, or sample that has not been treated or treated with a control (e.g., a buffer-only control or an inactive agent control).
In this context, the term "AGT" includes mRNA of AGT or its complete coding sequence, such as human AGT, which can be found in, for example, genBank accession No. gi:188595658 (NM-000029.3). Other examples of mRNA sequences for AGT are obtained using publicly available databases, such as the genomic project website of GenBank and UniProt. The term "AGT" may also refer to naturally occurring DNA sequence variants of the AGT gene, such as Single Nucleotide Polymorphisms (SNPs) in the AGT gene.
The terms "expression vector" and "construct" are used interchangeably herein and are capable of delivering one or more genes or sequences of interest into a host cell, and preferably expressing the genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.
As used herein, "pharmaceutical composition" may refer to a composition useful for the treatment of a disease, as well as in vitro culture experiments of cells. For the treatment of diseases, the term "pharmaceutical composition" generally refers to a unit dosage form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of combining the active ingredient with adjuvants that constitute one or more adjunct ingredients. Typically, the compositions are prepared by uniformly and sufficiently combining the active siRNA or siRNA conjugate with a liquid adjuvant, a finely divided solid adjuvant, or both.
As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and/or toxicologically compatible with the other ingredients comprising the formulation and/or the mammal being treated therewith. Preferably, the term "pharmaceutically acceptable" as used herein refers to use in animals, particularly humans, approved by the federal regulatory agency or a state government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia.
Herein, the term "pharmaceutically acceptable adjuvant" is art-recognized and includes pharmaceutically acceptable materials, compositions or carriers suitable for administration of the siRNA or siRNA conjugates of the present disclosure to a mammal. The adjuvant includes a liquid or solid filler, diluent, excipient, solvent or encapsulating material that participates in carrying the subject substance or transferring it from one organ or body part to another organ or body part. The excipients must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the patient. Some examples of materials that may be used as pharmaceutically acceptable excipients include: sugars such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cotton seed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; ringer's solution; ethanol; phosphate buffer; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preserving and antioxidant agents, may also be present in the compositions.
In addition to any conventional adjuvants, the scope of incompatibility with the siRNA or siRNA conjugate of the invention, e.g., any adverse biological effects produced or interactions with any other component of the pharmaceutically acceptable composition in a deleterious manner, is also contemplated by the present invention.
Pharmaceutical compositions of the present disclosure include formulations suitable for oral, nasal, topical, buccal, sublingual, rectal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. The amount of active ingredient that can be combined with the adjuvant materials to prepare a single dosage form is generally that amount of siRNA or siRNA conjugate that produces a therapeutic effect. Generally, the amount is from about 1% to about 99% active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%, in one percent.
In this context, the term "treatment" is intended to mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing the disease or symptoms thereof, and/or may be therapeutic in terms of partially or completely curing the disease and/or adverse effects caused by the disease. As used herein, "treating" encompasses diseases in mammals, particularly humans, including: (a) Preventing the occurrence of a disease or disorder in an individual susceptible to the disease but not yet diagnosed with the disease; (b) inhibiting disease, e.g., arresting disease progression; or (c) alleviating a disease, e.g., alleviating symptoms associated with a disease. As used herein, "treating" encompasses any administration of an siRNA, siRNA conjugate or drug to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including, but not limited to, administration of a drug comprising an siRNA or siRNA conjugate as described herein to an individual in need thereof.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: synthesis of siRNA
According to standard oligonucleotide solid phase synthesis protocols, commercially available 5'-DMT-2' -TBDMS-rU phosphoramidite monomers, 5'-DMT-2' -TBDMS-rA (Bz) phosphoramidite monomers, 5'-DMT-2' -TBDMS-rC (Ac) phosphoramidite monomers, 5'-DMT-2' -TBDMS-rG (iBu) phosphoramidite monomers are used. RNA was synthesized on a scale of 500 nmol. Phosphoramidite solutions were prepared at a concentration of 50mM, and 0.3M Benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During the synthesis, 0.1M oxidizing reagent (I 2 : pyridine: THF: water) converts trivalent phosphorus into a pentavalent phosphorus stable phosphate backbone. After synthesis is complete, the sequence is ammonolyzed from the solid support and allowed to settle. 2 'O-tert-butyldimethylsilyl protecting group was removed from 2' with triethylamine trihydrofluoride.
