CN111378656A - Nucleic acid for inhibiting Ebola virus, pharmaceutical composition containing nucleic acid and application of pharmaceutical composition - Google Patents

Nucleic acid for inhibiting Ebola virus, pharmaceutical composition containing nucleic acid and application of pharmaceutical composition Download PDF

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CN111378656A
CN111378656A CN201811625241.XA CN201811625241A CN111378656A CN 111378656 A CN111378656 A CN 111378656A CN 201811625241 A CN201811625241 A CN 201811625241A CN 111378656 A CN111378656 A CN 111378656A
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nucleotide sequence
sirna
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sense strand
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CN111378656B (en
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张鸿雁
高山
康代武
刘涛
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Suzhou Ribo Life Science Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Abstract

A siRNA for inhibiting Ebola virus gene expression, the siRNA 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

Nucleic acid for inhibiting Ebola virus, pharmaceutical composition containing nucleic acid and application of pharmaceutical composition
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.
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 the clinical manifestations are acute onset fever, myalgia, bleeding rash and liver and kidney function damage.
EBOV genus filoviridae genus filovirus, being 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), the death rate after infection can reach 50%; 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, containing 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, which accomplishes the 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. Small interfering RNA (siRNA) can inhibit or block the expression of any target gene of interest in a sequence-specific manner based on the mechanism of RNA interference (RNAi), 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 approach is to chemically modify siRNA to improve its stability in blood. To date, the skilled artisan has conducted extensive research on siRNA, however, the degradation process and mechanism of siRNA in blood is still poorly understood, and the choice 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 comprises 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 complementary in reverse direction to form a double-stranded region, the nucleotide sequence 2 is at least partially complementary in reverse direction to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in EBOVmRNA 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, 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 as 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 which 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 a graph of the results of stability testing of sirnas provided by the present disclosure in vitro lysosomal lysates.
Detailed Description
The following describes in detail specific embodiments of the present disclosure. 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" means the same meaning 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 has the same meaning and refers to mRNA transcribed from the Ebola virus gene.
In the above and below, capital C, G, U, A, T represents the base composition of nucleotides, 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 alphanumeric combination P1 indicates that the nucleotide adjacent to the right of the alphanumeric combination P1 is a 5' -phosphate nucleotide or a 5' -phosphate analog modified nucleotide, the alphanumeric combination VP indicates that the nucleotide adjacent to the right of the alphanumeric combination VP is a vinyl phosphate modified nucleotide, the alphanumeric combination Ps indicates that the nucleotide adjacent to the right of the alphanumeric combination Ps is a phosphorothioate modified nucleotide, and the capital letter P indicates that the nucleotide adjacent to the right of the alphanumeric 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 a purine and a 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 perfectly reverse complementary means that no more than 1 base mismatch exists 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, when a nucleotide in situ is replaced with a nucleotide without a base or its equivalent, it is also believed that a nucleotide difference is created at that position.
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 another 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, 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 sirnas of the present disclosure contain 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 each 19 nucleotides in length, the nucleotide sequence 1 and the nucleotide sequence 2 are at least partially complementary in reverse direction to form a double-stranded region, the nucleotide sequence 2 is at least partially complementary in reverse direction to a first nucleotide sequence, and the first nucleotide sequence is a nucleotide sequence in ebovr mrna that is the same as the nucleotide sequence 2 in length; 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 as SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 6; or
The nucleotide sequence 1 is a sequence shown as SEQ ID NO. 7, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 8; or
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;
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 non-fluorinated modified nucleotides, 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 the 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 to 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 the 5 'end to the 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 fluorinated modified nucleotide, and the remaining nucleotides in the sense strand are non-fluorinated modified nucleotides; in the antisense strand, the nucleotides at the 2 nd, 6 th, 14 th and 16 th positions or 2 nd, 6 th, 8 th, 9 th, 14 th and 16 th positions of the nucleotide sequence 2 are fluorine-modified nucleotides, 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, 2 '-amino modified nucleotides (2' -NH)2) As shown in equation (110). In some embodiments, the 2' -Deoxynucleotide (DNA) is according to formula (111):
Figure BDA0001927838770000061
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 to provide a 2',4' -BNA nucleotide. In some embodiments, the BNA may be LNA, ENA, cET BNA, etc., where LNA is shown as equation (112), ENA is shown as equation (113), and cET BNA is shown as equation (114):
Figure BDA0001927838770000062
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):
Figure BDA0001927838770000063
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).
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):
Figure BDA0001927838770000071
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 all 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 further contain other modified nucleotide groups that do not result in a significant impairment or loss of the function of the siRNA to modulate expression of a target gene.
Currently, there are various 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, for example, Watts, J.K., G.F.Delevavey and M.J.Damha, chemical modified siRNA: tools and applications. drug discovery, 2008.13 (19-20): p.842-55, the entire contents of which are incorporated herein by reference).
