CN111378656B - Nucleic acid for inhibiting Ebola virus, pharmaceutical composition containing nucleic acid and application of pharmaceutical composition - Google Patents
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Abstract
A siRNA for inhibiting Ebola virus gene expression, which comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, and the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reversely complemented to form a double-stranded region. The length of the nucleotide sequence 1 and the length of the nucleotide sequence 2 are both 19 nucleotides, the nucleotide sequence 2 is at least partially complementary with a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence with the same length as the nucleotide sequence 2 in the Ebola virus mRNA. According to the direction from the 5 'end to the 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence 1 are fluorine modified nucleotides, and the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions of the nucleotide sequence 2 are fluorine modified nucleotides. The siRNA and the pharmaceutical composition containing the siRNA have good activity of inhibiting Ebola virus gene expression.
Description
Technical Field
The present disclosure relates to a siRNA, a pharmaceutical composition containing the siRNA and their uses. Specifically, the disclosure relates to a siRNA for inhibiting Ebola virus gene expression, a pharmaceutical composition containing the siRNA as an active ingredient, and applications of the siRNA and the pharmaceutical composition in preparation of medicines for preventing and/or treating Ebola virus diseases.
Background
Ebola virus disease is an acute hemorrhagic infection caused by Ebola virus (EBOV) of filoviridae. It is mainly transmitted through the blood and excrement of patients, and is clinically manifested by acute onset fever, myalgia, bleeding, rash and liver and kidney function damage.
EBOV genus filoviridae genus filovirus, which is a single-stranded non-segmented negative-strand RNA virus; it is divided into four subtypes: zaire Ebola Virus (ZEBOV), has the strongest virulence, with a mortality rate of up to 90% after human infection; sudan Ebola Virus (Sudan Ebola Virus, SEBOV) with a mortality rate of 50% after infection; cote d' Ivore Ebola Virus (CEBOV) and Leston Ebola Virus (Reston Ebola Virus, REBOV) are lethal to non-human primates and less virulent to humans.
The EBOV genome is a negative-strand RNA 18.9kb in length and contains 7 open reading frames arranged in the order: 3 '-NP-VP 35-VP40-GP-VP30-VP 24-L-5', each product being encoded by a separate mRNA. The L protein and VP35 together constitute a polymerase, completing replication of Ebola RNA. Both are ideal therapeutic targets, where inhibition completely prevents RNA synthesis, and both proteins are not present in mammalian cells. VP24 may also be a therapeutic target as it inhibits the type I interferon response of the host.
If the gene expression of the virus can be silenced from the gene level, and the generation and the replication of the Ebola virus can be blocked, thereby fundamentally reducing the virus metabolism and the infection to cells, the gene expression vector is undoubtedly the most ideal Ebola virus treatment means. The small interfering RNA (siRNA) can suppress or block the expression of any target gene of interest in a sequence-specific manner based on the RNA interference (RNAi) mechanism, thereby achieving the purpose of treating diseases.
siRNA has been previously studied as a pharmaceutically active ingredient for treating ebola virus disease, however, siRNA itself has poor stability and is easily degraded by nuclease in vivo (in particular, siRNA first enters the blood circulation system of the body after systemic administration in vivo, and blood is rich in endogenous nuclease). To overcome this obstacle, one of the approaches is to chemically modify siRNA to improve the stability of siRNA in blood. To date, the person skilled in the art has conducted extensive studies on siRNA, however, the degradation process and mechanism of siRNA in blood is still poorly understood, and the selection of modification mode and modification site is still an empirically defined process that must be verified by repeated experiments.
Therefore, for siRNA with different nucleotide sequences, the skilled person still needs to specifically analyze the siRNA, find out a specific modification scheme suitable for the siRNA sequence, in order to develop modified siRNA with good stability, good biological activity or and low cytotoxicity.
Disclosure of Invention
The object of the present disclosure is to provide a siRNA molecule capable of selectively and effectively inhibiting expression of ebola virus genes, and a pharmaceutical composition comprising the siRNA as an active ingredient, which is effective in preventing and/or treating pathological conditions and diseases caused by ebola virus infection, such as acute onset fever, myalgia, bleeding rash, and liver and kidney function impairment.
The inventors found that, surprisingly, the sirnas provided by the present disclosure have significantly higher ebola virus gene suppression activity and exhibit excellent blood stability.
Therefore, the present disclosure provides a siRNA that can specifically target an ebola virus gene to significantly inhibit ebola virus EBOV gene expression.
The siRNA provided by the present disclosure contains a sense strand and an antisense strand, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are both 19 nucleotides in length, the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence 2 is at least partially reverse-complementary to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in ebovr mrna with the same length as the nucleotide sequence 2; in the direction from 5 'end to 3' end, the nucleotides at positions 7, 8 and 9 of the nucleotide sequence 1 are fluorine-modified nucleotides; the first nucleotide at the 5 'end of the nucleotide sequence 2 is the first nucleotide at the 5' end of the antisense strand, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluoro-modified nucleotides.
In some embodiments, the present disclosure provides an siRNA composition comprising a first siRNA and a second siRNA; the first siRNA is selected from the siRNA as described above, and the first nucleotide sequence in the first siRNA is a nucleotide sequence in EBOV VP 35; the second siRNA is the siRNA described above, and the first nucleotide sequence in the second siRNA is a nucleotide sequence in EBOVL mRNA.
In some embodiments, the present disclosure provides a pharmaceutical composition comprising an active ingredient that is an siRNA as described above or an siRNA composition as described above and a pharmaceutically acceptable carrier.
In one embodiment, the weight ratio of the effective ingredient to the pharmaceutically acceptable carrier is 1 (1-500), and may be, for example, 1 (1-50).
In some embodiments, the present disclosure provides the use of an siRNA as described above, an siRNA composition as described above, and/or a pharmaceutical composition as described above, in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by an infection by the ebola virus.
In some embodiments, the present disclosure provides a kit comprising an siRNA as described above, an siRNA composition as described above, and/or a pharmaceutical composition as described above.
It is another object of the present disclosure to provide an siRNA based on a specific modification scheme, the siRNA having the modification scheme having higher stability of the siRNA, higher activity of the siRNA and/or lower off-target effect.
Advantageous effects
The siRNA provided by the present disclosure has higher mRNA inhibitory activity.
The siRNA and the siRNA pharmaceutical composition provided by the disclosure have high plasma stability and intracellular stability, and also have high mRNA (messenger ribonucleic acid) inhibition activity.
Furthermore, the sirnas provided by the present disclosure also exhibit very low immunotoxicity, representing good in vivo safety.
The modified siRNA unexpectedly has higher stability in blood, higher stability in lysosomes, lower off-target effects, lower immunotoxicity and/or higher activity in inhibiting gene expression.
Additional features and advantages of the disclosure will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:
figure 1 is the results of stability testing of sirnas provided by the present disclosure in lysosomal lysates in vitro.
Detailed Description
Specific embodiments of the present disclosure are described in detail below. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.
Definition of
In this disclosure, ebola virus and its english abbreviation EBOV are sometimes used alone and sometimes in combination. For example, "Ebola virus", "EBOV" or "Ebola virus EBOV" are intended to mean the same and all refer to Ebola virus.
In the present disclosure, the Ebola virus gene refers to a gene whose DNA sequence is shown in Genbank accession No. KR063670.1(Sudan), AF086833.2(Zaire-1976), MF102255.1(Zaire-Makona) and KC242790.1 (Zaire-Luebo). Accordingly, the target mRNA or ebola virus mRNA is synonymous and refers to the mRNA transcribed from the ebola virus gene.
