TW202400187A - Oligonucleotide - Google Patents
Oligonucleotide Download PDFInfo
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- TW202400187A TW202400187A TW112116791A TW112116791A TW202400187A TW 202400187 A TW202400187 A TW 202400187A TW 112116791 A TW112116791 A TW 112116791A TW 112116791 A TW112116791 A TW 112116791A TW 202400187 A TW202400187 A TW 202400187A
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- oligonucleotide
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- nucleobase
- mrna
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Classifications
-
- 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
-
- 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
- C12N2310/11—Antisense
-
- 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
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
-
- 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
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
Abstract
Description
本發明係關於與多瘤病毒RNA結合的寡核苷酸領域。此類寡核苷酸可用於治療由此類病毒引起或與此類病毒相關的任何疾病或病症。The present invention relates to the field of oligonucleotides that bind to polyomavirus RNA. Such oligonucleotides may be used to treat any disease or condition caused by or associated with such viruses.
多瘤病毒係小型無包膜雙股DNA病毒,其天然宿主通常是哺乳動物和鳥類。成人感染大多無症狀,但當免疫系統受損時,可能會成為病理性的。人多瘤病毒之實例係BK病毒(BKV)、JC病毒(JCV)和梅克爾細胞病毒(Merkel cell virus)(MCV)。Polyomaviruses are small, non-enveloped double-stranded DNA viruses whose natural hosts are usually mammals and birds. Infections in adults are mostly asymptomatic but may become pathological when the immune system is compromised. Examples of human polyomaviruses are BK virus (BKV), JC virus (JCV) and Merkel cell virus (MCV).
JCV和BKV均為在兒童早期感染人的機會病原體(Leploeg, M.D.等人, Clinical Infectious Diseases [臨床傳染病], 2001)。成人的血清陽性率很高。該兩種病毒都被認為潛伏在宿主的腎細胞中(Wunderink, H.F.等人, American Journal of Transplantation [美國移植雜誌], 2017)。例如,在免疫抑制的個體中可能會發生再活化(Wunderink, H.F.等人, American Journal of Transplantation [美國移植雜誌], 2017;Parajuli, S.等人, Clinical Transplantation [臨床移植], 2018;Gard, L.等人, PLoS One [公共科學圖書館綜合], 2017)。Both JCV and BKV are opportunistic pathogens that infect humans in early childhood (Leploeg, M.D. et al., Clinical Infectious Diseases, 2001). Seroprevalence in adults is high. Both viruses are thought to lie dormant in the host's kidney cells (Wunderink, H.F. et al., American Journal of Transplantation, 2017). For example, reactivation may occur in immunosuppressed individuals (Wunderink, H.F. et al., American Journal of Transplantation, 2017; Parajuli, S. et al., Clinical Transplantation, 2018; Gard, L. et al., PLoS One [PLoS One], 2017).
多瘤病毒具有共同的基因組結構。它們具有在感染週期的早期和晚期均有表現的基因。早期和晚期基因均產生RNA,經由差異剪接,可以從中翻譯出多種蛋白質。晚期基因典型地編碼三種衣殼蛋白,而早期基因編碼小T抗原和大T抗原,並且通常包括一或多個可變剪接編碼區(Helle, F.等人, Viruses [病毒], (2017), 3; 9(17): 327, 1-18)。Polyomaviruses share a common genome structure. They have genes that are expressed in both early and late stages of the infection cycle. Both early and late genes produce RNA from which a variety of proteins can be translated through differential splicing. Late genes typically encode three capsid proteins, whereas early genes encode small and large T antigens and often include one or more alternatively spliced coding regions (Helle, F. et al., Viruses, (2017) , 3; 9(17): 327, 1-18).
WO 2019/168402描述了調節多瘤病毒大T抗原前mRNA(pre-mRNA)的剪接的反義寡核苷酸。此類反義寡核苷酸可以具有與所述前mRNA中的剪接供體位點和/或剪接受體位點互補的序列。WO 2019/168402 describes antisense oligonucleotides that modulate the splicing of polyomavirus large T antigen pre-mRNA (pre-mRNA). Such antisense oligonucleotides may have sequences complementary to splice donor sites and/or splice acceptor sites in the pre-mRNA.
然而,仍然需要新的和改進的反義寡核苷酸,以治療由多瘤病毒引起或與多瘤病毒相關的疾病或病症。However, there remains a need for new and improved antisense oligonucleotides to treat diseases or conditions caused by or associated with polyomaviruses.
在第一態樣中,本發明係關於一種寡核苷酸,該寡核苷酸包含根據SEQ ID NO: 1至11中的一個的核鹼基序列或包含類似於SEQ ID NO: 1至11中的任一個的核鹼基序列,其特徵在於所述SEQ ID NO的至少一個核鹼基被具有與被替換的核鹼基相同的鹼基配對特異性的核鹼基類似物替換。In a first aspect, the invention relates to an oligonucleotide comprising a nucleobase sequence according to one of SEQ ID NO: 1 to 11 or comprising a sequence similar to SEQ ID NO: 1 to 11 The nucleobase sequence of any one of the SEQ ID NOs, characterized in that at least one nucleobase of the SEQ ID NO is replaced by a nucleobase analog having the same base pairing specificity as the replaced nucleobase.
在第二態樣中,本發明係關於一種載體,其包含 (i) 如第一態樣所定義的寡核苷酸,(ii) 如第一態樣所定義的寡核苷酸之反向互補序列,或 (iii) 能夠被轉錄為如第一態樣所定義的寡核苷酸的DNA。In a second aspect, the invention relates to a vector comprising (i) an oligonucleotide as defined in the first aspect, (ii) the reverse of an oligonucleotide as defined in the first aspect. complementary sequence, or (iii) DNA capable of being transcribed into an oligonucleotide as defined in the first aspect.
在第三態樣中,本發明係關於一種藥物組成物,該藥物組成物包含如第一態樣所定義的寡核苷酸或如第二態樣所定義的載體。In a third aspect, the invention relates to a pharmaceutical composition comprising an oligonucleotide as defined in the first aspect or a vector as defined in the second aspect.
在第四態樣中,本發明係關於如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物,用於作為藥品使用,特別地用於在治療受試者的多瘤病毒感染中使用。In a fourth aspect, the present invention relates to an oligonucleotide as defined in the first aspect, a carrier as defined in the second aspect or a pharmaceutical composition as defined in the third aspect, for use as a medicine Use, in particular for use in the treatment of polyomavirus infection in a subject.
在第五態樣中,本發明係關於離體方法,該等方法包括抑制細胞中多瘤病毒複製的離體方法和產生移植物的離體方法,該兩種方法都包括使用如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物。In a fifth aspect, the invention relates to ex vivo methods, including ex vivo methods for inhibiting polyomavirus replication in cells and ex vivo methods for generating grafts, both of which include using, as in the first aspect, An oligonucleotide as defined in the first aspect, a vector as defined in the second aspect or a pharmaceutical composition as defined in the third aspect.