In some embodiments, at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand 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):
Figure BDA0001927838770000072
in some embodiments, the siRNA wherein the phosphorothioate linkage is present at least one of the group consisting of:
between the 1 st and 2 nd nucleotides at the 5' terminal end of the sense strand;
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):
Figure BDA0001927838770000081
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：
sense strand: GmGmCmUfGmGfAfUmGmUmGmUmGmUmGmGmAmAmAmdTdT (SEQ ID NO:11)
Antisense strand: UmUmGmCmAfAmCfAmUmCumCumCmCmCmCmCmCmCmCmMemCmCmMemCmMemCmAmGfAmcdTdT (SEQ ID NO:12)
siP2：
Sense strand: CmAmGmUfUmAFGfUfAmAmAmAmAmaGmGmGmAmUmdTdT (SEQ ID NO:13)
Antisense strand: AmAfUmCMAmmCumUmUfGfUmAMmAMmUmAmcFcUmGmGmdTdT (SEQ ID NO:14)
siP3：
Sense strand: GmCMCMGfUmGfAfCfAmcAmmAmAmmUmUmUmmdTdT (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: CmCmUmAmafCfAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: AmCfAmAmcFAmcFcCfUmMemGmUfUmGmGmGmdTdT (SEQ ID NO:20)
siP6：
Sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: UmUmGmGmAmCmAmCmAmUmCumCmCmCmCmCmCmMemCmMemCmAmGfCmdTdT (SEQ ID NO:22)
siP7：
Sense strand: CmAmGmUmUmAFGfUfAmmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: AmAfUmCMAmmAmmCumUmGmAmmUmAmfAmCfUmGmGmdTdT (SEQ ID NO:24)
siP8：
Sense strand: GmAmCmGmGfAfCfAmGmAmAmAmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:25)
Antisense strand: UmAmUmAmUfUmGmGmUmmmmmmmmmmMmCmCmGfUmGmCmdTdT (SEQ ID NO:26)
siP9：
Sense strand: GmCMAmCmCmGfAfCfAmGmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: UmAFAmUmAmUfUmGmGmGmGmGmGmGmGmdTdT (SEQ ID NO:28)
siP10：
Sense strand: CmCmUMAMmAFfAfGfAmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: AmCfAmAmaCfAmCmCmUmMemGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1S：
sense strand: GmUmsGmCmUfGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: UmsUfsUmGmCmAmCfCfAmUmCumCumCmCmCmCmCmMemCmCmAmGmCmdTdT (SEQ ID NO:12)
siP2S：
Sense strand: CmsMGmUfUmGfUfAmCmAmAmGmGmAmUmUmdTsdT (SEQ ID NO:13)
Antisense strand: AmsAfsUmCMAmmCumUmUfGfUmAMmAMmUmAmfAmCfUmGmGmdTsdT (SEQ ID NO:14)
siP3S：
Sense strand: GmsCMAmmGfUmGfAfCfAmcGmAmAmAmmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: UmsAfsAmUmAmUfUmGfCfUmGmGmmmmmmmmmmmmmmmMcMcMcMcGdT (SEQ ID NO:16)
siP4S：
Sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: UmsAfsAmUmUfUmGfCfUmGmGmGmGmGmGmGmGmGmdTmdTdT (SEQ ID NO:18)
siP5S：
Sense strand: CmsCmUmAmfAmCfAfGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: AmsCfsAmAmaCfAmCfCfUmUmGmUfUmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6S：
Sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:21)
Antisense strand: UmsUfsUmGmCmAmCmAmCmUmCumCmCmMemCmMemCmAmGfCmdTdT (SEQ ID NO:22)
siP7S：
Sense strand: CmsMmGmUmUmAFGfUfAmmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:23)
Antisense strand: AmsAfsUmCMAmmMemCumUmGmAmmUmAmfAmCfUmGmGmdTsdT (SEQ ID NO:24)
siP8S：
Sense strand: GmsMcmmGmGmGfAfCfAmGmAmAmAmAmAmmUmUmAmdTsdT (SEQ ID NO:25)
Antisense strand: UmsAfsAmUmAmUfUmGmGmUmGmmmmmmmmmmmmmMmCmCmGmCmdTsdT (SEQ ID NO:26)
siP9S：
Sense strand: GmsCMAmmGmGmGmGfAfCfAmmAmmAmmAmmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: UmsAfsAmUmAmUfUmGmCmUmGmGmGmGmGmGmGmdTmdT (SEQ ID NO:28)
siP10S：
Sense strand: CmsCmUmAmmAmmMefAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: AmsCfsAmAmaCfAmCmCmMemCumGmUfUmMafGmGmGmdTsdT (SEQ ID NO: 30);
siP1P1：
sense strand: GmGmCmUfGmGfAfUmGmUmGmUmGmUmGmGmAmAmAmdTdT (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: GmCMCMGfUmGfAfCfAmcAmmAmAmmUmUmUmmdTdT (SEQ ID NO:15)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmmGmCmCmdTdT (SEQ ID NO:16)
siP4P1：
Sense strand: GmCMAmCmGfCmGfAfCfAmcAmAmAmAmAmAmUmUmUmmdTdT (SEQ ID NO:17)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmGmGmCmGmGmCmdTdT (SEQ ID NO:18)
siP5P1：
Sense strand: CmCmUmAmafCfAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: P1-AmCfAmAmAmCfAmCfCfUmUmGmUfUmGmGmGmGmdTdT (SEQ ID NO:20)
siP6P1：
Sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: P1-UmUfUmGmCmAmCmAmAmUmCumCumCmCmCmCmMemCmCmCmdTdT (SEQ ID NO:22)
siP7P1：
Sense strand: CmAmGmUmUmAFGfUfAmmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: P1-AmAfUmCMAmmMemCumUmGmAmmMemUmAmcCfUmGmGmdTdT (SEQ ID NO:24)
siP8P1：
Sense strand: GmAmCmGmGfAfCfAmGmAmAmAmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:25)
Antisense strand: P1-UmAFAmUmUfUmGmCmUmGmmmmmmmmmmMmMmMmMmCmGmCmdTdT (SEQ ID NO:26)
siP9P1：
Sense strand: GmCMAmCmCmGfAfCfAmGmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: P1-UmAFAmUmUfUmGmCmUmGmGmGmGmGmCmdTdT (SEQ ID NO:28)
siP10P1：
Sense strand: CmCmUMAMmAFfAfGfAmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmCfAmAmAmAmCfAmCmUmUmGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1SP1：
sense strand: GmUmsGmCmUfGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: P1-UmsUfsUmGmCmAmCfCfAmUmCumCmCmMemCmdTdT (SEQ ID NO:12)
siP2SP1：
Sense strand: CmsMGmUfUmGfUfAmCmAmAmGmGmAmUmUmdTsdT (SEQ ID NO:13)
Antisense strand: P1-AmsAfsUmCMAmmAmmCumUfGfUmAmmAmmMemUmAmcFaGmGmdTsdT (SEQ ID NO:14)
siP3SP1：