For the RNA sequence after the synthesis, ammonia was used at 55℃for the synthesis: ammonia hydrolysis of methylamine=1:1 for 40min, after the ammonia hydrolysis is completed, CPG powder is removed, and the supernatant is taken and pumped out. Adding triethylamine and hydrofluoric acid: triethylamine: nmp=6:4:3 protecting group removal agent, reacted at 60 ℃ for 2h according to 1: adding n-butanol at a ratio of 5, standing at-20deg.C for 30min, and centrifuging to obtain precipitate. RNase-free water was added for dissolution and purification by reverse phase chromatography (0.1M TEAA and acetonitrile). The purified sample was desalted by ultrafiltration with PBS. And annealing to obtain siRNAs (52 siRNAs in this example, respectively named NPD011-1 to NPD 011-52), and the inventors verified the obtained siRNAs, and found that the target siRNAs were successfully prepared, see the following table:
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example 2: in vitro cell model (Hep 3B cells) for testing the Activity of Small interfering nucleic acids (siRNAs)
Cell treatment: taking human liver cancer Hep 3B cells (Hep 3B cells for short) from Shanghai cell bank of China academy of sciences, and waiting for confluency rate of Hep 3B cell lines>70%, cell plating was performed at a rate of 2X 10 5 Cells/well were plated in 12-well plates, each well was added with 900. Mu.l of DMEM medium (Gibco, US) containing 10% Fetal Bovine Serum (FBS) (Gibco, US) and placed at 37℃in 5% CO 2 Is cultured in an incubator.
Preparing transfection reagent: preparing an siRNA mother liquor containing the siRNA prepared in the embodiment 1, and diluting the siRNA mother liquor by adopting DEPC (diethyl pyrocarbonate ) water to obtain a 10 mu M siRNA system; diluting 10 mu M siRNA system by using Opti-MEM to obtain 0.2 mu M siRNA system solution, blowing and sucking for 3-5 times, and mixing uniformly. Diluting 0.2 mu M siRNA by adopting Opti-MEM to obtain 0.002 mu M siRNA system solution, blowing and sucking for 3-5 times, and mixing uniformly. Mu.l RNAiMAX transfection reagent (Invitrogen, 13778150) was diluted with 50. Mu.l Opti-MEM and mixed by pipetting 3-5 times. Mixing the siRNA diluent with the RNAiMAX diluent according to the standard transfection procedure of RNAiMAX, blowing and sucking for 3-5 times, mixing uniformly, and standing for 10min at room temperature to obtain the transfection complex.
After 24h plating of Hep 3B cells in 12-well plates, the transfection complexes containing different siRNAs (each siRNA transfected into Hep 3B cells at 0.1nM or 10nM concentration, respectively) were added to different 12-well plates, and placed in 37℃and 5% CO 2 Is transfected in the incubator of (a). After 24h of transfection, total RNA of Hep 3B cells in 12-well plates was extracted as an experimental group for 52 total groups (see for specific see nomenclature see siRNA serial number nomenclature of Table 1). The negative control group (noted as Blank) differs from the experimental group only in that the transfection complex of the negative control group does not contain any siRNA.