在以下詳細描述本發明之前,應理解的是本發明並不限於本文所述之特定方法、方案和試劑,因為該等可變。亦應理解,本文所用的術語僅用於描述特定實施方式之目的,而不旨在限制本發明之範圍,該範圍將僅受所附請求項的限制。除非另外定義,否則本文所用的所有技術性和科學性術語具有與本領域中普通技術者通常所理解的相同含義。Before the present invention is described in detail below, it is to be understood that this invention is not limited to the specific methods, protocols and reagents described herein, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which scope will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.
較佳的是,本文所用的術語如以下中所述之定義「A multilingual glossary of biotechnological terms: (IUPAC Recommendations) [生物技術術語的多語言詞彙表:(IUPAC建議)]」, Leuenberger, H.G.W, Nagel, B.和Kölbl, H.編輯 (1995), Helvetica Chimica Acta [瑞士化學學報], CH-4010 巴塞爾, 瑞士。Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel , B. and Kölbl, H., editors (1995), Helvetica Chimica Acta [Swiss Journal of Chemistry], CH-4010 Basel, Switzerland.
本說明書全文中引用了幾個文檔。本文引用的每個文檔(包括所有專利、專利申請、科學出版物、製造商的說明、使用說明等)(無論是在上文還是下文中)均以引用方式全文併入本文。Several documents are referenced throughout this manual. Each document (including all patents, patent applications, scientific publications, manufacturer's instructions, instructions for use, etc.) cited herein (whether supra or below) is hereby incorporated by reference in its entirety.
在下文中,將描述本發明之要素。該等要素與特定的實施方式一起列出,然而,應該理解,它們可以以任何方式和任何數量組合以創建另外的實施方式。不同描述的實例和較佳的實施方式不應被解釋為將本發明僅限於明確描述的實施方式。本說明書應理解為支持並涵蓋將明確描述的實施方式與任何數量的揭露和/或較佳的要素組合的實施方式。此外,除非上下文另外指示,否則本申請中所有描述的要素的任何排列和組合都應被認為由本申請的描述揭露。In the following, elements of the invention will be described. These elements are listed with particular embodiments, however, it is understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed as limiting the invention to the expressly described embodiments. This specification is to be understood as supporting and encompassing embodiments that combine the specifically described embodiments with any number of disclosed and/or preferred elements. Furthermore, unless the context dictates otherwise, any permutations and combinations of all described elements in this application shall be deemed to be disclosed by the description of this application.
貫穿本說明書和以下請求項,除非上下文另外要求,否則詞語「包含(comprise)」以及變化形式如「包含(comprises)」和「包含(comprising)」將理解為隱含包括所陳述的整體或步驟或整體或步驟的群組,但不排除任何其他整體或步驟或整體或步驟的群組。在較佳的實施方式中,「包含」可以意指「由……組成」。除非內容另外明確指示,如在本說明書和所附申請專利範圍中所用,單數形式「一個/一種(a/an)」以及「該(the)」包括複數指示物。Throughout this specification and the following claims, unless the context otherwise requires, the word "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of stated integers or steps. or a group of integers or steps, but not to the exclusion of any other integer or step or group of integers or steps. In a preferred embodiment, "comprising" can mean "consisting of." As used in this specification and the appended claims, the singular forms "a/an" and "the" include plural referents unless the content clearly dictates otherwise.
在第一態樣中,本發明係關於一種寡核苷酸,特別地包含核鹼基序列的寡核苷酸,該核鹼基序列具有根據SEQ ID NO: 1至11中的一個的核鹼基序列的鹼基配對特異性。In a first aspect, the invention relates to an oligonucleotide, in particular an oligonucleotide comprising a nucleobase sequence having a nucleobase according to one of SEQ ID NOs: 1 to 11 Base pairing specificity of the base sequence.
典型地,此寡核苷酸包含根據SEQ ID NO: 1至11中的一個的核鹼基序列或包含類似於SEQ ID NO: 1至11中的任一個的核鹼基序列,其特徵在於所述SEQ ID NO的至少一個核鹼基被具有與被替換的核鹼基相同的鹼基配對特異性的核鹼基類似物替換。 寡核苷酸,基本結構 Typically, the oligonucleotide comprises a nucleobase sequence according to one of SEQ ID NOs: 1 to 11 or a nucleobase sequence similar to any one of SEQ ID NOs: 1 to 11, characterized in that At least one nucleobase of the SEQ ID NO is replaced by a nucleobase analog having the same base pairing specificity as the replaced nucleobase. Oligonucleotide, basic structure
本發明之寡核苷酸能夠與人細胞感染多瘤病毒後產生的多瘤病毒前mRNA(「靶RNA」)特異性結合。因此,它亦可以被描述為多瘤病毒寡核苷酸或反義寡核苷酸(「ASO」)或多瘤病毒反義寡核苷酸。寡核苷酸能夠特異性結合的靶RNA的部分(亦即一段連續核鹼基)在本文中稱為「靶區域」。The oligonucleotide of the present invention can specifically bind to polyomavirus pre-mRNA ("target RNA") produced after human cells are infected with polyomavirus. Therefore, it may also be described as a polyomavirus oligonucleotide or antisense oligonucleotide ("ASO") or a polyomavirus antisense oligonucleotide. The portion of the target RNA that the oligonucleotide can specifically bind to (i.e., a stretch of contiguous nucleobases) is referred to herein as the "target region."
在較佳的實施方式中,至少一部分寡核苷酸與靶區域至少基本上互補。因此,寡核苷酸包含至少12個連續核鹼基,該等連續核鹼基的序列係靶區域的至少12個連續核鹼基序列之反向互補序列。較佳的是,靶區域的長度為12至30個,亦即12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個核鹼基,或12至28個核鹼基。更佳的是,靶區域的長度為17至26個,亦即17、18、19、20、21、22、23、24、25或26個核鹼基。甚至更佳的是,靶區域的長度為18至22個,亦即18、19、20、21或22個核鹼基。最較佳的是,靶區域的長度為20個核鹼基。In preferred embodiments, at least a portion of the oligonucleotides are at least substantially complementary to the target region. Thus, the oligonucleotide contains at least 12 contiguous nucleobases whose sequence is the reverse complement of the at least 12 contiguous nucleobase sequence of the target region. Preferably, the length of the target area is 12 to 30, that is, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29 or 30 nucleobases, or 12 to 28 nucleobases. More preferably, the length of the target region is 17 to 26, that is, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleobases. Even better, the target region is 18 to 22, ie 18, 19, 20, 21 or 22 nucleobases in length. Most preferably, the target region is 20 nucleobases in length.