Sense strand: GmsCMAmmGfUmGfAfCfAmcGmAmAmAmmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: P1-UmsAfsAmUmUfUmGfCfUmGmGmmmGmCmCmdTdTdT (SEQ ID NO:16)
siP4SP1：
Sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: P1-UmsAfsAmUmUfUmGmGfCfUmGmGmGmCmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmdTsdT (SEQ ID NO:18)
siP5SP1：
Sense strand: CmsCmUmAmfAmCfAfGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: P1-AmsCfsAmAmaCfAmCfCfUmUmGmUfUmGmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6SP1：
Sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (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: GmsMcmmGmGmGfAfCfAmGmAmAmAmAmAmmUmUmAmdTsdT (SEQ ID NO:25)
Antisense strand: P1-UmsAfsAmUmAmUfUmGmCmUmGmGmmmmGmCmdTdTdT (SEQ ID NO:26)
siP9SP1：
Sense strand: GmsCMAmmGmGmGmGfAfCfAmmAmmAmmAmmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: P1-UmsAfsAmUmUfUmGmCmUmGmGmGmGmGmGmCmCmdTmdTdT (SEQ ID NO:28)
siP10SP1：
Sense strand: CmsCmUmAmmAmmMefAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmsCfsAmAmaCfAmCmUmMemUmGmUfUmGmGmGmdTsdT (SEQ ID NO: 30);
the inventors of the present disclosure have surprisingly found that the sirnas provided by the present disclosure not only have significantly enhanced plasma and lysosomal stability, but also retain 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 NH4 +Is provided withOne of the organic ammonium cations. For solubility enhancement, in some embodiments, the cation is selected from one or more of alkali metal ions, tertiary amine forming ammonium cations, and quaternary ammonium cations. 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 sirnas described in 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 sirnas described in this disclosure are present 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 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(viralprotein-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 and the second siRNA is from 1:5 to 5: 1. In some embodiments, the molar ratio of the first siRNA and the second siRNA is 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 as SEQ ID NO. 1, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 2;
b) in the siRNA, 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;
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 as SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown as 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 a pharmaceutically acceptable salt or derivative 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:
Figure BDA0001927838770000121
wherein:
X1and X2Each independently is O, S, N-A orC-A, wherein A is hydrogen or C1-C20A hydrocarbon chain;
y and Z are each independently C O, C S, S O, CH OH or SO2；
R1、R2、R3、R4、R5、R6And R7Each 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, R2Is hydrogen;
and, if at least one of n or m is 2, then R3And the nitrogen in formula (201) forms a structure as shown in formula (202) or formula (203):
Figure BDA0001927838770000131
wherein g, e and f are each independently an integer of 1 to 6, "HCC" represents a hydrocarbon chain, and each of x N represents a nitrogen atom shown in formula (201).
Among them, the compound represented by formula (201) can be prepared according to the description 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):
Figure BDA0001927838770000132
Figure BDA0001927838770000141
the helper lipid is cholesterol, cholesterol analogue and/or cholesterol derivative; and is
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 of the present disclosure, siRNA composition, and use of pharmaceutical composition
The present disclosure also provides the use of the siRNA as described above, the siRNA composition as described above, the 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 ebola virus.
In some embodiments, the pathological condition or disease caused by infection with ebola virus is selected from at least one of acute onset fever, myalgia, bleeding skin 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 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, which indicates that the synthesized siRNA is the sense strand, antisense strand or double strand with the target sequence.
TABLE 1 sequences of siRNA
Figure BDA0001927838770000151
Figure BDA0001927838770000161
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 a siRNA composition
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% CO2Culture 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 was constructed according to the method described in Kumico Ui-Tei et al, Functional diagnosis of siRNA sequence based DNA stabilization, modified siRNA with a DNA seed area is a power full for a large gene sizing with a signaling reduced off-target effect, 2008.36(7),2136-2151, and co-transfected with the siRNA to be evaluated into HEK293A cells to reflect the on-target activity and off-target effect of the siRNA by the expression level of the dual luciferase reporter gene. The method comprises the following specific steps:
[1] construction of detection plasmids
Using psiCHECKTM-2(PromegaTM) 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 target sequence to psiCHECKTM-Xho I/Not I site of plasmid 2.