Hereinabove and hereinafter, the capital letter C, G, U, A, T represents the base composition of a nucleotide, unless otherwise specified; the lower case letter d indicates that one nucleotide adjacent to the right side of the letter d is a deoxyribonucleotide; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that two nucleotides adjacent to the left and right of the letter s are in phosphorothioate-based linkage; the letter number combination P1 indicates that a nucleotide adjacent to the right of the letter number combination P1 is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide, the letter combination VP indicates that a nucleotide adjacent to the right of the letter combination VP is a vinylphosphate modified nucleotide, the letter combination Ps indicates that a nucleotide adjacent to the right of the letter combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that a nucleotide adjacent to the right of the letter P is a 5' -phosphate nucleotide.
In the present context, the terms "complementary" or "reverse complementary" are used interchangeably and have the meaning well known to the skilled person, i.e. in a double stranded nucleic acid molecule, the bases of one strand pair with the bases on the other strand in a complementary manner. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA; the purine base guanine (C) always pairs with the pyrimidine base cytosine (G); each base pair includes one purine and one pyrimidine.
In the above and below, essentially reverse complementary means that there are no more than 3 base mismatches between the two nucleotide sequences involved, unless otherwise specified; substantially completely reverse complementary means that no more than 1 base mismatch occurs between two nucleotide sequences; perfect complementarity means that there is no base mismatch between two nucleotide sequences.
In the above and below, the nucleotide difference between one nucleotide sequence and the other nucleotide sequence means that the nucleotide at the same position has a change in the base type as compared with the latter, for example, in the case where one nucleotide base is A in the latter, in the case where the corresponding nucleotide base at the same position is U, C, G or T, it is considered that there is a nucleotide difference between the two nucleotide sequences at that position. In some embodiments, a nucleotide difference can also be considered to occur at a position when a nucleotide in the original position is replaced with an abasic nucleotide or its equivalent.
In the above and below, the "modified nucleotide" refers to a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with other group, or a nucleotide in which the base on the nucleotide is a modified base.
The term "subject", as used herein, refers to any animal, e.g., a mammal or a marsupial. Subjects of the present disclosure include, but are not limited to, humans, non-human primates (e.g., rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cows, sheep, rats, and any species of poultry.
As used herein, "treat," "alleviate," or "improve" may be used interchangeably herein. These terms refer to methods of achieving beneficial or desired results, including but not limited to therapeutic benefits. By "therapeutic benefit" is meant eradication or amelioration of the underlying disorder being treated. In addition, a therapeutic benefit is achieved by eradicating or ameliorating one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the subject, although the subject may still be afflicted with the underlying disorder.
As used herein, "prevent" and "prevention" are used interchangeably. These terms refer to methods of achieving beneficial or desired results, including but not limited to prophylactic benefits. To obtain a "prophylactic benefit," a composition can be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more pathological symptoms of a disease, even though a diagnosis of the disease may not have been made.
siRNA
The present disclosure provides a siRNA that is capable of selectively and effectively reducing expression of ebola virus EBOV genes.
The siRNA of the present disclosure contains a nucleotide group as a basic structural unit, which is well known to those skilled in the art, and the nucleotide group contains a phosphate group, a ribose group, and a base, which are not described in detail herein.
The siRNA of the present disclosure contains a sense strand and an antisense strand, each nucleotide of the sense strand and the antisense strand is a modified nucleotide, wherein the sense strand comprises a nucleotide sequence 1, the antisense strand comprises a nucleotide sequence 2, the nucleotide sequence 1 and the nucleotide sequence 2 are both 19 nucleotides in length, the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially reverse-complementary to form a double-stranded region, the nucleotide sequence 2 is at least partially reverse-complementary to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in ebovr mrna with the same length as the nucleotide sequence 2; in the direction from the 5 'end to the 3' end, the nucleotides at the 7 th, 8 th and 9 th positions of the nucleotide sequence 1 are fluorine-modified nucleotides; the first nucleotide at the 5 'end of the nucleotide sequence 2 is the first nucleotide at the 5' end of the antisense strand, and the nucleotides at positions 2, 6, 14 and 16 of the nucleotide sequence 2 are fluoro-modified nucleotides.
In some embodiments, nucleotide sequence 2 is substantially reverse complementary, or fully reverse complementary to the first stretch of nucleotide sequence; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
In some embodiments, at least nucleotides from positions 2-19 of the nucleotide sequence 2 are complementary to the first stretch of nucleotide sequence in the 5 'end to 3' end direction.
In some embodiments, the nucleotide at position 1 of the nucleotide sequence 2 is a or U in the 5 'to 3' direction.
In some embodiments, the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, or fully reverse complementary; by substantially reverse complementary is meant that no more than 3 base mismatches occur between two nucleotide sequences; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complementarity means that there is no mismatch between the two nucleotide sequences.
In some embodiments, the nucleotide sequence 1 is a sequence shown as SEQ ID NO. 1, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 2; or
The nucleotide sequence 1 is a sequence shown as SEQ ID NO. 3, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 4; or
The nucleotide sequence 1 is a sequence shown by SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown by SEQ ID NO. 6; or alternatively
The nucleotide sequence 1 is a sequence shown by SEQ ID NO. 7, and the nucleotide sequence 2 is a sequence shown by SEQ ID NO. 8; or alternatively
The nucleotide sequence 1 is a sequence shown by SEQ ID NO. 9, and the nucleotide sequence 2 is a sequence shown by SEQ ID NO. 10;
5'-GUGCUGAGAUGGUUGCAAA-3'(SEQ ID NO:1)
5'-UUUGCAACCAUCUCAGCAC-3'(SEQ ID NO:2)
5'-CCAGUUAGUACAAGUGAUU-3'(SEQ ID NO:3)
5'-AAUCACUUGUACUAACUGG-3'(SEQ ID NO:4)
5'-GCACGUGACAGCAAUAUUA-3'(SEQ ID NO:5)
5'-UAAUAUUGCUGUCACGUGC-3'(SEQ ID NO:6)
5'-GCACGCGACAGCAAUAUUA-3'(SEQ ID NO:7)
5'-UAAUAUUGCUGUCGCGUGC-3'(SEQ ID NO:8)
5'-CGCUAACAGAGGUGUUUGU-3'(SEQ ID NO:9)
5'-ACAAACACCUCUGUUAGCG-3'(SEQ ID NO:10)。
in some embodiments, the sense strand further comprises nucleotide sequence 3, the antisense strand further comprises nucleotide sequence 4, each nucleotide of nucleotide sequence 3 and nucleotide sequence 4 is independently one of a non-fluorinated modified nucleotide, the nucleotide sequence 3 and the nucleotide sequence 4 are each 1-4 nucleotides in length, the nucleotide sequence 3 and the nucleotide sequence 4 are equal in length and are substantially reverse complementary or fully reverse complementary, the nucleotide sequence 3 is linked to the 5 'end of the nucleotide sequence 1, and the nucleotide sequence 4 is linked to the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is substantially reverse complementary or fully reverse complementary to a second nucleotide sequence that is adjacent to the first nucleotide sequence in the target mRNA, And the length is the same as the nucleotide sequence 4; the substantially reverse complement refers to the presence of no more than 1 base mismatch between two nucleotide sequences; perfect reverse complement means that there is no mismatch between the two nucleotide sequences; each nucleotide of nucleotide sequence 3 and nucleotide sequence 4 is independently one of non-fluorinated modified nucleotides.
In some embodiments, the siRNA further comprises a nucleotide sequence 5 and/or a nucleotide sequence 6, each nucleotide of the nucleotide sequence 5 or the nucleotide sequence 6 is independently one of non-fluorinated modified nucleotides, the nucleotide sequence 5 or the nucleotide sequence 6 is 1 to 3 nucleotides in length, and is linked at the 3 'end of the antisense strand, thereby constituting a 3' overhang of the antisense strand; nucleotide sequence 6 is ligated to the 3 'end of the sense strand, thereby constituting the 3' overhang of the sense strand.