通常,靶區域包含多瘤病毒前RNA。靶區域較佳的是包含相應多瘤病毒的大T抗原的內含子1(或其部分)和/或外顯子1(或其部分)的剪接供體位點。較佳的是,靶區域包括內含子1和/或(較佳的是和)外顯子1的多達30個連續核鹼基,其中30個連續核鹼基中的0至30個,亦即0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個連續核鹼基屬於內含子1的剪接供體位點,並且剩餘連續核鹼基屬於外顯子1。在較佳的實施方式中,靶區域包含與剪接供體位點相鄰的一個或兩個內含子核鹼基。在較佳的實施方式中,靶區域包含與剪接供體位點相鄰的1-28個,亦即1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27或28個內含子核鹼基。在另一個較佳的實施方式中,靶區域包含與剪接供體位點相鄰的1-28個,亦即1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27或28個外顯子核鹼基。Typically, the target region contains polyomavirus pre-RNA. The target region is preferably a splice donor site including intron 1 (or part thereof) and/or exon 1 (or part thereof) of the large T antigen of the corresponding polyoma virus. Preferably, the target region includes up to 30 contiguous nucleobases of intron 1 and/or (preferably and) exon 1, wherein 0 to 30 of the 30 contiguous nucleobases, That is, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleobases belong to the splice donor site of intron 1, and the remaining contiguous nucleobases belong to exon 1. In preferred embodiments, the target region contains one or two intronic nucleobases adjacent to the splice donor site. In a preferred embodiment, the target region includes 1-28 adjacent splicing donor sites, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 intron nucleobases. In another preferred embodiment, the target region includes 1-28 adjacent to the splice donor site, that is, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 or 28 exon nucleobases.
應當理解,內含子和外顯子核鹼基數之和不超過寡核苷酸中核鹼基的總數。It is understood that the sum of the number of nucleobases in the introns and exons does not exceed the total number of nucleobases in the oligonucleotide.
在實施方式中,寡核苷酸可包含不能與靶RNA特異性結合,並且特別地不與靶RNA反向互補的一或多個核鹼基(在與靶RNA反向互補的連續序列的情況下)。In embodiments, the oligonucleotide may comprise one or more nucleobases that are incapable of specifically binding to the target RNA, and specifically are not reverse complementary to the target RNA (in the case of a contiguous sequence that is reverse complementary to the target RNA Down).
寡核苷酸的長度為多達200、多達175或多達150,較佳的是多達100,並且更佳的是多達50個核鹼基(包括核苷酸和核苷酸類似物)長,例如12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個核鹼基。當寡核苷酸的長度大於50個核苷酸時(例如當長度為75或100或150或200個核苷酸時),所述寡核苷酸亦可稱為多核苷酸。在較佳的實施方式中,寡核苷酸的長度為12至27個,較佳的是12至22個,亦即12、13、14、15、16、17、18、19、20、21或22個核鹼基,更佳的是從17、18、19或20至22個核鹼基,亦即17、18、19、20、21或22個核苷酸。在較佳的實施方式中,寡核苷酸的長度為20個核鹼基。Oligonucleotides are up to 200, up to 175, or up to 150, preferably up to 100, and more preferably up to 50 nucleobases (including nucleotides and nucleotide analogs) in length ) long, such as 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleobases. When the length of the oligonucleotide is greater than 50 nucleotides (for example, when the length is 75 or 100 or 150 or 200 nucleotides), the oligonucleotide may also be called a polynucleotide. In a preferred embodiment, the length of the oligonucleotide is 12 to 27, preferably 12 to 22, that is, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 Or 22 nucleobases, more preferably from 17, 18, 19 or 20 to 22 nucleobases, that is, 17, 18, 19, 20, 21 or 22 nucleotides. In a preferred embodiment, the oligonucleotide is 20 nucleobases in length.
特別地,第一態樣涉及寡核苷酸,其包含核鹼基序列(或核苷酸序列),該核鹼基序列(或核苷酸序列)具有根據SEQ ID NO: 1至11中的一個的核鹼基序列的鹼基配對特異性。此意指寡核苷酸的核鹼基序列包含根據SEQ ID NO: 1至11中的一個的核鹼基序列,或者其包含類似於SEQ ID NO: 1至11中的任一個的核鹼基序列。「類似」意指特徵在於所述SEQ ID NO的至少一個核鹼基被具有與被替換的核鹼基相同的鹼基配對特異性的核鹼基類似物替換。核鹼基類似物係以下解釋的修飾。較佳的實施方式涉及SEQ ID NO: 5、6、7、8和10,較佳的是SEQ ID NO: 8。上述寡核苷酸的實施方式都適用(只要相容)。在較佳的實施方式中,寡核苷酸的核鹼基序列(或核苷酸序列)由根據SEQ ID NO: 1至11中的一個的核鹼基序列組成,或其中該寡核苷酸的核鹼基序列由類似於SEQ ID NO: 1至11中的任一個的核鹼基序列組成。 寡核苷酸,修飾 In particular, the first aspect relates to an oligonucleotide comprising a nucleobase sequence (or nucleotide sequence) having a sequence according to SEQ ID NOs: 1 to 11 The base pairing specificity of a nucleobase sequence. This means that the nucleobase sequence of the oligonucleotide comprises a nucleobase sequence according to one of SEQ ID NOs: 1 to 11, or that it contains a nucleobase sequence similar to any one of SEQ ID NOs: 1 to 11 sequence. "Similar" means characterized in that at least one nucleobase of the SEQ ID NO is replaced by a nucleobase analog having the same base pairing specificity as the nucleobase being replaced. Nucleobase analogs are modifications explained below. Preferred embodiments relate to SEQ ID NO: 5, 6, 7, 8 and 10, with SEQ ID NO: 8 being preferred. The oligonucleotide embodiments described above are applicable (so long as they are compatible). In a preferred embodiment, the nucleobase sequence (or nucleotide sequence) of the oligonucleotide consists of a nucleobase sequence according to one of SEQ ID NOs: 1 to 11, or wherein the oligonucleotide The nucleobase sequence consists of a nucleobase sequence similar to any one of SEQ ID NOs: 1 to 11. Oligonucleotides, modifications
寡核苷酸可以被修飾(本文中稱為「經修飾的寡核苷酸」)。預期此改進穩定性,特別地對核酸酶的抗性。當將寡核苷酸原樣投與(亦即裸投與)於患者時,此係有利的。Oligonucleotides can be modified (referred to herein as "modified oligonucleotides"). This is expected to improve stability, particularly resistance to nucleases. This is advantageous when the oligonucleotide is administered unchanged (i.e., naked) to the patient.
因此,在實施方式中,寡核苷酸係經修飾的寡核苷酸。在實施方式中,將所述寡核苷酸的修飾與(天然)RNA寡核苷酸進行比較。經修飾的寡核苷酸包含經修飾的核苷酸間鍵和/或經修飾的核苷酸。經修飾的核苷酸係核苷酸類似物的同義詞。Thus, in embodiments, the oligonucleotide is a modified oligonucleotide. In embodiments, the modifications of the oligonucleotide are compared to (natural) RNA oligonucleotides. Modified oligonucleotides contain modified internucleotide linkages and/or modified nucleotides. Modified nucleotide is a synonym for nucleotide analog.