[2] Transfection
In 96-well plates, according to LipofectamineTM2000(Invitrogen corporation) were used to co-transfect siRNA and each of the above plasmids separately, one plasmid corresponding to several groups of siRNA at specific concentrations, where 10ng of plasmid was transfected per well using LipofectamineTM20000.2 μ L. Each group of 3 multiple wells.
For each siRNA in Table 1, its inhibitory efficiency against the target plasmid GSCM 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 determinedStarting at 50nM, diluting 11 concentrations to 0.0005nM, detecting the inhibition efficiency of each siRNA to GSCM, PSCM, GSSM and PSSM of 4 plasmids respectively at each concentration, and calculating IC50。
[3] Detection of
24 hours after co-transfection, the expression level of the Dual luciferase reporter was detected by using a Dual luciferase reporter assay kit (Dual luciferase reporter gene assay kit, Promega corporation, cat. E2940) by lysing HEK293A cells 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
Figure BDA0001927838770000171
Figure BDA0001927838770000181
[4]In-target active IC50And off-target activity detection
According to the activity results measured by adopting different siRNA concentrations, a dose-effect curve is fitted by utilizing Graphpad 5.0 software log (inhibitor) vs. response-Variable slope function, and the IC of the siRNA to be measured for targeting each plasmid is calculated according to the dose-effect curve50The values, calculated as follows,
Figure BDA0001927838770000182
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 Y value at the bottom of the steady state period,
top is the value of Y at the Top of the steady state period,
LogIC50when Y is halfway between the bottom and the topThe value of X, and HillSlope is the slope of the curve.
IC50The results are shown in Table 4.
TABLE 4 in-target IC of siRNA50And off-target activity
Figure BDA0001927838770000183
*1At 50nM, the inhibition of PSCM by D-siP1 was 27%;
*2at 50nM, D-siP1 inhibited GSSM by 24%.
The inhibition rate was (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 very high inhibitory activity.
As can be seen from Table 4, each siRNA has a lower IC for GSCM50The concentration of the siRNA 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 also has low off-target effect.
Experimental example 2 this example demonstrates the activity of the siRNA compositions of the present disclosure in an in vitro psiCHECK system.
This example examined the on-target activity (on-target activity) of the siRNA of preparation 1 (siP7S, siP6S, and siP9S) and the siRNA composition of preparation 2 (C1 and C2) in the psiCHECK system in vitro.
[1] Construction of detection plasmid T
The target sites targeting the antisense strands of siP7S, siP6S and siP9S were cloned sequentially into psiCHECKTM-2(PromegaTM) Xho I/Not I sites of the plasmid. The target sequence DNA fragment (designated target sequence T) used for cloning was custom made by shanghai bongo biotechnology limited, and its sequence is shown in table 5:
TABLE 5 target sequences
Figure BDA0001927838770000191
[2] Transfection
In 96-well plates, according to LipofectamineTM2000(Invitrogen corporation) with final concentrations of 0.1nM, 0.01nM and 0.001nM, 10ng plasmid per well, using LipofectamineTM20000.2 μ L. Each group of 3 multiple wells.
[3] Detection of
The detection method was the same as in test example 1[3 ]. The results of Ren/Fin are shown in Table 6.
TABLE 6 on-target Activity of siRNA and siRNA compositions
Figure BDA0001927838770000192
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 sirnas provided by the present disclosure in vitro lysosomal lysates
Preparation of test samples treated with lysosome lysate 6. mu.l each of the siRNAs (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 (RatLiver Tritosomes, Xenotech, Cat. No. R0610.LT, lot No. 1610069) respectively, and the final concentration of the acid phosphatase was incubated at a constant temperature of 0.2 mU/. mu.L.37. mu.C. 5. mu.l of the mixture was taken out at 0, 1,2, 4, 6, 8, 24 and 48 hours respectively, and the mixture was added to a 15. mu.L urea solution of 9M for denaturation, followed by addition of 4. mu.l of a 6 × loading buffer (Solebao, Cat. No. 20160830) and immediately frozen in a refrigerator at-80 ℃ to terminate the reaction at 0 hour.
Reference samples were prepared without treatment with lysosomal lysis solution by mixing equimolar amounts of siRNA (20. mu.M), 1.5. mu.l each, with 7.5. mu.L of aqueous sodium citrate (pH5.0), 1. mu.L of deionized water, denaturing by addition of 30. mu.L of 9M urea solution, mixing by addition of 8. mu.L of 6 × loading buffer, and immediately freezing at-80 ℃ to terminate the reaction, each siRNA reference sample being labeled Con in the electropherogram.
Preparing 16 wt% non-denatured polyacrylamide gel, loading 20 μ l of each of the test sample and the reference sample to the gel, performing electrophoresis under a constant current of 20mA for 10min, and performing electrophoresis under a constant current of 40mA 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 sirnas provided by the present disclosure are 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, tested according to table 7.
TABLE 7 animal grouping, dosing and testing
Figure BDA0001927838770000201
LPS (lipopolysaccharide) of a positive control group is administrated by intraperitoneal injection (i.p.), a vehicle control Saline (normal Saline) and each siRNA administration group (siP 9S and siP10S in preparation example 1) are administrated by intravenous injection (i.v.), animal groups and administration doses are described in Table 7, 3h, 24h and 72h after administration, each blood is centrifuged at 500. mu.L.3000rpm for 15min, and serum is obtained by separation, wherein 30. mu.L is used for detecting immune factors (TNF α, IL-1 β, IL-5, IL-6, MCP-1, GM-CSF, KC and IFN-gamma), and the remaining serum is used for detecting biochemical serum (ALT, AST, TBIL, ALP, CRE and BUN), 4 animals of 24h after administration of groups 2, 5 and 8 are selected, 4 animals of 72h after administration of groups 9, 10, 11 and 12 are selected, and liver, spleen, kidney and pathological tissue staining are taken.