In some embodiments, the nucleotide sequence 5 or the nucleotide sequence 6 is 2 nucleotides in length, and in the direction from the 5 'end to the 3' end, the nucleotide sequence 5 is 2 consecutive thymine deoxyribonucleotides, 2 consecutive uracil ribonucleotides, or is fully reverse complementary to a third nucleotide sequence that is adjacent to the first nucleotide sequence or the second nucleotide sequence in the target mRNA and that is equal in length to the nucleotide sequence 5; the nucleotide sequence 6 is 2 continuous thymine deoxyribonucleotides or 2 continuous uracil ribonucleotides.
In some embodiments, in the direction from 5 'end to 3' end, in the sense strand, the 7 th, 8 th, 9 th or 5 th, 7 th, 8 th, 9 th nucleotide of the nucleotide sequence 1 is a fluorine-containing modified nucleotide, and the nucleotides at the remaining positions in the sense strand are non-fluorine-containing modified nucleotides; in the antisense strand, the 2 nd, 6 th, 14 th, 16 th or 2 nd, 6 th, 8 th, 9 th, 14 th, 16 th nucleotide of the nucleotide sequence 2 is a fluorine-modified nucleotide, and the nucleotides at the rest positions in the antisense strand are non-fluorine-modified nucleotides.
In this context, "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with fluorine, and has a structure represented by the following formula (107). "non-fluorinated modified nucleotide" refers to a nucleotide in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group, or a nucleotide analog. In some embodiments, each non-fluorinated modified nucleotide is independently selected from one of a nucleotide or a nucleotide analog in which the hydroxyl group at the 2' -position of the ribosyl group of the nucleotide is substituted with a non-fluorinated group.
The nucleotide in which the hydroxyl group at the 2 '-position of the ribosyl group is substituted with a non-fluorine group is known to those skilled in the art, and the nucleotide may be one selected from the group consisting of a 2' -alkoxy-modified nucleotide, a 2 '-substituted alkoxy-modified nucleotide, a 2' -alkyl-modified nucleotide, a 2 '-substituted alkyl-modified nucleotide, a 2' -amino-modified nucleotide, a 2 '-substituted amino-modified nucleotide, and a 2' -deoxynucleotide.
In some embodiments, the 2 '-alkoxy modified nucleotide is a methoxy modified nucleotide (2' -OMe), as shown in formula (108). In some embodiments, the 2' -substituted alkoxy modified nucleotide, for example, can be a 2' -O-methoxyethyl modified nucleotide (2' -MOE), as shown in formula (109). In some embodiments, the 2 '-amino modified nucleotide (2' -NH) 2 ) As shown in equation (110). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (111):
a nucleotide analog refers to a group that can replace a nucleotide in a nucleic acid, but that differs in structure from adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, or thymine deoxyribonucleotide. In some embodiments, the nucleotide analog can be a heteronucleotide, a bridged nucleotide (BNA for short), or an acyclic nucleotide.
BNA refers to a constrained or inaccessible nucleotide. BNAs may contain five-membered, six-membered, or seven-membered ring bridged structures with "fixed" C3' -endo-sugar pull-down. The bridge is typically incorporated at the 2'-, 4' -position of the ribose sugar to provide a 2',4' -BNA nucleotide. In some embodiments, BNA may be LNA, ENA, cET BNA, etc., where LNA is represented by equation (112), ENA is represented by equation (113), and cET BNA is represented by equation (114):
acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is opened. In some embodiments, the acyclic nucleotide can be an Unlocked Nucleic Acid (UNA) or a Glycerol Nucleic Acid (GNA), wherein UNA is represented by formula (115) and GNA is represented by formula (116):
in the above formulae (115) and (116), R represents a group selected from the group consisting of H, OH and an alkoxy group (O-alkyl group).
An isonucleotide is a compound formed by changing the position of a base in a nucleotide on a ribose ring. In some embodiments, the isonucleotides can be compounds in which the base moves from the 1' -position to the 2' -position or the 3' -position of the ribose ring, as shown in formula (117) or (118):
in the compounds of the above formula (117) to formula (118), Base represents a Base selected from A, U, G, C or T; r represents a group selected from the group consisting of H, OH, F and a non-fluorine group as described above.
In some embodiments, the nucleotide analog is selected from one of a heteronucleotide, LNA, ENA, cET, UNA, and GNA. In some embodiments, each of the non-fluorinated modified nucleotides is a methoxy modified nucleotide, which refers to a nucleotide in which the 2' -hydroxyl group of the ribosyl group is substituted with a methoxy group, both supra and infra.
In the above and the following, the terms "fluoro-modified nucleotide", "2 '-fluoro-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with fluorine" and "2 '-fluoro-ribosyl group" are the same, and refer to a compound having a structure represented by formula (107) in which 2' -hydroxyl group of nucleotide is substituted with fluorine; the terms "methoxy-modified nucleotide", "2 '-methoxy-modified nucleotide", "nucleotide in which 2' -hydroxyl group of ribose group is substituted with methoxy group" and "2 '-methoxy ribosyl group" have the same meaning, and refer to a compound having a structure represented by formula (108) in which 2' -hydroxyl group of ribose group of nucleotide is substituted with methoxy group.
In some embodiments, the sirnas of the present disclosure also contain other modified nucleotide groups that do not result in a significant impairment or loss of the function of the siRNA to regulate expression of a target gene.
Currently, there are a variety of ways available in the art for modifying siRNA, including, in addition to the ribose group modifications mentioned above, backbone modifications (e.g., phosphate group modifications), base modifications, etc. (see, e.g., Watts, J.K., G.F.Delevavey and M.J.Dama, chemical modified siRNA: tools and applications. drug discovery Today, 2008.13 (19-20): p.842-55, which is incorporated herein by reference in its entirety).
In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one of the sense strand and the antisense strand is a phosphate group having a modifying group.
In some embodiments, the phosphate group having a modifying group is a phosphorothioate group formed by substitution of at least one oxygen atom in a phosphodiester bond in the phosphate group with a sulfur atom.
In some embodiments, the phosphate group having a modifying group is a phosphorothioate group having a structure as shown in formula (121):
in some embodiments, the siRNA wherein the phosphorothioate linkage is present at least one of the group consisting of:
the 5' terminal end of the sense strand is between the 1 st nucleotide and the 2 nd nucleotide;
between the 2 nd and 3 rd nucleotides at the 5' terminal end of the sense strand;
between the 1 st and 2 nd nucleotides at the 3' terminal end of the sense strand;
between the 2 nd and 3 rd nucleotides at the 3' terminal end of the sense strand;
between the 1 st and 2 nd nucleotides at the 5' terminal end of the antisense strand;
between the 2 nd and 3 rd nucleotides at the 5' terminal end of the antisense strand;
between the 1 st and 2 nd nucleotides at the 3' terminal end of the antisense strand; and
the 3' terminal end of the antisense strand is between the 2 nd and 3 rd nucleotides.
In some embodiments, the 5' terminal nucleotide of the antisense strand is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide.
In some embodiments, the nucleotide 5' -phosphate has the structure shown in formula (122); in some embodiments, the 5' -phosphate analog modified nucleotide is a nucleotide represented by one of formula (123) -formula (126):
wherein R represents a group selected from the group consisting of H, OH, F and methoxy; base represents a Base selected from A, U, C, G or T.