在較佳的實施方式中,寡核苷酸包含能夠使RNA雙鏈體對核酸酶(較佳的是核酸外切酶,特別地RNA酶H)具有抗性的修飾,其中該RNA雙鏈體包含該寡核苷酸和與其至少部分互補的寡核苷酸(互補寡核苷酸,亦即靶RNA)。換言之,寡核苷酸提供了RNA雙鏈體對核酸酶降解的抗性,該RNA雙鏈體包含該寡核苷酸和與其至少部分互補的寡核苷酸。此較佳的是藉由如上所述之核苷酸間鍵修飾(例如硫代磷酸酯修飾的核苷酸和/或糖(支架)修飾(例如2'-O-修飾糖修飾))來提供。因此寡核苷酸較佳的是包含具有提供雙鏈體核酸酶抗性的經修飾的區域(亦即至少2、3、4、5、6、7、8、9或10個經修飾的核苷酸)。In a preferred embodiment, the oligonucleotide comprises modifications that render the RNA duplex resistant to nucleases, preferably exonucleases, in particular RNase H, wherein the RNA duplex Comprised of the oligonucleotide and an oligonucleotide that is at least partially complementary to it (complementary oligonucleotide, ie target RNA). In other words, the oligonucleotide provides resistance to nuclease degradation of an RNA duplex comprising the oligonucleotide and an oligonucleotide that is at least partially complementary thereto. This is preferably provided by inter-nucleotide bond modifications (e.g. phosphorothioate modified nucleotides and/or sugar (scaffold) modifications (e.g. 2'-O-modified sugar modifications)) as described above . Therefore oligonucleotides preferably comprise regions with modifications that provide duplex nuclease resistance (i.e. at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nuclei). glycosides).
經修飾的寡核苷酸可包含核苷酸類似物和/或經修飾的核苷酸間鍵。當每個核苷酸間鍵被修飾時,寡核苷酸被稱為具有「骨架修飾」。Modified oligonucleotides may contain nucleotide analogs and/or modified internucleotide linkages. When each internucleotide bond is modified, an oligonucleotide is said to have "backbone modifications."
核苷酸類似物較佳的是包含鹼基修飾(「經修飾的鹼基」)和/或糖修飾(「經修飾的糖」,亦稱為支架修飾)的核苷酸。在實施方式中,鹼基修飾係天然嘌呤和嘧啶鹼基(例如腺嘌呤、尿嘧啶、鳥嘌呤、胞嘧啶和胸腺嘧啶)的經修飾的版本,如次黃嘌呤、假尿嘧啶、假胞嘧啶(pseudocytosine)、1-甲基假尿嘧啶、乳清酸、胍丁胺、離胺酸、2-硫代嘧啶(例如2-硫代尿嘧啶、2-硫代胸腺嘧啶)、G形夾(G-clamp)及其衍生物、5-取代的嘧啶(例如5-鹵代尿嘧啶、5-鹵代甲基尿嘧啶、5-三氟甲基尿嘧啶、5-丙炔基尿嘧啶、5-丙炔基胞嘧啶、5-胺基甲基尿嘧啶、5-羥甲基尿嘧啶、5-胺基甲基胞嘧啶、5'-甲基胞嘧啶、5'-甲基胞苷、5-羥甲基胞嘧啶、Super T或如例如Kumar等人 J. Org. Chem.[有機化學雜誌] 2014, 79, 5047;Leszczynska等人 Org. Biol. Chem.[有機生物化學] 2014, 12, 1052中所述)、吡唑并[1,5-a]-1,3,5-三𠯤C-核苷(如例如Lefoix等人 J. Org. Chem.[有機化學雜誌] 2014, 79, 3221中的)、7-脫氮鳥嘌呤、7-脫氮腺嘌呤、7-氮雜-2,6-二胺基嘌呤、8-氮雜-7-脫氮鳥嘌呤、8-氮雜-7-脫氮腺嘌呤、8-氮雜-7-脫氮-2,6-二胺基嘌呤、Super G、Super A、硼化胞嘧啶(如例如Nizioł等人 Bioorg. Med. Chem.[生物有機與藥物化學] 2014, 22, 3906中的)、假異胞苷、C(Pyc)(如例如Yamada等人 Org. Biomol. Chem.[有機與生物分子化學] 2014, 12, 2255中的)和N4-乙基胞嘧啶或其衍生物; N 2 -環戊基鳥嘌呤(cPent-G)、 N 2 -環戊基-2-胺基嘌呤(cPent-AP)和 N 2 -丙基-2-胺基嘌呤(Pr-AP)、碳水化合物修飾的尿嘧啶(如例如Kaura等人 Org. Lett.[有機快報] 2014, 16, 3308中的)、胺基酸修飾的尿嘧啶(如例如Guenther等人 Chem. Commun.[化學通訊] 2014, 50, 9007中的);或其衍生物;或簡併或通用鹼基(像2,6-二氟甲苯),或不存在鹼基(像無鹼基位點)(例如1-去氧核糖、1,2-二去氧核糖、1-去氧-2-O-甲基核糖;或吡咯啶衍生物,其中環氧被氮替換(氮雜核糖(azaribose)))。可以在美國專利6,683,173(時代生物科學公司(Epoch Biosciences))中找到Super A、Super G和Super T的衍生物之實例。當摻入siRNA時,cPent-G、cPent-AP和Pr-AP顯示降低免疫刺激作用(Peacock H.等人 J. Am. Chem. Soc.[美國化學會誌] 2011, 133, 9200)。例如WO 2014/093924中描述了經修飾的鹼基的另外的實例。 Nucleotide analogs preferably include base modifications ("modified bases") and/or sugar modifications ("modified sugars", also known as scaffold modifications). In embodiments, the base modifications are modified versions of natural purine and pyrimidine bases (e.g., adenine, uracil, guanine, cytosine, and thymine), such as hypoxanthine, pseudouracil, pseudocytosine (pseudocytosine), 1-methylpseudouracil, orotic acid, agmatine, lysine, 2-thiopyrimidines (such as 2-thiouracil, 2-thiothymine), G-clip ( G-clamp) and its derivatives, 5-substituted pyrimidines (such as 5-halogenated uracil, 5-halogenated methyluracil, 5-trifluoromethyluracil, 5-propynyluracil, 5 -Proparnylcytosine, 5-aminomethyluracil, 5-hydroxymethyluracil, 5-aminomethylcytosine, 5'-methylcytosine, 5'-methylcytosine, 5 -Hydroxymethylcytosine, Super T or, for example, Kumar et al. J. Org. Chem . [Organic Chemistry Journal] 2014, 79 , 5047; Leszczynska et al. Org. Biol. Chem . [Organic Biochemistry] 2014, 12 , 1052), pyrazolo[1,5-a]-1,3,5-tri𠯤C-nucleoside (such as Lefoix et al. J. Org. Chem . [Journal of Organic Chemistry] 2014, 79 , 3221), 7-deazaguanine, 7-deazaadenine, 7-aza-2,6-diaminopurine, 8-aza-7-deazaguanine, 8-aza- 7-deazaadenine, 8-aza-7-deaza-2,6-diaminopurine, Super G, Super A, boronated cytosine (e.g. Nizioł et al . Bioorg. Med. Chem . [Biology] Organic and Medicinal Chemistry] 2014, 22, 3906), pseudoisocytidine, C(Pyc) (as e.g. Yamada et al. Org. Biomol. Chem . [Organic and Biomolecular Chemistry] 2014, 12, 2255) and N4-ethylcytosine or its derivatives; N2 -cyclopentylguanine (cPent-G), N2 -cyclopentyl-2-aminopurine (cPent-AP) and N2 -propyl- 2-aminopurine (Pr-AP), carbohydrate-modified uracil (e.g., Kaura et al. Org. Lett . [Organic Letters] 2014, 16, 3308), amino acid-modified uracil (e.g., e.g. Guenther et al. Chem. Commun. [Chemical Communications] 2014, 50, 9007); or derivatives thereof; or degenerate or universal bases (like 2,6-difluorotoluene), or no bases (like abasic site) (e.g. 1-deoxyribose, 1,2-dideoxyribose, 1-deoxy-2-O-methylribose; or pyrrolidine derivatives in which the epoxy is replaced by nitrogen (nitrogen azaribose))). Examples of derivatives of Super A, Super G and Super T can be found in US Patent 6,683,173 (Epoch Biosciences). When incorporated into siRNA, cPent-G, cPent-AP, and Pr-AP were shown to reduce immunostimulatory effects (Peacock H. et al. J. Am. Chem. Soc . [Journal of the American Chemical Society] 2011, 133 , 9200). Further examples of modified bases are described, for example, in WO 2014/093924.