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 were elevated in each treatment group compared with Saline for 3h after administration, and were most significantly elevated in LPS group, but were significantly reduced to levels equivalent to those of Saline 24h after administration. Suggesting that 3h is a transient increase in liver function, the animal can restore itself to normal levels.
BUN levels, increased in LPS group at 3h of administration and increased to the maximum at 24h, and decreased to levels comparable to those in Saline group at 72 h. There was no significant difference between each siRNA administration group and the Saline group at each time point.
The CRE level was slightly increased in each siRNA-administered group, but was not significantly different from that in Saline group except for the case that LPS group was significantly increased 24h after administration.
(2) The expression level of the immune factors 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, each siRNA administration group showed no significant morphological structural changes in the liver, kidney and spleen.
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 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 spirit of the present disclosure.
Sequence listing
<110> Sa Ribo Biotechnology Ltd
<120> nucleic acid for inhibiting Ebola virus, pharmaceutical composition containing the same and use thereof
<130>11490RIBO
<160>44
<170>SIPOSequenceListing 1.0
<210>1
<211>19
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<213> Artificial Sequence (Artificial Sequence)
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gugcugagau gguugcaaa 19
<210>2
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
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uuugcaacca ucucagcac 19
<210>3
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
ccaguuagua caagugauu 19
<210>4
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
aaucacuugu acuaacugg 19
<210>5
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>5
gcacgugaca gcaauauua 19
<210>6
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>6
uaauauugcu gucacgugc 19
<210>7
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>7
gcacgcgaca gcaauauua 19
<210>8
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>8
uaauauugcu gucgcgugc 19
<210>9
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>9
cgcuaacaga gguguuugu 19
<210>10
<211>19
<212>RNA
<213> Artificial Sequence (Artificial Sequence)
<400>10
acaaacaccu cuguuagcg 19
<210>11
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
gugcugagau gguugcaaat t 21
<210>12
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
uuugcaacca ucucagcact t 21
<210>13
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
ccaguuagua caagugauut t 21
<210>14
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
aaucacuugu acuaacuggt t 21
<210>15
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gcacgugaca gcaauauuat t 21
<210>16
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
uaauauugcu gucacgugct t 21
<210>17
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
gcacgcgaca gcaauauuat t 21
<210>18
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
uaauauugcu gucgcgugct t 21
<210>19
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
cgcuaacaga gguguuugut t 21
<210>20
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
acaaacaccu cuguuagcgt t 21
<210>21
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
gugcugagau gguugcaaat t 21
<210>22
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
uuugcaacca ucucagcact t 21
<210>23
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
ccaguuagua caagugauut t 21
<210>24
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
aaucacuugu acuaacuggt t 21
<210>25
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
gcacgugaca gcaauauuat t 21
<210>26
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
uaauauugcu gucacgugct t 21
<210>27
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
gcacgcgaca gcaauauuat t 21
<210>28
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
uaauauugcu gucgcgugct t 21
<210>29
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
cgcuaacaga gguguuugut t 21
<210>30
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
acaaacaccu cuguuagcgt t 21
<210>31
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
gugcugagau gguugcaaat t 21
<210>32
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
uuugcaacca ucucagcact t 21
<210>33
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
ccaguuagua caagugauut t 21
<210>34
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
aaucacuugu acuaacuggt t 21
<210>35
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>35
gcacgugaca gcaauauuat t 21
<210>36
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>36
uaauauugcu gucacgugct t 21
<210>37
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>37
gcacgcgaca gcaauauuat t 21
<210>38
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>38
uaauauugcu gucgcgugct t 21
<210>39
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>39
cgcuaacaga gguguuugut t 21
<210>40
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>40
acaaacaccu cuguuagcgt t 21
<210>41
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>41
uucuccgaac gugucacgut t 21
<210>42
<211>21
<212>DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400>42
acgugacacg uucggagaat t 21
<210>43
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>43
tcgagccagt tagtacaagt gattttctcc gaacgtgtca cgtttgtgct gag 53
<210>44
<211>53
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>44
atggttgcaa attctccgaa cgtgtcacgt ttgcacgcga cagcaatatt agc 53

Claims (16)

1. An siRNA capable of inhibiting expression of an ebola virus gene, said siRNA comprising a sense strand and an antisense strand, each nucleotide of said sense strand and said antisense strand being a modified nucleotide, wherein said sense strand comprises nucleotide sequence 1 and said antisense strand comprises nucleotide sequence 2, each of said nucleotide sequences 1 and 2 being 19 nucleotides in length, said nucleotide sequences 1 and 2 being at least partially reverse complementary to form a double-stranded region, said nucleotide sequence 2 being at least partially reverse complementary to a first nucleotide sequence, said first nucleotide sequence being a nucleotide sequence in an ebola virus mRNA that is the same length as said 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.