In some embodiments, the siRNA provided by the present disclosure is any one of the sirnas shown in siP1-siP10, siP1S-siP10S, and siP1P1-siP10P1 below:
siP1：
a sense strand: GmGmCmUfGmGfAfUmGmUmGmUmGmUmGmGmAmAmAmdTdT (SEQ ID NO:11)
Antisense strand: UmUmGmCmAfAmfCfAmUmCumCumCmCmCmMemCmMemCmGfAmcdTdT (SEQ ID NO:12)
siP2：
A sense strand: CmAmGmUfUmAFGfUfAmAmAmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:13)
Antisense strand: AmAfUmCMAmmCumUmUfGfUmAMmAMmUmAmcFcUmGmGmdTdT (SEQ ID NO:14)
siP3：
Sense strand: GmCMCMCmGfUmGfAFCmAmmAmmUmUmAMdTdT (SEQ ID NO:15)
Antisense strand: UmAmUmAmUfUmUmGfCfUmGmUmmmmmmmmmmmMmCmGmGmGmCmdTdT (SEQ ID NO:16)
siP4：
Sense strand: GmCMAmCmGfCmGfAfCfAmcAmAmAmAmAmAmUmUmUmmdTdT (SEQ ID NO:17)
Antisense strand: UmAmUmAmUfUmGfCfUmGmUmGmGmGmGmGmGmGmGmCmdTdT (SEQ ID NO:18)
siP5：
Sense strand: CmGmCMUmAFAmfAfaGfAmGmGmGmGmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: AmCfAmAmcAmfAmfCfUmMemGmUfUmGmGmGmGmdTdT (SEQ ID NO:20)
siP6：
A sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: UmUmGmCmAfAmCmAmUmCumCumCmCmCmCmCmCmMemCmAmgmdTdT (SEQ ID NO:22)
siP7：
Sense strand: CmAmGmUmUmAFGfUfAmmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: AmAfUmCMAmmAmmCumUmGmAmmUmAmfAmCfUmGmGmdTdT (SEQ ID NO:24)
siP8：
A sense strand: GmCMmGmGmGfAFCfAmmAmAmAmUmUmUmmdTdT (SEQ ID NO:25)
Antisense strand: UmAmUmAmUfUmGmGmUmmmmmmmmmmMmCmCmGfUmGmCmdTdT (SEQ ID NO:26)
siP9：
A sense strand: GmCMmCmGmGfAFCfAmmAmAmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: UmAFAmUmAmmUfUmGmUmGmUmUmmmGmGfCmGmCmdTdT (SEQ ID NO:28)
siP10：
A sense strand: CmGmUmAMAMmAFAfGfAmGmGmGmUmUmGmdTdT (SEQ ID NO:29)
Antisense strand: AmCfAmAmAmCfAmCmCmCumMemGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1S：
sense strand: GmUmsGmCmUfGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: UmsUfsUmGmCmAfAmmCumCumCumCmMemCmMemCmGfCmdTdT (SEQ ID NO:12)
siP2S：
Sense strand: CmsmsAmGmUfUmGfUfAmmAmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:13)
Antisense strand: AmsAfsUmCMAmmCumUmUfGfUmAMmAMmUmAmfAmCfUmGmGmdTsdT (SEQ ID NO:14)
siP3S：
A sense strand: GmbCmCMmGfUmGfAfCfAmGmAmAmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: UmsAfsAmUmUfUmGfCfUmGmUmmMemCMAFCmGmGmCmdTsdT (SEQ ID NO:16)
siP4S：
A sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: UmsAfsAmUmUfUmGfCfUmGmUmGmGmGmGmCmCmdTdT (SEQ ID NO:18)
siP5S：
A sense strand: cmms GmCMUmAmfAFGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: AmsCfsAmAmaCfAmCfCfUmUmGmUfUmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6S：
Sense strand: GmUmsGmUmGmGmAfGfAfUmGmUmUmGmGmAmAmAmdTsdT (SEQ ID NO:21)
Antisense strand: UmsUfsUmGmCmAfAmCmAmmUmCumCmCmMemCmAmgGfCmdTsdT (SEQ ID NO:22)
siP7S：
Sense strand: CmsmsAmGmUmUmAFGfUfAmmAmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:23)
Antisense strand: AmsAfsUmCMAmmAmmUmGmAMmAMmAmmAmmCfUmGmGmdTsdT (SEQ ID NO:24)
siP8S：
A sense strand: GmsMcmmGmGmGfAfCfAmGmAmAmAmAmAmmUmUmAmdTsdT (SEQ ID NO:25)
Antisense strand: UmsAfsAmUmAmUfUmGmGmUmGmmmmmmmmmmmmmMmCmCmGmCmdTsdT (SEQ ID NO:26)
siP9S：
A sense strand: GmCmSAMmGmCmGmGmGfCfAmmAmUmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: UmsAfsAmUmAmUfUmGmCmUmGmGmGmGmGmGmGmdTmdT (SEQ ID NO:28)
siP10S：
A sense strand: cmms Gmms mUmAMAMmAFAfGfAmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:29)
Antisense strand: AmsCfsAmAmaCfAmCmCmMemCumGmUfUmMafGmGmGmdTsdT (SEQ ID NO: 30);
siP1P1：
a sense strand: GmGmCmUfGmGmGmUfGmUmGmUmGmAmAmadTdT (SEQ ID NO:11)
Antisense strand: P1-UmUfUmGmCmAmCfCfAmUmCumCmCmCmdTmMemCmMemCmAmGfCmdT (SEQ ID NO:12)
siP2P1：
Sense strand: CmAmGmUfUmAFGfUfAmAmAmAmAmaGmGmGmAmUmdTdT (SEQ ID NO:13)
Antisense strand: P1-AmAfUmCMAmmAmmCumUfGfUmAmmmmAMamAmCfUmGmGmdTdT (SEQ ID NO:14)
siP3P1：
Sense strand: GmCMCMCmGfUmGfAFCmAmmAmmUmUmAMdTdT (SEQ ID NO:15)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmGmCmCmdTdT (SEQ ID NO:16)
siP4P1：
Sense strand: GmCMAmCmGfCmGfAfCfAmcAmAmAmAmAmAmUmUmUmmdTdT (SEQ ID NO:17)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmGmGmCmGmGmCmdTdT (SEQ ID NO:18)
siP5P1：
Sense strand: CmGmCMUmAFAmfAfaGfAmGmGmGmGmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: P1-AmCfAmAmaCfAmfCfUmMcUmGmUfUmGmGmGmGmdTdT (SEQ ID NO:20)
siP6P1：
A sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: P1-UmUfUmGmCmAmCmAmAmUmCumCumCmCmCmCmMemCmCmCmdTdT (SEQ ID NO:22)
siP7P1：
A sense strand: CmAmGmUmUmUmGfUfAmmAmAmmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: P1-AmAfUmCMAmmMemCumUmGmAmmMemUmAmcCfUmGmGmdTdT (SEQ ID NO:24)
siP8P1：
A sense strand: GmCMmGmGmGfAFCfAmmAmAmAmUmUmUmmdTdT (SEQ ID NO:25)
Antisense strand: P1-UmAFAmUmUfUmGmUmGmUmGmUmmmmmCMdTdT (SEQ ID NO:26)
siP9P1：
A sense strand: GmCMmCmGmGfAFCfAmmAmAmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: P1-UmAFAmUmUfUmGmCmUmGmGmGmGmGmCmdTdT (SEQ ID NO:28)
siP10P1：
A sense strand: CmCmUMAMmAFfAfGfAmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmCfAmAmAmAmCfAmCmUmUmGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1SP1：
a sense strand: GmUmsGmCmUfGfAfUmGmUmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: P1-UmsUfsUmGmCmAmCfCfAmUmCumCmCmMemCmdTdT (SEQ ID NO:12)
siP2SP1：
A sense strand: CmsmsAmGmUfUmGfUfAmmAmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:13)
Antisense strand: P1-AmsAfsUmCMAmmAmmCumUfGfUmAmmAmmMemUmAmcFaGmGmdTsdT (SEQ ID NO:14)
siP3SP1：
Sense strand: GmsCMAmmGfUmGfAfCfAmcGmAmAmAmmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: P1-UmsAfsAmUmUfUmGfCfUmGmGmGmCmCmdTsdT (SEQ ID NO:16)
siP4SP1：
Sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: P1-UmsAfsAmUmUfUmGmGfCfUmGmGmGmCmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmdTsdT (SEQ ID NO:18)
siP5SP1：
A sense strand: cmms GmCMUmAmfAFGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: P1-AmsCfsAmAmfAmfCfUmCumGmUfUmUmUmGmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6SP1：
A sense strand: GmUmsGmUmGmGmAfGfAfUmGmUmUmGmGmAmAmAmdTsdT (SEQ ID NO:21)
Antisense strand: P1-UmsUfsUmGmCmAmCmAmmAmUmCumCumCmCmMemCmCmCmMemCmCmdTdT (SEQ ID NO:22)
siP7SP1：
Sense strand: CmsMmGmUmUmAFGfUfAmmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:23)
Antisense strand: P1-AmsAfsUmCMAmmMemCumUmGmAmmMemUmAmcCfUmGmGmdTsdT (SEQ ID NO:24)
siP8SP1：
Sense strand: GmCmSAMmGmGmGmGfAfCfAmGmAmAmmUmUmAmmdTsdT (SEQ ID NO:25)
Antisense strand: P1-UmsAfsAmUmAmUfUmGmCmUmGmGmmmmGmCmdTdTdT (SEQ ID NO:26)
siP9SP1：
Sense strand: GmCmSAMmGmCmGmGmGfCfAmmAmUmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: P1-UmsAfsAmUmUfUmGmCmUmGmGmGmGmGmGmCmCmdTmdTdT (SEQ ID NO:28)
siP10SP1：
Sense strand: CmsCmUmAmmAmmMefAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmsCfsAmAmfAmCmUmMemUmGmUfUmGmGmGmGmdTsdT (SEQ ID NO: 30);
the inventors of the present disclosure surprisingly found that the siRNA provided by the present disclosure not only has significantly enhanced plasma and lysosomal stability, but also retains very high gene suppression activity.