較佳的經修飾的鹼基係5'-甲基胞嘧啶和5'-甲基胞苷。在較佳的實施方式中,寡核苷酸的所有胞嘧啶被修飾為(亦即被替換為)5'-甲基胞嘧啶或較佳的是5'-甲基胞苷。Preferred modified bases are 5'-methylcytosine and 5'-methylcytosine. In preferred embodiments, all cytosines of the oligonucleotide are modified (i.e., replaced with) 5'-methylcytosine or preferably 5'-methylcytosine.
通常,至少在與靶區域互補的寡核苷酸部分中使用核鹼基類似物來替換具有相同鹼基配對特異性的核鹼基。「鹼基配對」係指兩個核鹼基藉由氫鍵相互結合。特別地,替換胞嘧啶的核鹼基類似物能夠與鳥嘌呤鹼基配對,替換鳥嘌呤的核鹼基類似物能夠與胞嘧啶鹼基配對,替換腺嘌呤的核鹼基類似物能夠與尿嘧啶鹼基配對,並且替換尿嘧啶的核鹼基類似物能夠與腺嘌呤鹼基配對。Typically, nucleobase analogs are used to replace nucleobases with the same base pairing specificity in at least the portion of the oligonucleotide that is complementary to the target region. "Base pairing" means that two nucleobases are bonded to each other through hydrogen bonds. In particular, nucleobase analogs that replace cytosine can base pair with guanine, nucleobase analogs that replace guanine can base pair with cytosine, and nucleobase analogs that replace adenine can base pair with uracil. Base pairing, and nucleobase analogues that replace uracil are able to base pair with adenine.
寡核苷酸可包含至少1個,例如至少1、2、3、4、5、6、7、8、9或至少10個鹼基修飾。該等可以都是相同類型的修飾,或者它們可以包含兩種或更多種不同的修飾。The oligonucleotide may comprise at least 1, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 base modifications. These may all be the same type of modification, or they may contain two or more different modifications.
糖(支架)修飾可為核糖基部分的修飾,例如2’- O-修飾的RNA核苷酸,如2’- O-烷基或2’- O-(取代的)烷基,例如2’- O-甲基、2’- O-(2-氰乙基)、2’- O-(2-甲氧基)乙基(2'-MOE)、2’- O-(2-硫代甲基)乙基、2’- O-丁醯基、2’- O-炔丙基、2’- O-縮醛酯(如例如Biscans等人 Bioorg. Med. Chem.[生物有機與藥物化學] 2015, 23, 5360)、2’- O-烯丙基、2’- O-(2S-甲氧基丙基)、2’- O-(N-(胺基乙基)胺基甲醯基)甲基)(2'-AECM)、2’- O-(2-羧乙基)和胺基甲醯基衍生物(Yamada等人 Org. Biomol. Chem.[有機與生物分子化學] 2014, 12, 6457)、2’- O-(2-胺基)丙基、2’- O-(2-(二甲基胺基)丙基)、2’- O-(2-胺基)乙基、2’- O-(2-(二甲基胺基)乙基);2'-去氧(DNA);2’- O-(鹵代烷氧基)甲基(Arai K.等人 Bioorg. Med. Chem.[生物有機與藥物化學] 2011, 21, 6285)例如2’- O-(2-氯乙氧基)甲基(MCEM)、2’- O-(2,2-二氯乙氧基)甲基(DCEM);2’- O-烷氧基羰基,例如2’- O-[2-(甲氧基羰基)乙基](MOCE)、2’- O-[2-(N-甲基胺基甲醯基)乙基](MCE)、2’- O-[2-(N,N-二甲基胺基甲醯基)乙基](DCME)、2’- O-[2-(甲硫基)乙基](2'-MTE)、2'-(ω-O-絲胺醇);2'-鹵代,例如2'-F、FANA(2'-F阿拉伯糖基核酸);2’,4’-二氟-2’-去氧;碳環糖和氮雜糖修飾;其他經修飾的RNA核苷酸係3'-O-取代的(例如3'-O-甲基、3'-O-丁醯基、3'-O-炔丙基)、4'-取代的(例如4'-胺基甲基-2'-O-甲基或4'-胺基甲基-2'-氟;5'-取代的,例如5'-甲基)或CNA(Østergaard等人 ACS Chem. Biol.[ACS生物化學雜誌] 2014, 22, 6227)。亦設想了上述的衍生物。 The sugar (scaffold) modification may be a modification of the ribosyl moiety, such as a 2'- O -modified RNA nucleotide, such as a 2'- O -alkyl or a 2'- O- (substituted)alkyl, such as a 2' - O -methyl, 2'- O -(2-cyanoethyl), 2'- O -(2-methoxy)ethyl (2'-MOE), 2'- O -(2-thio Methyl)ethyl, 2'- O -butyl, 2'- O -propargyl, 2'- O -acetal ester (such as Biscans et al . Bioorg. Med. Chem . [Bioorganic and Medicinal Chemistry] 2015 , 23 , 5360), 2'- O -allyl, 2'- O -(2S-methoxypropyl), 2'- O -(N-(aminoethyl)aminomethyl) Methyl) (2'-AECM), 2'- O -(2-carboxyethyl) and aminoformyl derivatives (Yamada et al. Org. Biomol. Chem . [Organic and Biomolecular Chemistry] 2014, 12 , 6457), 2'- O -(2-amino)propyl, 2'- O -(2-(dimethylamino)propyl), 2'- O -(2-amino)ethyl , 2'- O -(2-(dimethylamino)ethyl); 2'-deoxy (DNA); 2'- O -(haloalkoxy)methyl (Arai K. et al . Bioorg. Med . Chem . [Bioorganic and Medicinal Chemistry] 2011, 21 , 6285) For example, 2'- O -(2-chloroethoxy)methyl (MCEM), 2'- O -(2,2-dichloroethoxy methyl)methyl (DCEM); 2'- O -alkoxycarbonyl, such as 2'- O -[2-(methoxycarbonyl)ethyl] (MOCE), 2'- O -[2-(N -methylaminoformyl)ethyl] (MCE), 2'- O -[2-(N,N-dimethylaminoformyl)ethyl] (DCME), 2'- O - [2-(Methylthio)ethyl] (2'-MTE), 2'-(ω-O-serinol);2'-halo, such as 2'-F, FANA (2'-F Arabinol) Glycosyl nucleic acid); 2',4'-difluoro-2'-deoxy; carbocyclic sugar and aza sugar modification; other modified RNA nucleotides are 3'-O-substituted (such as 3'- O-methyl, 3'-O-butyl, 3'-O-propargyl), 4'-substituted (e.g. 4'-aminomethyl-2'-O-methyl or 4'-amino Methyl-2'-fluoro;5'-substituted, e.g. 5'-methyl) or CNA (Østergaard et al. ACS Chem. Biol . 2014, 22, 6227). Derivatives of the above are also contemplated.