Based on the determined RNA concentration values, 500ng RNA was taken for subsequent experiments. cDNA samples were prepared using a reverse transcription kit (HiScript RII 1st Strand cDNA Synthesis Kit R211-01/02, northenzan) according to standard methods. The required volume of RNA solution was determined using RNase-free ddH 2 The O was replenished to 3. Mu.l. According to the recommended reagent dosage of the kit, a mixture of reaction solutions is prepared as follows: 2 xRT mix 5. Mu.l, hiScript II Enzyme Mix. Mu.l, random hexamers (50 ng/ul) 0.5ul, oligo (dT) 23VN (50. Mu.M) 0.5. Mu.l per reaction. PCR (A300, langmuir) was performed by reverse transcription procedure, set up according to the following reaction conditions: 25 ℃ for 5min,50 ℃ for 15min and 85 ℃ for 2min.
Post-reverse transcription cDNA samples were diluted 20-fold (190 ulH O added) and QPCR using SYBR green (ChamQ Universal SYBR qPCR Master Mix Q-02/03, norpran) was used to quantify the change in gene expression levels of interest. The reaction system is as follows: 2X ChamQ Universal SYBR qPCR Master Mix. Mu.l, forword Primer (10. Mu.M) 0.2. Mu.l, reverse Primer (10. Mu.M) 0.2. Mu.l, ddH2O 2.6. Mu.l, cDNA template 2. Mu.l. RT-PCR reactions were performed on-machine using a QPCR instrument (Analytik Jena qTOWER 384G) according to the following procedure: 95 ℃ for 30s;95℃10s,60℃30s 40 cycles, dissolution profile program. As a result of the experiment, the delta deltaCT method was used to analyze the target gene expression change of the test samples and the control samples (siNC group). Among them, PCR amplification primers of the internal reference genes PPIB and AGT are shown in Table 2.
Table 2: amplification primers for PPIB and AGT
The inhibition ratio of the expression level of small interfering nucleic acid (siRNA) mRNA was calculated as follows: inhibition ratio = [1- (expression amount of AGT mRNA in experimental group/expression amount of PPIB mRNA in experimental group)/(expression amount of AGT mRNA in negative control group/expression amount of PPIB mRNA in negative control group) ]x100%. The activity of each siRNA sequence is shown in Table 3 below.
Table 3: activity of the respective siRNA sequences
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Example 3: siRNA preparation of modified and conjugated GalNAc vector:
according to standard oligonucleotide solid phase synthesis protocols, commercially available 2'-OMe-U phosphoramidite monomers, 2' -OMe-a (Bz) phosphoramidite monomers, 2'-OMe-C (Ac) phosphoramidite monomers, 2' -OMe-G (ibu) phosphoramidite monomers, 2'-F-dU phosphoramidite monomers, 2' -F-Ac-dC phosphoramidite monomers, 2'-F-ibu-dG phosphoramidite monomers, 2' -F-Bz-dA phosphoramidite monomers, and synthetic GalNAc phosphoramidite monomers (compound 1048, compound 1048 were prepared according to the methods described in patent CN 202110008013.3). RNA was synthesized on a scale of 500nmol, a 50mM RNA phosphoramidite solution and a 200mM GalNAc monomer solution were prepared, and a 0.3M Benzylthiotetrazole (BTT) acetonitrile solution was used as an activator. During the synthesis, the trivalent phosphorus is converted into a pentavalent phosphorus stable phosphate backbone using 0.1M oxidizing reagent (I2: pyridine: THF: water). After synthesis is complete, the sequence is ammonolyzed from the solid support and allowed to settle. Subsequent purification was the same as for siRNA synthesis in example 1. The inventors validated the resulting conjugate (compound 1048 attached to the 5' end of the sense strand) and the mass spectrum data of each modified siRNA sequence is shown in table 4.
Table 4: nucleotide sequence of siRNA
aM represents 2 '-O-methyl adenine nucleoside, uM represents 2' -O-methyl uracil nucleoside, gM represents 2 '-O-methyl guanine nucleoside, cM represents 2' -O-methyl cytosine nucleoside, aF represents 2 '-fluoro adenine nucleoside, uF represents 2' -fluoro uracil nucleoside, gF represents 2 '-fluoro guanine nucleoside, cF represents 2' -fluoro cytosine nucleoside, two nucleotides (or compound 1048 and nucleotide) are connected by phosphorothioate diester bonds, and no symbol is present between directly adjacent nucleotides.