In the siRNA of the present disclosure, each adjacent nucleotide is connected by a phosphodiester bond or a phosphorothioate diester bond, and the non-bridging oxygen atom or sulfur atom in the phosphodiester bond or phosphorothioate diester bond has a negative charge, and can exist in the form of a hydroxyl group or a thiol group, and the hydrogen ion in the hydroxyl group or the thiol group can be partially or completely replaced by a cation. The cation may be any cation, such as a metal cation, ammonium NH 4 + One of organic ammonium cations. In some embodiments, the cation is selected from the group consisting of alkali gold, for enhanced solubilityOne or more of a metal ion, a tertiary amine-forming ammonium cation, and a quaternary ammonium cation. The alkali metal ion may be K + And/or Na + The cation formed by the tertiary amine may be an ammonium ion formed by triethylamine and/or an ammonium ion formed by N, N-diisopropylethylamine. Thus, the siRNA of the present disclosure may be present, at least in part, in the form of a salt. In one mode, the non-bridging oxygen or sulfur atoms in the phosphodiester or phosphorothioate linkages are at least partially bound to sodium ions, and the siRNA of the present disclosure is in the form of a sodium salt or a partial sodium salt.
The sirnas of the present disclosure can be prepared using conventional methods, such as solid phase phosphoramidite synthesis, which is well known in the art, or can be prepared using commercially available custom synthesis.
It is clear to one skilled in the art that modified nucleotide groups can be introduced into the sirnas described in the present disclosure by using nucleoside monomers with corresponding modifications. Methods for preparing nucleoside monomers with corresponding modifications and methods for introducing modified nucleotide groups into siRNA are also well known to those skilled in the art. All modified nucleoside monomers are commercially available or can be prepared by known methods.
siRNA compositions
The present disclosure also provides a siRNA composition comprising a first siRNA and a second siRNA; the first siRNA is selected from one or more siRNAs, each siRNA in the one or more siRNAs is an siRNA described previously in the disclosure, and the first nucleotide sequence is a nucleotide sequence in the VP35mRNA of Ebola virus; the second siRNA is selected from one or more sirnas, each siRNA of the one or more sirnas is an siRNA described previously in this disclosure, and the first nucleotide sequence is a nucleotide sequence in ebola virus L mRNA.
The disclosed siRNA compositions are capable of simultaneously inhibiting 2 transcripts of Ebola virus, VP35(viral protein-35) and L (L polymerase) mRNA. In some embodiments, the siRNA compositions of the present disclosure are capable of simultaneously inhibiting different subtypes of EBOV virus, with a broader spectrum of inhibitory potency. The ratio of the two siRNAs can be flexibly controlled according to actual conditions. In some embodiments, the molar ratio of the first siRNA and the second siRNA is from 1:10 to 10: 1. In some embodiments, the molar ratio of the first siRNA to the second siRNA is from 1:5 to 5: 1. In some embodiments, the molar ratio of the first siRNA to the second siRNA is from 1:2 to 2: 1. In some embodiments, the siRNA compositions of the present disclosure have a synergistic effect compared to the use of one of the sirnas alone.
In some embodiments, the first siRNA is one or more of the following a) to c):
a) in the siRNA, the nucleotide sequence 1 is a sequence shown in SEQ ID NO. 1, and the nucleotide sequence 2 is a sequence shown in SEQ ID NO. 2;
b) in the siRNA, the nucleotide sequence 1 is a sequence shown in SEQ ID NO. 3, and the nucleotide sequence 2 is a sequence shown in SEQ ID NO. 4;
c) in the siRNA, the nucleotide sequence 1 is a sequence shown as SEQ ID NO. 9, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 10;
in some embodiments, the second siRNA is one or more of the sirnas described in d) and e) below:
d) in the siRNA, the nucleotide sequence 1 is a sequence shown by SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown by SEQ ID NO. 6;
e) in the siRNA, the nucleotide sequence 1 is a sequence shown by SEQ ID NO. 7, and the nucleotide sequence 2 is a sequence shown by SEQ ID NO. 8.
In some embodiments, the first siRNA is selected from one or more of siP1, siP2, siP5, siP6, siP7, siP10, siP1S, siP2S, siP5S, siP6S, siP7S, siP10S, siP1P1, siP2P1, siP5P1, siP6P1, siP7P1, siP10SP1, siP1SP1, siP2SP1, siP5SP1, siP6SP1, siP7SP1, and siP10SP 1; the second siRNA is selected from one or more of siP3, siP4, siP8, siP9, siP3S, siP4S, siP8S, siP9S, siP3P1, siP4P1, siP8P1, siP9SP1, siP3SP1, siP4SP1, siP8SP1 and siP9SP 1.
In some embodiments, the first siRNA is selected from one or more of siP1, siP2, siP6, siP7, siP1S, siP2S, siP6S, siP7S, siP1P1, siP2P1, siP6P1, siP7P1, siP1SP1, siP2SP1, siP6SP1, and siP7SP 1; the second siRNA is one or more of siP4, siP9, siP4S, siP9S, siP4P1, siP9P1, siP4SP1 and siP9SP 1.
Pharmaceutical composition
The present disclosure also provides a pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the siRNA or the siRNA composition as described above.
In some embodiments, the weight ratio of the effective ingredient to the pharmaceutically acceptable carrier is 1 (1-500).
In some embodiments, the weight ratio of the effective ingredient to the pharmaceutically acceptable carrier is 1 (1-50).
In some embodiments, the pharmaceutical composition may be in the form of a liposomal formulation. In some embodiments, the pharmaceutically acceptable carrier used in the liposome formulation comprises an amine-containing transfection compound (which may also be referred to hereinafter as an organic amine), a helper lipid, and/or a pegylated lipid. Wherein the organic amine, helper lipid, and pegylated lipid may be selected from one or more of the amine-containing transfection compounds described in CN103380113A (herein incorporated by reference in its entirety), or pharmaceutically acceptable salts or derivatives thereof, helper lipid, and pegylated lipid, respectively.