此外,糖(支架)修飾可以包括雙環核酸單體(BNA),該雙環核酸單體可為橋接的核酸單體。所述BNA的每次出現都可能導致獨立地選自由以下組成之群組的單體:構象限制性核苷酸(CRN)單體、鎖核酸(LNA)單體、木-LNA單體、α-LNA單體、α-L-LNA單體、β-D-LNA單體、2'-胺基-LNA單體、2'-(烷基胺基)-LNA單體、2'-(醯胺基)-LNA單體、2'-N-取代的-2'-胺基-LNA單體、2'-硫代-LNA單體、(2’-O,4’-C)約束的乙基(cEt)BNA單體、(2’-O,4’-C)約束的甲氧基乙基(cMOE)BNA單體、2’,4’-BNA NC(N-H)單體、2’,4’-BNA NC(N-Me)單體、2’,4’-BNA NC(N-Bn)單體、乙烯橋接核酸(ENA)單體、carba LNA(cLNA)單體、3,4-二氫-2H-哌喃核酸(DpNA)單體、2'-C-橋接雙環核苷酸(CBBN)單體、雜環橋接BNA單體(如三唑基或四唑基連接的)、醯胺基橋接BNA單體、尿素橋接BNA單體、磺胺橋接BNA單體、雙環碳環核苷酸單體、TriNA單體、α-L-TriNA單體、雙環DNA(bcDNA)單體、F-bcDNA單體、三環DNA(tcDNA)單體、F-tcDNA單體、氧雜環丁烷核苷酸單體、衍生自2'-胺基-LNA的鎖PMO單體、胍橋接核酸(GuNA)單體、螺環丙烯橋接核酸(scpBNA)單體及其衍生物。 Additionally, sugar (scaffold) modifications may include bicyclic nucleic acid monomers (BNA), which may be bridged nucleic acid monomers. Each occurrence of said BNA may result in a monomer independently selected from the group consisting of: conformationally restricted nucleotide (CRN) monomer, locked nucleic acid (LNA) monomer, wood-LNA monomer, alpha -LNA monomer, α-L-LNA monomer, β-D-LNA monomer, 2'-amino-LNA monomer, 2'-(alkylamino)-LNA monomer, 2'-(遯Amino)-LNA monomer, 2'-N-substituted-2'-amino-LNA monomer, 2'-thio-LNA monomer, (2'-O,4'-C) constrained ethyl (cEt) BNA monomer, (2'-O,4'-C) constrained methoxyethyl (cMOE) BNA monomer, 2',4'-BNA NC (NH) monomer, 2', 4'-BNA NC (N-Me) monomer, 2',4'-BNA NC (N-Bn) monomer, ethylene bridged nucleic acid (ENA) monomer, carba LNA (cLNA) monomer, 3,4- Dihydro-2H-piranucleic acid (DpNA) monomer, 2'-C-bridged bicyclic nucleotide (CBBN) monomer, heterocyclic bridged BNA monomer (such as triazolyl or tetrazolyl linked), chelate Amino-bridged BNA monomer, urea-bridged BNA monomer, sulfonamide-bridged BNA monomer, bicyclic carbocyclic nucleotide monomer, TriNA monomer, α-L-TriNA monomer, bicyclic DNA (bcDNA) monomer, F- bcDNA monomer, tricyclic DNA (tcDNA) monomer, F-tcDNA monomer, oxetane nucleotide monomer, locked PMO monomer derived from 2'-amino-LNA, guanidine bridged nucleic acid (GuNA ) monomer, spirocyclopropene bridged nucleic acid (scpBNA) monomer and its derivatives.
較佳的糖修飾選自由以下組成之群組:2’- O-修飾,較佳的是2’- O-烷基或2’- O-(取代的)烷基,更佳的是2’- O-甲基或2’- O-(2-甲氧基)乙基(2'-MOE)和對BNA單體(較佳的是CRN單體或鎖核酸(LNA)單體)的修飾。更佳的是2’- O-甲基糖修飾和對LNA的修飾的組合。甚至更佳的是2’- O-甲基作為唯一的糖修飾。 Preferred sugar modifications are selected from the group consisting of: 2'- O -modification, preferably 2'- O -alkyl or 2'- O- (substituted)alkyl, more preferably 2' -O -methyl or 2'- O- (2-methoxy)ethyl (2'-MOE) and modification of BNA monomer (preferably CRN monomer or locked nucleic acid (LNA) monomer) . Even more preferred is a combination of 2'- O -methyl sugar modification and modification of LNA. Even better is 2'- O -methyl as the only sugar modification.
寡核苷酸可包含至少1個,例如至少1、2、3、4、5、6、7、8、9或至少10個糖修飾。該等可以都是相同類型的修飾,或者它們可以包含兩種或更多種不同的修飾。The oligonucleotide may comprise at least 1, for example at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or at least 10 sugar modifications. These may all be the same type of modification, or they may contain two or more different modifications.
核苷酸間鍵修飾可以選自由以下組成之群組:RNA的經修飾的磷酸二酯,如硫代磷酸酯(PS)、手性純硫代磷酸酯、(R)-硫代磷酸酯、(S)-硫代磷酸酯、二硫代磷酸酯(PS2)、磷醯基乙酸酯(PACE)、磷醯基乙醯胺(PACA),、硫代磷醯基乙酸酯(硫代PACE)、硫代磷醯基乙醯胺、硫代磷酸酯前驅藥、H-膦酸酯、膦酸甲酯、硫代膦酸甲酯、磷酸甲酯、硫代磷酸甲酯、磷酸乙酯、硫代磷酸乙酯、硼磷酸酯、硼硫代磷酸酯、硼磷酸甲酯、硼硫代磷酸甲酯、硼膦酸甲酯、硼硫代磷酸甲酯、磷酸酯、磷酸三酯、胺基烷基磷酸三酯及其衍生物。另一種修飾包括磷酸胍、亞磷醯胺、胺基磷醯酯、N3’àP5’胺基磷醯酯、二胺基磷酸酯、硫代二胺基磷酸酯、胺基磺酸酯、二亞甲基亞碸、醯胺、磺酸酯、矽氧烷、硫化物、碸、甲乙醯基、硫代甲乙醯基、亞甲基甲乙醯基、烯基、亞甲基肼基、磺醯胺、醯胺、三唑、草醯基、胺基甲酸酯、亞甲基亞胺基(MMI)和硫代乙醯胺基核酸(TANA)以及它們的衍生物。手性純硫代磷酸酯鍵之實例描述於例如WO 2014/010250或WO 2017/062862(浪潮生命科學公司(WaVe Life Sciences))中。磷酸胍鍵之實例描述於WO 2016/028187(Noogen公司)中。亦包括各種鹽、混合鹽和游離酸形式,以及3’à3’和2’à5’鍵。Internucleotide bond modifications may be selected from the group consisting of: modified phosphodiesters of RNA such as phosphorothioate (PS), chiral pure phosphorothioate, (R)-phosphorothioate, (S)-Phosphorothioate, phosphorodithioate (PS2), phosphoryl acetate (PACE), phosphoryl acetamide (PACA), phosphorothioate (thio PACE), phosphorothioate acetamide, phosphorothioate prodrug, H-phosphonate, methyl phosphonate, methyl thiophosphonate, methyl phosphate, methyl phosphorothioate, ethyl phosphate , Ethyl phosphorothioate, borophosphate ester, phosphorothioate ester, methyl phosphorothioate, methyl phosphorothioate, methyl phosphorothioate, methyl phosphorothioate, phosphoric acid ester, triester phosphate, amine Alkyl phosphate trysters and their derivatives. Another modification includes guanidine phosphate, phosphoramidite, aminophosphonate, N3'àP5'aminophosphatide, diaminophosphate, thiodiaminophosphate, amidosulfonate, diacetate Methylene, amide, sulfonate, siloxane, sulfide, sulfonate, methylacetyl, thiomethylacetyl, methylenemethylacetyl, alkenyl, methylenehydrazino, sulfonamide , amide, triazole, oxalyl, carbamate, methyleneimine (MMI) and thioacetamide-based nucleic acids (TANA) and their derivatives. Examples of chirally pure phosphorothioate bonds are described, for example, in WO 2014/010250 or WO 2017/062862 (WaVe Life Sciences). Examples of guanidine phosphate bonds are described in WO 2016/028187 (Noogen). Also included are various salts, mixed salts and free acid forms, as well as 3’à3’ and 2’à5’ linkages.