Example 4: in vitro cell model (Hep 3B cells) testing of the Activity of the conjugates
Human liver cancer Hep 3B cells (Shanghai cell bank of China academy of sciences) were cultured in DMEM medium (Gibco, US) supplemented with 10% fbs (Gibco, US) at 37 ℃ and 5% CO 2 Culturing in an incubator (il 60, thermo Fisher). The transfection experiments were performed on the same day, cells were digested with 0.25% trypsin (Trysin) (Gibco, US), counted and seeded at a density of 20 ten thousand per well in 12-well plates. After 24h, the conjugates prepared in example 2 were transfected according to standard procedures of RNAiMAX reagent instructions, the final concentration of siRNA in the different conjugates was 10nM/1nM/0.5nM/0.25nM/0.1nM/0.05nM/0.01nM, respectively. The transfected group was negative control with siNC (sense strand 5'-UUCUCCGAACGUGUCACGUTT-3' (SEQ ID NO: 121), antisense strand 5'-ACGUGACACGUUCGGAGAATT-3' (SEQ ID NO: 122)).
After 24h transfection, total RNA of the cells was extracted and the expression of AGT mRNA sequence in the cells was detected by qRT PCR, the reference gene was PPIB, for specific detection procedures, see example 1. The activity of each modified siRNA is shown in Table 5.
Table 5: activity of the respective siRNA sequences
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. An siRNA comprising a sense strand and an antisense strand, said antisense strand comprising a region of complementarity complementary to said sense strand, wherein said sense strand is selected from the group consisting of nucleotide sequences differing by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID nos. 1-52, 105-110, and said antisense strand is selected from the group consisting of nucleotide sequences differing by NO more than 2 nucleotides from the nucleotide sequence of each of SEQ ID nos. 53-104, 111-116.
2. The siRNA of claim 1, wherein the sense strand comprises one of the following nucleotide sequences, or a nucleotide sequence differing therefrom by no more than 2 nucleotides:
SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 39, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 48, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109 and SEQ ID NO. 110. And/or
The antisense strand comprises one of the following nucleotide sequences, or a nucleotide sequence differing therefrom by no more than 2 nucleotides:
SEQ ID NO 56, SEQ ID NO 57, SEQ ID NO 58, SEQ ID NO 63, SEQ ID NO 65, SEQ ID NO 66, SEQ ID NO 71, SEQ ID NO 73, SEQ ID NO 75, SEQ ID NO 76, SEQ ID NO 77, SEQ ID NO 80, SEQ ID NO 81, SEQ ID NO 82, SEQ ID NO 83, SEQ ID NO 84, SEQ ID NO 85, SEQ ID NO 86, SEQ ID NO 91, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 100, SEQ ID NO 103, SEQ ID NO 104, SEQ ID NO 111, SEQ ID NO 112, SEQ ID NO 113, SEQ ID NO 114, SEQ ID NO 115 and 4SEQ ID NO 116.