In some embodiments, the organic amine is a compound described in CN103380113A and represented by formula (201) and/or a pharmaceutically acceptable salt thereof:
wherein:
X 1 and X 2 Each independently O, S, N-A or C-A, wherein A is hydrogen or C 1 -C 20 A hydrocarbon chain;
y and Z are each independently C-O, C-S, S-O, CH-OH or SO 2 ；
R 1 、R 2 、R 3 、R 4 、R 5 、R 6 And R 7 Each independently is hydrogen, a cyclic or acyclic, substituted or unsubstituted, branched or linear aliphatic group, a cyclic or acyclic, substituted or unsubstituted, branched or linear heteroaliphatic group, a substituted or unsubstituted, branched or linear acyl group, a substituted or unsubstituted, branched or linear aryl group, a substituted or unsubstituted, branched or linear heteroaryl group;
x is an integer from 1 to 10;
n is an integer of 1 to 3, m is an integer of 0 to 20, p is 0 or 1; and wherein, when m and p are both 0, R 2 Is hydrogen;
and, if at least one of n or m is 2, then R 3 And the nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):
wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each x N represents a nitrogen atom shown in formula (201).
Among them, the compound represented by formula (201) can be prepared as described in CN 103380113A.
In some embodiments, the organic amine is an organic amine according to formula (214) and/or an organic amine according to formula (215):
the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative; and is provided with
The pegylated lipid is 1, 2-dipalmitoamide-sn-glycerol-3-phosphatidylethanolamine-N- [ methoxy (polyethylene glycol) ] -2000.
In some embodiments, the molar ratio between the organic amine, the helper lipid, and the pegylated lipid is (19.7-80): (0.3-50).
In some embodiments, the molar ratio between the organic amine, the helper lipid, and the pegylated lipid in the pharmaceutical composition is (50-70): (20-40): (3-20).
siRNA, siRNA composition and application of pharmaceutical composition
The present disclosure also provides the use of an siRNA as described above, an siRNA composition as described above, a pharmaceutical composition as described above for the preparation of a medicament for the treatment and/or prevention of a pathological condition or disease caused by infection by an ebola virus.
In some embodiments, the pathological condition or disease caused by ebola virus infection is selected from at least one of an acute onset fever, a myalgia hemorrhagic rash, and liver renal function impairment.
Reagent kit
The present disclosure also provides a kit comprising an siRNA as described above, an siRNA composition as described above, and/or a pharmaceutical composition as described above.
In some embodiments, the kits described herein can provide a modified siRNA or siRNA composition in one container. In some embodiments, a kit described herein may comprise one container providing a pharmaceutically acceptable excipient. In some embodiments, the kit may further comprise other ingredients, such as stabilizers or preservatives and the like. In some embodiments, the kits described herein can comprise at least one additional therapeutic agent in a container other than the container providing the modified siRNA or siRNA composition described herein. In some embodiments, the kit can comprise instructions for mixing the modified siRNA or siRNA composition with a pharmaceutically acceptable carrier and/or adjuvant or other ingredient (if any).
In the kits of the present disclosure, the modified siRNA or siRNA composition and pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and/or pharmaceutically acceptable adjuvant may be provided in any form, such as a liquid form, a dried form, or a lyophilized form. In some embodiments, the modified siRNA or siRNA composition and a pharmaceutically acceptable carrier and/or adjuvant and the pharmaceutical composition and optional pharmaceutically acceptable adjuvant are substantially pure and/or sterile. In some embodiments, sterile water may be provided in the kits of the present disclosure.
The present disclosure is further illustrated by the following examples, but is not to be construed as being limited thereby.
Examples
The present disclosure will be described in detail below by way of examples. Unless otherwise specified, reagents and media used in the following examples are commercially available, and nucleic acid electrophoresis and real-timePCR were carried out according to a conventional protocol. For example, the method can be carried out as described in molecular cloning (Cold spring harbor laboratory Press (1989)).
Preparation example 1 this example illustrates the preparation of siRNA and control siRNA provided by the present disclosure
In this preparation example, siRNA sequences in Table 1 were synthesized. Among these, examples 1-10 are modified sirnas specifically targeting ebola virus of the present disclosure, wherein siP1S, siP2S, siP5S, siP6S, siP7S, and siP10S target EBOV VP35mRNA, siP3S, siP4S, siP8S, and siP9S target EBOV L mRNA; comparative examples 1-5 are bare sequences of unmodified siRNA; comparative example 6 is a negative control siRNA without inhibiting the Ebola virus gene.
In the present preparation example, siRNAs listed in Table 1 below were obtained by the conventional solid-phase phosphoramidite method. Equimolar sense and antisense strands were dissolved in DEPC water (available from Amresco under code No. E174), heated to 70-95 deg.C, and then cooled at room temperature, and the two single strands were annealed to form a double-stranded structure by hydrogen bonding.
The molecular weight of single and double strands was analyzed by liquid chromatography-mass spectrometry (LC-MS). The observed value is consistent with the theoretical value, indicating that the synthesized siRNA is sense strand, antisense strand or double strand with the target sequence.
TABLE 1 sequences of siRNA
Note: capital C, G, U, A, T indicates the base composition of the nucleotide; the lower case letter d indicates that one nucleotide adjacent to the right side of the letter d is a deoxyribonucleotide; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the linkage between two nucleotides adjacent to the left and right of the letter s is a phosphorothioate linkage. In the left column of the sequence, S represents the sense strand and AS represents the antisense strand.
Preparation example 2 this example illustrates the preparation of siRNA compositions
Mixing the first siRNA and the second siRNA in equimolar amount to obtain an siRNA composition; the kinds of the first siRNA and the second siRNA used are shown in Table 2.
TABLE 2 classes of first siRNA and second siRNA used in siRNA compositions
| siRNA compositions | First siRNA | Second siRNA |
| C1 | siP6S | siP9S |
| C2 | siP7S | siP9S |
Experimental example 1 this example demonstrates that the siRNA of the present disclosure has low off-target effects while having higher activity in vitro.
The HEK293A cells used in this example were supplied by the institute of molecular medicine, university of Beijing, nucleic acid technology laboratory, and cultured in DMEM complete medium (Hyclone) containing 20% fetal bovine serum (FBS, Hyclone) and 0.2 vol% of a streptavidin-antibody (penicilin-Streptomycin, Gibco, Invitrogen) at 37 ℃ in 5% CO 2 Culture in 95% air incubator.
This example examined the siRNA of preparation example 1 for on-target activity and off-target effects in an in vitro psiCHECK system. That is, the activity of siRNA targeting a perfect match target sequence (whose nucleotide sequence is perfectly complementary to the full-length nucleotide sequence of the antisense/sense strand of siRNA) or a seed region matching target sequence (whose nucleotide sequence is complementary to the nucleotide sequence at positions 1-8 of the antisense/sense strand of siRNA) was determined.
A detection plasmid is constructed according to the method described in Kumico Ui-Tei et al, Functional diagnosis of siRNA sequence by systematic DNA subscription, modified siRNA with a DNA sequence arm is a power full tool for a large gene transforming with a signaling reduced off-target effect, nucleic Acids Research,2008.36(7),2136-2151, and is co-transfected into HEK293A cells with the siRNA to be evaluated, and the on-target activity and off-target effect of the siRNA are reflected by the expression level of a dual-luciferase reporter gene. The method comprises the following specific steps:
[1] construction of detection plasmids
Using psiCHECK TM -2(Promega TM ) Plasmid 4 recombinant plasmids were constructed, where GSCM represents the target plasmid, PSCM, GSSM, PSSM represent off-target plasmids:
(1) GSCM, containing a target sequence that is fully complementary to all 19 nucleotide sequences of the antisense strand in the siRNA to be tested;
(2) PSCM, containing a target sequence which is completely consistent with all 19 nucleotide sequences of the antisense strand in the siRNA to be detected;
(3) GSSM, containing a target sequence, the target sequence is completely complementary with 1-8 bit nucleotide sequence from 5' end of antisense chain in siRNA to be detected, the rest part of the target sequence is corresponding to 9-19 bit nucleotide sequence from 5' end of antisense chain in siRNA to be detected, the sequence is not complementary completely, namely when any one of 9-19 bit nucleotide from 5' end of antisense chain in siRNA to be detected is G, C, A or U, the corresponding position nucleotide of target sequence is T, A, C or G respectively.