2. The siRNA of claim 1, wherein in the 5 'to 3' direction, the nucleotides at positions 7, 8, 9 or 5, 7, 8, 9 of said nucleotide sequence 1 are fluoro-modified nucleotides, and the nucleotides at the remaining positions in said sense strand are non-fluoro-modified nucleotides; 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 fluorinated modified nucleotide, and the rest positions of the nucleotide in the antisense strand are non-fluorinated modified nucleotides;
alternatively, 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.
3. The siRNA of claim 1, wherein the nucleotide sequence 1 and the nucleotide sequence 2 are substantially reverse complementary, or fully reverse complementary, and/or the nucleotide sequence 2 is substantially reverse complementary, or fully reverse complementary to the first stretch of nucleotide sequence;
optionally, at least the nucleotides at positions 2-19 of the nucleotide sequence 2 are complementary to the first stretch of nucleotide sequence in the 5 'to 3' direction.
4. The siRNA of any one of claims 1-3, wherein,
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 as SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 6; or
The nucleotide sequence 1 is a sequence shown as SEQ ID NO. 7, and the nucleotide sequence 2 is a sequence shown as SEQ ID NO. 8; or
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;
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 these, the capital letters C, G, U, A indicate the base composition of nucleotides.
5. The siRNA of any one of claims 1-4, wherein said sense strand further comprises nucleotide sequence 3 and said antisense strand further comprises nucleotide sequence 4, each nucleotide of nucleotide sequence 3 and nucleotide sequence 4 being independently one of a non-fluorinated modified nucleotide; said nucleotide sequence 3 and said nucleotide sequence 4 are each 1-4 nucleotides in length, said nucleotide sequence 3 and said nucleotide sequence 4 being of equal length and being substantially reverse complementary or fully reverse complementary; the nucleotide sequence 3 is connected to the 5 'end of the nucleotide sequence 1, the nucleotide sequence 4 is connected to the 3' end of the nucleotide sequence 2, the nucleotide sequence 4 is substantially reverse complementary or completely reverse complementary with a second nucleotide sequence which is adjacent to the first nucleotide sequence in the Ebola virus mRNA and has the same length with the nucleotide sequence 4.
6. The siRNA of any one of claims 1-5, wherein said siRNA further comprises a nucleotide sequence 5 and/or a nucleotide sequence 6, each nucleotide of said nucleotide sequence 5 or nucleotide sequence 6 being independently one of a non-fluorinated modified nucleotide; the length of the nucleotide sequence 5 or the nucleotide sequence 6 is 1 to 3 nucleotides, the nucleotide sequence 5 is connected to the 3 'terminal of the antisense chain to form a 3' overhang of the antisense chain; nucleotide sequence 6 is ligated to the 3 'end of the sense strand, thereby constituting the 3' overhang of the sense strand;
alternatively, the nucleotide sequence 5 or nucleotide sequence 6 is 2 nucleotides in length; the nucleotide sequence 5 is a sequence of 2 consecutive thymidylate ribonucleotides, a sequence of 2 uracil ribonucleotides, or is completely reverse complementary to the third nucleotide sequence, in the 5 'to 3' direction; the third nucleotide sequence is a nucleotide sequence which is adjacent to the first nucleotide sequence or the second nucleotide sequence in the Ebola virus mRNA and has the length equal to the length of the nucleotide sequence 5; the nucleotide sequence 6 is 2 continuous thymine deoxyribonucleotides or 2 continuous uracil ribonucleotides.
7. siRNA according to any one of claims 1-6, wherein at least 1 of the phosphate groups in the phosphate-sugar backbone of at least one single strand of said sense strand and said antisense strand is a phosphate group having a modifying group, and/or the 5' terminal nucleotide of said antisense strand is a 5' -phosphonucleotide or a 5' -phosphoanalogue modified nucleotide;
optionally, the phosphate group having a modifying group is a phosphorothioate group in which at least one oxygen atom in a phosphodiester bond in the phosphate group is replaced with a sulfur atom, and the phosphorothioate group linkage is present at least one position in the group consisting of:
between the 1 st and 2 nd nucleotides at the 5' terminal end of the sense strand;
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
between the 2 nd and 3 rd nucleotides at the 3' terminal end of the antisense strand;
alternatively, the nucleotide 5 '-phosphate or nucleotide 5' -phosphate analogue modified is a nucleotide represented by one of formula (122) to formula (126):
Figure FDA0001927838760000031
formula (122), formula (123), formula (124), formula (125), 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.