Optionally, the siRNA comprises at least one modified nucleotide;
optionally, the modification comprises at least one selected from the group consisting of phosphorylation, methoxylation, thio, cholesterol, vitamins, folic acid, cholic acid, and PEG;
optionally, the modified nucleotide is selected from at least one of:
a 5 '-phosphorothioate diester modified nucleotide, a 5' -methylated cytosine nucleotide, a 2 '-O-methyl modified nucleotide, a 2' -O-2-methoxyethyl modified nucleotide, a 2 '-fluoro modified nucleotide, a 3' -nitrogen substituted modified nucleotide, a 2 '-deoxy-2' -fluoro modified nucleotide, a 2 '-deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2' -amino modified nucleotide, a morpholino nucleotide, a polypeptide nucleotide, an phosphoramidate, a nucleotide comprising a non-natural base;
optionally, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides, each independently selected from at least one of a 2' -methoxy modified nucleotide, a 2' -fluoro modified nucleotide, and a 5' -phosphorothioate modified nucleotide;
optionally, in the sense strand and/or the antisense strand, the 2' -fluoro modified nucleotide is present at a position consisting of:
2 '-fluoro modified nucleotides at positions 9, 10 and 11 of the 5' -terminal nucleotide of the sense strand as a starting point; and/or
2 '-fluoro modified nucleotides at positions 2, 6, 14 and 16 of the 5' -terminal nucleotide of the antisense strand as a starting point;
optionally, the siRNA comprises one or more of the following:
wherein, in the sense strand and the antisense strand, aM, gM, cM and uM are respectively 2 '-O-methyl a, 2' -O-Me G, 2'-O-Me C and 2' -O-Me U; aF. gF, cF and uF are 2 '-fluoro A, 2' -fluoro C, 2 '-fluoro G and 2' -fluoro U, respectively; * Indicating that the nucleotide has a 5' -phosphorothioate modification.
3. An siRNA conjugate comprising: the siRNA and ligand of claim 1 or 2, wherein said siRNA is covalently linked to said ligand.
4. The siRNA conjugate of claim 3, wherein the ligand is linked to the sense strand in the siRNA;
optionally, the ligand is linked to the 5 'end or the 3' end of the sense strand in the siRNA via a phosphoester bond or a phosphorothioate bond.
5. The siRNA conjugate of claim 3 or 4, wherein the ligand is a GalNAc targeting compound.
6. The siRNA conjugate according to claim 5, wherein said GalNAc targeting compound comprises at least one selected from 1043, 1046 and 1048;
7. a pharmaceutical composition comprising: the siRNA of claim 1 or 2; or the siRNA conjugate of any one of claims 3 to 6;
optionally, further comprising: pharmaceutically acceptable auxiliary materials.
8. Use of the siRNA of claim 1 or 2, the siRNA conjugate of any one of claims 3 to 6 or the pharmaceutical composition of claim 8 in the manufacture of a medicament for inhibiting expression of angiotensinogen.
9. Use of the siRNA of claim 1 or 2, the siRNA conjugate of any one of claims 3 to 6 or the pharmaceutical composition of claim 8 for the preparation of a medicament for the prevention and/or treatment of a related disease caused by an excessive amount of angiotensinogen.
10. The use according to claim 9, wherein the related diseases caused by the excessive angiotensinogen are selected from the group consisting of hypertension;
optionally, the hypertension includes borderline hypertension, primary hypertension, secondary hypertension, hypertension risk, hypertension urge status, isolated systolic and diastolic hypertension, pregnancy-related hypertension, diabetic hypertension, refractory hypertension, paroxysmal hypertension, renal vascular hypertension, gordbat hypertension, pulmonary arterial hypertension, portal venous hypertension, systemic venous hypertension, systolic hypertension, and unstable hypertension.
CN202310460609.6A 2022-07-08 2023-04-26 SiRNA for inhibiting expression of angiotensinogen and application thereof Pending CN117363614A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117568350A (en) * 2024-01-15 2024-02-20 苏州时安生物技术有限公司 Double-stranded RNA for regulating expression of angiotensinogen gene, conjugate, pharmaceutical composition and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117568350A (en) * 2024-01-15 2024-02-20 苏州时安生物技术有限公司 Double-stranded RNA for regulating expression of angiotensinogen gene, conjugate, pharmaceutical composition and application thereof
CN117568350B (en) * 2024-01-15 2024-04-30 苏州时安生物技术有限公司 Double-stranded RNA for regulating expression of angiotensinogen gene, conjugate, pharmaceutical composition and application thereof

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