寡核苷酸可包含至少1個,例如至少1、2、3、4、5、6、7、8、9或至少10個核苷酸間鍵修飾。該等可以都是相同類型的修飾,或者它們可以包含兩種或更多種不同的修飾。The oligonucleotide may comprise at least 1, eg, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10, internucleotide linkage modifications. These may all be the same type of modification, or they may contain two or more different modifications.
較佳的核苷酸間鍵修飾係PS、PS2、胺基磷醯酯或二胺基磷酸酯,較佳的是PS。在較佳的實施方式中,寡核苷酸的所有核苷酸間鍵都是PS鍵。換言之,寡核苷酸的骨架係PS。Preferred inter-nucleotide bond modifications are PS, PS2, aminophosphonate or diaminophosphate, with PS being preferred. In a preferred embodiment, all internucleotide linkages of the oligonucleotide are PS linkages. In other words, the backbone of the oligonucleotide is PS.
在一個實施方式中,寡核苷酸5'端的多個核苷酸中的一個和/或寡核苷酸3'端的多個核苷酸中的一個被修飾(亦即係核苷酸類似物和/或具有經修飾的核苷酸間鍵),並且寡核苷酸中心部分的核苷酸未被修飾。「5'或3'端的核苷酸」涵蓋相應端寡核苷酸的20%核苷酸,並且「中心部分」涵蓋寡核苷酸的剩餘核苷酸。例如,如果寡核苷酸具有由20個核苷酸組成的序列,則寡核苷酸5'端的4個核苷酸係5'端的核苷酸,寡核苷酸3'端的4個核苷酸係3'端的核苷酸,並且其餘12個核苷酸係寡核苷酸中心部分的核苷酸。如果根據百分比的核苷酸數不是整數,則將其四捨五入到最接近的整數(最多為4的第一個小數向下舍,至少為5的第一個小數向上入)。因此,在一個實施方式中,存在於寡核苷酸5'端和/或3'端的至少一個核苷酸和/或至少一個核苷酸間鍵被修飾,而其他核苷酸和其他核苷酸間鍵未被修飾。In one embodiment, one of the nucleotides at the 5' end of the oligonucleotide and/or one of the nucleotides at the 3' end of the oligonucleotide is modified (i.e., is a nucleotide analog and/or have modified internucleotide linkages), and the nucleotides in the central portion of the oligonucleotide are not modified. The "5' or 3' end nucleotides" cover 20% of the nucleotides of the corresponding end of the oligonucleotide, and the "central portion" covers the remaining nucleotides of the oligonucleotide. For example, if the oligonucleotide has a sequence consisting of 20 nucleotides, then the 4 nucleotides at the 5' end of the oligonucleotide are the 5' end nucleotides and the 4 nucleotides at the 3' end of the oligonucleotide are The acid is the nucleotide at the 3' end, and the remaining 12 nucleotides are the nucleotides in the central portion of the oligonucleotide. If the number of nucleotides based on a percentage is not a whole number, it is rounded to the nearest whole number (the first decimal point up to 4 is rounded down, the first decimal point at least 5 is rounded up). Thus, in one embodiment, at least one nucleotide and/or at least one internucleotide linkage present at the 5' end and/or 3' end of the oligonucleotide is modified, while other nucleotides and other nucleosides The interacid bond is not modified.
所涵蓋的修飾均已在本文中定義。預期在此類位置修飾寡核苷酸可能有助於改進其穩定性或對核酸外切酶的抗性。當將寡核苷酸原樣投與(亦即裸投與)於患者時,此係有利的。Covered modifications are defined herein. It is expected that modification of the oligonucleotide at such positions may help improve its stability or resistance to exonucleases. This is advantageous when the oligonucleotide is administered unchanged (i.e., naked) to the patient.
在實施方式中,寡核苷酸5'處的最後1、2、3、4個核苷酸和/或核苷酸間鍵被修飾。In embodiments, the last 1, 2, 3, 4 nucleotides and/or inter-nucleotide linkages 5' to the oligonucleotide are modified.
在實施方式中,寡核苷酸3'處的最後1、2、3、4個核苷酸和/或核苷酸間鍵被修飾。In embodiments, the last 1, 2, 3, 4 nucleotides and/or inter-nucleotide linkages 3' to the oligonucleotide are modified.
在實施方式中,寡核苷酸5'和3'處的最後1、2、3、4個核苷酸和/或核苷酸間鍵被修飾。較佳的是,寡核苷酸5'和3'處的2個核苷酸和/或2個核苷酸間鍵被修飾。較佳的是,寡核苷酸5'和3'處的3個核苷酸和/或核苷酸間鍵被修飾。較佳的是,寡核苷酸5'和3'處的4個核苷酸和/或核苷酸間鍵被修飾。In embodiments, the last 1, 2, 3, 4 nucleotides and/or inter-nucleotide linkages at the 5' and 3' of the oligonucleotide are modified. Preferably, 2 nucleotides and/or 2 inter-nucleotide linkages at 5' and 3' of the oligonucleotide are modified. Preferably, the three nucleotides 5' and 3' of the oligonucleotide and/or the inter-nucleotide linkage are modified. Preferably, the four nucleotides 5' and 3' of the oligonucleotide and/or the inter-nucleotide linkage are modified.