(4) PSSM contains a target sequence, the target sequence is completely complementary with 1-8 bit nucleotide sequence from the 5' end of the sense strand in the siRNA to be detected, the rest part of the target sequence corresponds to 9-19 bit nucleotide sequence from the 5' end of the sense strand in the siRNA to be detected, and the sequence is not completely complementary, namely when the nucleotide at any position of 9-19 bit from the 5' end of the sense strand in the siRNA to be detected is G, C, A or U, the nucleotide at the corresponding position of the target sequence is T, A, C or G respectively.
Cloning of the target sequence to psiCHECK TM -2 Xho I/Not I site of plasmid.
[2] Transfection
In 96-well plates, according to Lipofectamine TM 2000(Invitrogen corporation) Co-transfecting siRNA and each of the above plasmids, one plasmid corresponding to several groups of siRNA at specific concentrations, where 10ng of plasmid was transfected per well, using Lipofectamine TM 20000.2 μ L. Each group of 3 multiple wells.
For each siRNA in Table 1, the inhibitory efficiency against the GSCM at the target plasmid was examined at final concentrations of 0.1nM, 0.01nM and 0.001 nM.
For the siRNA to be tested in Table 4, the final concentration of siRNA was from 50nM, diluted 11 times to 0.0005nM, and the inhibition efficiency of each siRNA on 4 plasmids GSCM, PSCM, GSSM and PSSM was determined at each concentration, and IC was calculated 50 。
[3] Detection
24 hours after co-transfection, the expression level of the Dual luciferase reporter gene was detected by lysing HEK293A cells using a Dual luciferase reporter assay kit (Dual luciferase reporter assay kit, Promega Corp., cat. E2940) according to the instructions for use. Each test group at a specific concentration was treated with no siRNA as a control (con). Renilla luciferase protein levels (Ren) were normalized to firefly luciferase protein levels (Fir). The results are shown in Table 3.
TABLE 3 on-target Activity of siRNA
[4]In-target active IC 50 And off-target activity detection
According to the activity results measured by different siRNA concentrations, a Graphpad 5.0 software log (inhibitor) vs. response-Variable slope function is utilized to fit a dose-effect curve, and the IC of the siRNA to be measured targeting each plasmid is calculated according to the dose-effect curve 50 The values, calculated as follows,
in the formula:
y is the expression level of the residual mRNA,
x is the logarithm value of the concentration of the transfection siRNA,
bot is the value of Y at the bottom of the steady state period,
top is the value of Y at the Top of the steady state period,
LogIC 50 is the value of X when Y is halfway between the bottom to the top, and HillSlope is the slope of the curve.
IC 50 The results are shown in Table 4.
TABLE 4 in-target IC of siRNA 50 And off-target activity
*1 At 50nM, D-siP1 inhibited PSCM by 27%;
*2 at 50nM, D-siP1 inhibited GSSM by 24%.
The inhibition ratio is (1-Ren/Fir) × 100%.
When the inhibition rate is less than 10%, no inhibition is indicated.
As can be seen from table 3, the various modified sirnas provided by the present disclosure all have high inhibitory activity.
As can be seen from Table 4, each siRNA has a lower IC for GSCM 50 The siRNA concentration is between 0.0047nM and 0.014nM, and no obvious inhibition effect is seen at each siRNA concentration corresponding to PSCM, GSSM and PSSM, which shows that the siRNA disclosed by the invention has high activity in vitro and low off-target effect.
Experimental example 2 this example illustrates the activity of siRNA compositions of the present disclosure in an in vitro psiCHECK system.
This example examined the on-target activity (on-target activity) of the sirnas in preparation example 1 (siP7S, siP6S, and siP9S) and the siRNA compositions in preparation example 2 (C1 and C2) in the in vitro psiCHECK system.
[1] Construction of detection plasmid T
Target sites targeting siP7S, siP6S and siP9S antisense chains were cloned sequentially into psiCHECK in sequence TM -2(Promega TM ) Xho I/Not I site of the plasmid. The DNA fragment of the target sequence used for cloning (named target sequence T) is customized in ShanghaiThe sequence of the company Boshang Biotechnology GmbH is shown in Table 5:
TABLE 5 target sequences
[2] Transfection
In 96-well plates, according to Lipofectamine TM 2000(Invitrogen corporation) with final concentrations of 0.1nM, 0.01nM and 0.001nM, 10ng plasmid per well, using Lipofectamine TM 20000.2 μ L. Each group of 3 multiple wells.
[3] Detection of
The detection method was the same as in test example 1[3 ]. The Ren/Fir results are shown in Table 6.
TABLE 6 in-target Activity of siRNA and siRNA compositions
As can be seen from table 6, the siRNA compositions provided by the present disclosure are capable of significantly inhibiting expression of a target plasmid.
Experimental example 3 this example demonstrates the stability of siRNA provided by the present disclosure in lysosomal lysates in vitro
Preparation of test samples treated with lysosomal lysis solution: mu.l of each siRNA (20. mu.M) obtained in preparation example 1 was mixed with 27.2. mu.L of an aqueous sodium citrate solution (pH5.0), 4.08. mu.L of deionized water and 2.72. mu.L of a murine lysosome lysate (Rat Liver Tritosomes, Xenotech, Inc., cat 0610.LT, lot 1610069) to give a final concentration of 0.2 mU/. mu.L of acid phosphatase. Incubation was carried out at constant temperature of 37 ℃. Mu.l of each mixture was taken out at 0, 1,2, 4, 6, 8, 24 and 48 hours, denatured by adding to 15. mu.L of 9M urea solution, followed by 4. mu.l of 6 Xloading buffer (Solebao, cat. 20160830) and immediately frozen in a freezer at-80 ℃ to terminate the reaction. 0 hour represents the time when the sample to be tested is immediately taken out after being mixed with the lysosome lysis solution.
Preparation of reference samples not treated with lysosomal lysate: equimolar amounts of siRNA (20. mu.M) each in 1.5. mu.l were mixed with 7.5. mu.L of an aqueous sodium citrate solution (pH5.0) and 1. mu.L of deionized water, denatured by adding 30. mu.L of a 9M urea solution, mixed by adding 8. mu.L of 6 Xloading buffer, and immediately frozen in a freezer at-80 ℃ to terminate the reaction. Each siRNA reference sample was labeled Con in the electropherogram.
Preparing 16 wt% non-denatured polyacrylamide gel, loading 20 μ l of each of the above test sample and reference sample to the gel, performing electrophoresis under 20mA constant current for 10min, and performing electrophoresis under 40mA constant current for 30 min. After the electrophoresis was completed, the gel was placed on a shaker and stained with Gelred dye (BioTium Co., Ltd., cat. No. 13G1203) for 10 min. The gel was observed by imaging and photographed, and the results are shown in FIG. 1.
As can be seen in fig. 1, the modified siRNA provided by the present disclosure is stable in murine lysosomes for at least 48 hours.
Experimental example 4 this example demonstrates that the present disclosure provides results of an in vivo immunotoxicity assay for siRNA
siP9S and siP10S were tested for immunotoxicity in mice.
124 CD-1 mice (purchased from Wittingle) with the age of 6-8 weeks were selected, and male and female half of the mice were selected. All animals were grouped, dosed, and tested according to table 7.