8. The siRNA of any one of claims 1-7, wherein said siRNA is any one of the siRNAs set forth in siP1-siP10, siP1S-siP10S, siP1P1-siP10P1 and siP1SP1-siP10SP 1:
siP1：
sense strand: GmGmCmUfGmGfAfUmGmUmGmUmGmUmGmGmAmAmAmdTdT (SEQ ID NO:11)
Antisense strand: UmUmGmCmAfAmCfAmUmCumCumCmCmCmCmCmCmCmCmMemCmCmMemCmMemCmAmGfAmcdTdT (SEQ ID NO:12)
siP2：
Sense strand: CmAmGmUfUmAFGfUfAmAmAmAmAmaGmGmGmAmUmdTdT (SEQ ID NO:13)
Antisense strand: AmAfUmCMAmmCumUmUfGfUmAMmAMmUmAmcFcUmGmGmdTdT (SEQ ID NO:14)
siP3：
Sense strand: GmCMCMGfUmGfAfCfAmcAmmAmAmmUmUmUmmdTdT (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: CmCmUmAmafCfAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: AmCfAmAmcFAmcFcCfUmMemGmUfUmGmGmGmdTdT (SEQ ID NO:20)
siP6：
Sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: UmUmGmGmAmCmAmCmAmUmCumCmCmCmCmCmCmMemCmMemCmAmGfCmdTdT (SEQ ID NO:22)
siP7：
Sense strand: CmAmGmUmUmAFGfUfAmmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: AmAfUmCMAmmAmmCumUmGmAmmUmAmfAmCfUmGmGmdTdT (SEQ ID NO:24)
siP8：
Sense strand: GmAmCmGmGfAfCfAmGmAmAmAmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:25)
Antisense strand: UmAmUmAmUfUmGmGmUmmmmmmmmmmMmCmCmGfUmGmCmdTdT (SEQ ID NO:26)
siP9：
Sense strand: GmCMAmCmCmGfAfCfAmGmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: UmAFAmUmAmUfUmGmGmGmGmGmGmGmGmdTdT (SEQ ID NO:28)
siP10：
Sense strand: CmCmUMAMmAFfAfGfAmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: AmCfAmAmaCfAmCmCmUmMemGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1S：
sense strand: GmUmsGmCmUfGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: UmsUfsUmGmCmAmCfCfAmUmCumCumCmCmCmCmCmMemCmCmAmGmCmdTdT (SEQ ID NO:12)
siP2S：
Sense strand: CmsMGmUfUmGfUfAmCmAmAmGmGmAmUmUmdTsdT (SEQ ID NO:13)
Antisense strand: AmsAfsUmCMAmmCumUmUfGfUmAMmAMmUmAmfAmCfUmGmGmdTsdT (SEQ ID NO:14)
siP3S：
Sense strand: GmsCMAmmGfUmGfAfCfAmcGmAmAmAmmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: UmsAfsAmUmAmUfUmGfCfUmGmGmmmmmmmmmmmmmmmMcMcMcMcGdT (SEQ ID NO:16)
siP4S：
Sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: UmsAfsAmUmUfUmGfCfUmGmGmGmGmGmGmGmGmGmdTmdTdT (SEQ ID NO:18)
siP5S：
Sense strand: CmsCmUmAmfAmCfAfGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: AmsCfsAmAmaCfAmCfCfUmUmGmUfUmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6S：
Sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:21)
Antisense strand: UmsUfsUmGmCmAmCmAmCmUmCumCmCmMemCmMemCmAmGfCmdTdT (SEQ ID NO:22)
siP7S：
Sense strand: CmsMmGmUmUmAFGfUfAmmAmAmAmmGmGmAmUmdTsdT (SEQ ID NO:23)
Antisense strand: AmsAfsUmCMAmmMemCumUmGmAmmUmAmfAmCfUmGmGmdTsdT (SEQ ID NO:24)
siP8S：
Sense strand: GmsMcmmGmGmGfAfCfAmGmAmAmAmAmAmmUmUmAmdTsdT (SEQ ID NO:25)
Antisense strand: UmsAfsAmUmAmUfUmGmGmUmGmmmmmmmmmmmmmMmCmCmGmCmdTsdT (SEQ ID NO:26)
siP9S：
Sense strand: GmsCMAmmGmGmGmGfAfCfAmmAmmAmmAmmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: UmsAfsAmUmAmUfUmGmCmUmGmGmGmGmGmGmGmdTmdT (SEQ ID NO:28)
siP10S：
Sense strand: CmsCmUmAmmAmmMefAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: AmsCfsAmAmaCfAmCmCmMemCumGmUfUmMafGmGmGmdTsdT (SEQ ID NO: 30);
siP1P1：
sense strand: GmGmCmUfGmGfAfUmGmUmGmUmGmUmGmGmAmAmAmdTdT (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: GmCMCMGfUmGfAfCfAmcAmmAmAmmUmUmUmmdTdT (SEQ ID NO:15)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmmGmCmCmdTdT (SEQ ID NO:16)
siP4P1：
Sense strand: GmCMAmCmGfCmGfAfCfAmcAmAmAmAmAmAmUmUmUmmdTdT (SEQ ID NO:17)
Antisense strand: P1-UmAFAmUmUfUmGfCfUmGmGmGmGmCmGmGmCmdTdT (SEQ ID NO:18)
siP5P1：
Sense strand: CmCmUmAmafCfAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:19)
Antisense strand: P1-AmCfAmAmAmCfAmCfCfUmUmGmUfUmGmGmGmGmdTdT (SEQ ID NO:20)
siP6P1：
Sense strand: GmGmCmGmGmAfGfAfUmGmUmGmUmGmGmGmAmAmAmdTdT (SEQ ID NO:21)
Antisense strand: P1-UmUfUmGmCmAmCmAmAmUmCumCumCmCmCmCmMemCmCmCmdTdT (SEQ ID NO:22)
siP7P1：
Sense strand: CmAmGmUmUmAFGfUfAmmAmAmmGmGmGmAmUmdTdT (SEQ ID NO:23)
Antisense strand: P1-AmAfUmCMAmmMemCumUmGmAmmMemUmAmcCfUmGmGmdTdT (SEQ ID NO:24)
siP8P1：
Sense strand: GmAmCmGmGfAfCfAmGmAmAmAmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:25)
Antisense strand: P1-UmAFAmUmUfUmGmCmUmGmmmmmmmmmmMmMmMmMmCmGmCmdTdT (SEQ ID NO:26)
siP9P1：
Sense strand: GmCMAmCmCmGfAfCfAmGmAmAmAmAmmUmUmUmdTdT (SEQ ID NO:27)
Antisense strand: P1-UmAFAmUmUfUmGmCmUmGmGmGmGmGmCmdTdT (SEQ ID NO:28)
siP10P1：
Sense strand: CmCmUMAMmAFfAfGfAmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmCfAmAmAmAmCfAmCmUmUmGmUfUmGmGmGmdTdT (SEQ ID NO: 30);
siP1SP1：
sense strand: GmUmsGmCmUfGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (SEQ ID NO:11)
Antisense strand: P1-UmsUfsUmGmCmAmCfCfAmUmCumCmCmMemCmdTdT (SEQ ID NO:12)
siP2SP1：
Sense strand: CmsMGmUfUmGfUfAmCmAmAmGmGmAmUmUmdTsdT (SEQ ID NO:13)
Antisense strand: P1-AmsAfsUmCMAmmAmmCumUfGfUmAmmAmmMemUmAmcFaGmGmdTsdT (SEQ ID NO:14)
siP3SP1：
Sense strand: GmsCMAmmGfUmGfAfCfAmcGmAmAmAmmAmmUmUmAmmdTsdT (SEQ ID NO:15)
Antisense strand: P1-UmsAfsAmUmUfUmGfCfUmGmGmmmGmCmCmdTdTdT (SEQ ID NO:16)
siP4SP1：
Sense strand: GmsCMAmmGfCmGfAfCfAmmAmmAmmAmmUmUmAmmdTsdT (SEQ ID NO:17)
Antisense strand: P1-UmsAfsAmUmUfUmGmGfCfUmGmGmGmCmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmGmdTsdT (SEQ ID NO:18)
siP5SP1：
Sense strand: CmsCmUmAmfAmCfAfGfAmGmGmGmGmUmUmUmGmUmdTsdT (SEQ ID NO:19)
Antisense strand: P1-AmsCfsAmAmaCfAmCfCfUmUmGmUfUmGmGmGmGmGmdTsdT (SEQ ID NO:20)
siP6SP1：
Sense strand: GmUmsGmUmGmGmGfAfUmGmUmGmUmGmGmAmAmAmdTsdT (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: GmsMcmmGmGmGfAfCfAmGmAmAmAmAmAmmUmUmAmdTsdT (SEQ ID NO:25)
Antisense strand: P1-UmsAfsAmUmAmUfUmGmCmUmGmGmmmmGmCmdTdTdT (SEQ ID NO:26)
siP9SP1：
Sense strand: GmsCMAmmGmGmGmGfAfCfAmmAmmAmmAmmUmUmAmdTsdT (SEQ ID NO:27)
Antisense strand: P1-UmsAfsAmUmUfUmGmCmUmGmGmGmGmGmGmCmCmdTmdTdT (SEQ ID NO:28)
siP10SP1：
Sense strand: CmsCmUmAmmAmmMefAfGfAmGmGmGmGmUmUmUmGmUmdTdT (SEQ ID NO:29)
Antisense strand: P1-AmsCfsAmAmaCfAmCmUmMemUmGmUfUmGmGmGmdTsdT (SEQ ID NO: 30);
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 the two nucleotides adjacent to the left and right of the letter s are phosphorothioate-based linkages, and the alphanumeric combination P1 indicates that the adjacent nucleotide to the right of the alphanumeric combination P1 is a 5' -phospho-nucleotide.
9. An 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 the siRNA of any one of claims 1-8, and the first nucleotide sequence is a nucleotide sequence in Ebola virus VP35 mRNA;
the second siRNA is selected from one or more siRNAs, each siRNA in the one or more siRNAs is the siRNA in any one of claims 1-8, and the first nucleotide sequence is a nucleotide sequence in Ebola virus LmRNA.
10. An siRNA composition according to claim 9, wherein the molar ratio of said first siRNA and said second siRNA is from 1:10 to 10: 1;
optionally, the molar ratio of the first siRNA and the second siRNA is 1:5 to 5: 1.
11. An siRNA composition according to claim 9 or 10 wherein said first siRNA is one or more of the following a) to c) said siRNA:
a) in the siRNA, 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;
b) in the siRNA, 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;
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;
and/or the second siRNA is one or more of the siRNAs of d) and e) below:
d) in the siRNA, the nucleotide sequence 1 is a sequence shown as SEQ ID NO. 5, and the nucleotide sequence 2 is a sequence shown as 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.
12. An siRNA composition according to claim 11 wherein said 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.
Optionally, 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.
13. A pharmaceutical composition comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the siRNA of any one of claims 1 to 8 or the siRNA composition of any one of claims 9 to 12.
14. The pharmaceutical composition according to claim 13, wherein the weight ratio of the effective ingredient to the pharmaceutically acceptable carrier is 1 (1-500).
Optionally, the weight ratio of the effective component to the pharmaceutically acceptable carrier is 1 (1-50).
15. Use of an siRNA according to any one of claims 1 to 8, an siRNA composition according to any one of claims 9 to 12 and/or a pharmaceutical composition according to claim 13 or 14 for the manufacture of a medicament for the treatment and/or prevention of a pathological condition or disease caused by infection by an ebola virus.
16. A kit comprising the siRNA of any one of claims 1 to 8, the siRNA composition of any one of claims 9 to 12, and/or the pharmaceutical composition of claim 13 or 14.
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