TABLE 7 animal grouping, dosing and testing
LPS (lipopolysaccharide) of the positive control group was administered by intraperitoneal injection (i.p.), while vehicle control, Saline (physiological Saline) and each siRNA administration group (siP 9S and siP10S in preparation example 1) were administered by intravenous injection (i.v.), and the animal groups and administration doses were as described in table 7. At 3h, 24h and 72h after administration, 500 μ L of blood was taken from each. Centrifuging at 3000rpm for 15min, and separating to obtain serum. Wherein 30 μ L is used to detect immune factors (TNF α, IL-1 β, IL-5, IL-6, MCP-1, GM-CSF, KC and IFN- γ), and the remaining serum is used to detect serum biochemistry (ALT, AST, TBIL, ALP, CRE, BUN). 4 animals 24h after the drug administration in groups 2, 5 and 8, and 4 animals 72h after the drug administration in groups 9, 10, 11 and 12 were selected, and the liver, spleen and kidney were subjected to HE staining to detect histopathology.
The results show that:
(1) serum biochemical indicators showed that no significant abnormalities were observed at 3 detection time points for each of the indicators, except for the transient elevation of ALT and AST at 3 h. The concrete expression is as follows:
ALP levels were not significantly different between each time point and the Saline group for each siRNA-administered group, except that LPS group was significantly reduced after 24h and 72h treatment compared to the Saline group.
TBIL levels were not significantly different between the various time points and the Saline group in the treatment groups, except for LPS group and siP9S100mg/kg, which were slightly elevated at 3h of administration.
ALT and AST levels are increased in each treatment group compared with Saline within 3h of administration, and are increased most obviously in LPS group, but are reduced obviously within 24h after administration and are reduced to levels equivalent to that of Saline group. Suggesting that 3h is a transient increase in liver function, the animal can restore itself to normal levels.
BUN level, LPS group rose at 3h and rose to the maximum at 24h, 72h decreased to a level comparable to that of Saline group. There was no significant difference between each siRNA administration group and the Saline group at each time point.
CRE level, except LPS group is obviously increased 24h after administration, each siRNA administration group is slightly increased, but has no significant difference with Saline group.
(2) The immune factor expression level showed that each siRNA-administered group did not cause a significant increase in each immune factor at each time point, except that LPS group caused a significant increase in each immune factor at 3h after administration.
(3) Pathological sections showed that, compared with the Saline group, no significant change in morphological structure occurred in the liver, kidney and spleen in each siRNA administration group.
Therefore, the modified siRNA provided by the disclosure has extremely low immune toxicity in vivo and high biological safety.
Some embodiments of the present disclosure are described in detail above, however, the present disclosure is not limited to the specific details in the above embodiments, and many simple modifications may be made to the technical solution of the present disclosure within the scope of the technical idea of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.
It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.
In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure as long as it does not depart from the gist of the present disclosure.
Sequence listing
<110> Sa Ribo Biotechnology Ltd
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gcacgugaca gcaauauuat t 21
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Claims (9)
1. An siRNA capable of inhibiting Ebola virus gene expression, wherein the siRNA is any one of the following siRNAs siP8S and siP 9S:
siP8S：
a sense strand: GmsmCMAmmGmGfAfCfAmGmAmmAmAmmUmUmAmmdTsdT,
antisense strand: UmsAfsAmUmAmUfUmGmGmUmGmmmmmmmmmmmmMmCmCmGmGmCmdTsdT;
siP9S：
sense strand: GmsMcmAmmGmGmGmGfCfAmCmAmAmAmmUmUmAmmdTsdT,
antisense strand: UmsAfsAmUmAmUfUmGmGmGmUmGmGmGmGmGmGmdTmdTsdT;
wherein, the capital letters C, G, U, A, T represent the base composition of nucleotides, dT represents thymine deoxyribonucleotide; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that the two nucleotides adjacent to the letter s are phosphorothioate-based linked.
2. An siRNA composition comprising a first siRNA and a second siRNA;
the first siRNA is selected from one or more of siP6, siP7, siP6S, siP7S, siP6P1, siP7P1, siP6SP1 and siP7SP 1;
siP6：
a sense strand: GmGmGmUmGmAfGfAfUmGmUmUmGmUmGmAmAmAmdTdT,
antisense strand: UmUmGmCmAfAmCmAmUmCumMemCmCmMemCmMemCmAmGfCmdTdT;
siP7：
sense strand: CmAmGmUmUmUmGfUfAmmAmAmmGmGmAmUmdTdT,
antisense strand: AmAfUmMemAmmMemUmGmAmmUmAmmAmmCfUmGmGmdTdT;
siP6S：
sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT,
antisense strand: UmsUfsUmGmCmAfAmCmAmUmCumCmMemCmMemCmMefAmGfCmdTsdT;
siP7S：
sense strand: CmsAmmGmUmUmAFGfUfAmmAmAmAmAmGmGmAmUmdTsdT,
antisense strand: AmsAfsUmMemMemAmmUmGmAmmUmAmmAmmCfUmGmGmdTsdT;
siP6P1：
sense strand: GmGmGmUmGmAfGfAfUmGmUmUmGmUmGmAmAmAmdTdT,
antisense strand: P1-UmUfUmGmAfAmCmAmmAmmUmCumMemCmCmMemCmGfAmmdTdT;
siP7P1：
a sense strand: CmAmGmUmUmUmGfUfAmmAmAmmGmGmAmUmdTdT,
antisense strand: P1-AmAfUmMemMemCumGmUmUmmmmmmmmmmAmcUmGmGmdTdT;
siP6SP1：
a sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT,
antisense strand: P1-UmsUfsUmGmCmAmCmAmCmAmUmCumMemCmCmMemCmMemCmMemCmdTdT;
siP7SP1：
a sense strand: CmsmsAmGmUmUmAFGfUfAmmAmAmAmAmmGmAmUmUmdTsdT,
antisense strand: P1-AmsAfsUmCMAmfUmGmUmAMmmmmUmAmmGmGmdTsdT;
wherein the capital letters C, G, U, A, T represent the base composition of nucleotides, and dT represents thymidine; the lower case letter m indicates that one nucleotide adjacent to the left side of the letter m is a methoxy-modified nucleotide; the lower case letter f indicates that one nucleotide adjacent to the left side of the letter f is a fluoro-modified nucleotide; the lower case letter s indicates that two nucleotides adjacent to the left and right of the letter s are phosphorothioate-based linkages, and the alphanumeric combination P1 indicates that one nucleotide adjacent to the right of the alphanumeric combination P1 is a 5 '-phosphonucleotide or a 5' -phosphate analog-modified nucleotide, which 5 '-phosphonucleotide or 5' -phosphate analog-modified nucleotide is a nucleotide represented by one of formulae 122 to 126:
wherein R represents a group selected from the group consisting of H, OH, F and methoxy; base represents a modified Base;
the second siRNA is selected from one or more siRNAs of claim 1.
3. The siRNA composition of claim 2, wherein the molar ratio of said first siRNA to said second siRNA is from 1:10 to 10: 1.
4. An siRNA composition according to claim 3 wherein the molar ratio of said first siRNA to said second siRNA is from 1:5 to 5: 1.
5. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the siRNA of claim 1 or the siRNA composition of any one of claims 2 to 4.
6. The pharmaceutical composition according to claim 5, wherein the weight ratio of the effective component to the pharmaceutically acceptable carrier is 1 (1-500).
7. The pharmaceutical composition according to claim 6, wherein the weight ratio of the effective component to the pharmaceutically acceptable carrier is 1 (1-50).
8. Use of an siRNA of claim 1, an siRNA composition of any one of claims 2-4, or a pharmaceutical composition of any one of claims 5-7 in the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by an ebola virus infection.
9. A kit comprising the siRNA of claim 1, the siRNA composition of any one of claims 2 to 4, or the pharmaceutical composition of any one of claims 5 to 7.
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| WO2023109932A1 (en) * | 2021-12-16 | 2023-06-22 | 上海拓界生物医药科技有限公司 | Dsrna and preparation method therefor and application thereof |
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