例如,對於SEQ ID NO: 1至10,寡核苷酸包含SEQ ID NO: 1至10的核苷酸5至16之間的未經修飾的核苷酸間鍵,以及該寡核苷酸的至少兩個最5'端核苷酸之間和該寡核苷酸的至少兩個最3'端核苷酸之間的經修飾的核苷酸間鍵。例如,對於SEQ ID NO: 11,寡核苷酸包含SEQ ID NO: 11的核苷酸5至15之間的未經修飾的核苷酸間鍵,以及該寡核苷酸的至少兩個最5'端核苷酸之間和該寡核苷酸的至少兩個最3'端核苷酸之間的經修飾的核苷酸間鍵。For example, for SEQ ID NO: 1 to 10, the oligonucleotide includes an unmodified internucleotide linkage between nucleotides 5 to 16 of SEQ ID NO: 1 to 10, and the oligonucleotide A modified internucleotide linkage between at least two 5'most nucleotides and between at least two 3'most nucleotides of the oligonucleotide. For example, for SEQ ID NO: 11, the oligonucleotide includes an unmodified internucleotide linkage between nucleotides 5 to 15 of SEQ ID NO: 11, and at least two of the last two nucleotides of the oligonucleotide. Modified internucleotide linkages between the 5' terminal nucleotides and between the at least two 3' terminal nucleotides of the oligonucleotide.
較佳的是,寡核苷酸的5'和/或3'端的至少1個,並且較佳的是2至4個,更佳的是4個核苷酸,以及視需要所有核苷酸被修飾,特別地藉由具有經修飾的核苷酸間鍵。Preferably, at least 1, and preferably 2 to 4, more preferably 4 nucleotides at the 5' and/or 3' end of the oligonucleotide, and optionally all nucleotides are Modified, particularly by having modified internucleotide linkages.
修飾(特別地5'和/或3'端的核苷酸的修飾)較佳的是包含經修飾的核苷酸間鍵,更佳的是PS。此外,核苷酸較佳的是核苷酸類似物。此核苷酸類似物的特徵在於包含經修飾的糖(較佳的是2’- O-甲基)、經修飾的鹼基(較佳的是5'-甲基胞嘧啶)和/或係LNA單體。例如,設想了表徵核苷酸類似物的以下組合:僅經修飾的糖;經修飾的糖和經修飾的鹼基;僅LNA單體;LNA單體和經修飾的鹼基。 Modifications (especially modifications of the 5' and/or 3' end nucleotides) preferably comprise modified inter-nucleotide linkages, more preferably PS. Furthermore, the nucleotide is preferably a nucleotide analog. This nucleotide analog is characterized by containing a modified sugar (preferably 2'- O -methyl), a modified base (preferably 5'-methylcytosine) and/or a system LNA monomer. For example, the following combinations are contemplated to characterize nucleotide analogs: modified sugar only; modified sugar and modified base; LNA monomer only; LNA monomer and modified base.
在較佳的實施方式中,寡核苷酸的所有核苷酸間鍵都是PS鍵,並且寡核苷酸的所有核苷酸都具有2’- O-甲基鹼基。其中,較佳的是,寡核苷酸的所有胞苷被修飾為5-甲基胞苷(亦即寡核苷酸不包含胞苷,而是包含5-甲基胞苷,特別地在與靶RNA中鳥苷配對的位置處)。 In a preferred embodiment, all internucleotide linkages of the oligonucleotide are PS linkages, and all nucleotides of the oligonucleotide have 2'- O -methyl bases. Among them, preferably, all cytidines of the oligonucleotide are modified to 5-methylcytidine (that is, the oligonucleotide does not contain cytidine, but contains 5-methylcytidine, especially when combined with at the position where the guanosine pairs in the target RNA).
因此,在實施方式中,當提到寡核苷酸5'和/或3'端的修飾時,此類修飾係: -經修飾的核苷酸間鍵為PS,和/或 -經修飾的糖為2'-O-甲基 -經修飾的鹼基為5-甲基胞嘧啶和/或 -經修飾的核苷酸為鎖核酸(LNA)單體。 Therefore, in embodiments, when referring to modifications at the 5' and/or 3' end of an oligonucleotide, such modifications are: - the modified internucleotide bond is PS, and/or -Modified sugar is 2'-O-methyl -The modified base is 5-methylcytosine and/or -The modified nucleotides are locked nucleic acid (LNA) monomers.
因此,在實施方式中,當提到寡核苷酸5'和/或3'端的修飾時,此類修飾係: -經修飾的核苷酸間鍵為PS, -經修飾的糖為2'-O-甲基, -經修飾的鹼基為5-甲基胞嘧啶和/或 -經修飾的核苷酸為鎖核酸(LNA)單體。 Therefore, in embodiments, when referring to modifications at the 5' and/or 3' end of an oligonucleotide, such modifications are: -The modified internucleotide bond is PS, -The modified sugar is 2'-O-methyl, -The modified base is 5-methylcytosine and/or -The modified nucleotides are locked nucleic acid (LNA) monomers.
較佳的是,此類修飾係: -經修飾的核苷酸間鍵為PS並且 -經修飾的糖為2'-O-甲基。 Preferably, such modifications are: - the modified internucleotide bond is PS and -The modified sugar is 2'-O-methyl.
較佳的是,此類修飾係: -經修飾的核苷酸間鍵為PS, -經修飾的糖為2'-O-甲基並且 -經修飾的鹼基為5-甲基胞嘧啶。 Preferably, such modifications are: -The modified internucleotide bond is PS, -The modified sugar is 2'-O-methyl and -The modified base is 5-methylcytosine.
較佳的是,此類修飾係: -經修飾的核苷酸間鍵為PS並且 -經修飾的核苷酸為鎖核酸(LNA)單體。 Preferably, such modifications are: - the modified internucleotide bond is PS and -The modified nucleotides are locked nucleic acid (LNA) monomers.
較佳的是,此類修飾係: -經修飾的核苷酸間鍵為PS, -經修飾的核苷酸為鎖核酸(LNA)單體並且 -經修飾的鹼基為5-甲基胞嘧啶。 Preferably, such modifications are: -The modified internucleotide bond is PS, - the modified nucleotide is a locked nucleic acid (LNA) monomer and -The modified base is 5-methylcytosine.
在實施方式中,寡核苷酸如下:寡核苷酸中心部分的核苷酸間鍵未被修飾,並且較佳的是寡核苷酸的2至4個最5'端和/或2至4個最3'端的核苷酸間鍵被修飾,較佳的是該等核苷酸間鍵為硫代磷酸酯核苷酸間鍵。In embodiments, the oligonucleotide is as follows: the internucleotide linkages in the central portion of the oligonucleotide are unmodified, and preferably the 2 to 4 most 5' ends of the oligonucleotide and/or the 2 to The 4 most 3' end inter-nucleotide bonds are modified, preferably these inter-nucleotide bonds are phosphorothioate inter-nucleotide bonds.
在實施方式中,寡核苷酸被如下修飾:每個核苷酸間鍵均是硫代磷酸酯鍵,並且所有核苷酸都具有2'-O-甲基鹼基。In embodiments, the oligonucleotide is modified such that each internucleotide linkage is a phosphorothioate linkage and all nucleotides have a 2'-O-methyl base.
在較佳的實施方式中,寡核苷酸被如下修飾:每個核苷酸間鍵均是硫代磷酸酯鍵,所有胞苷都是5'-甲基胞苷,並且所有核苷酸都具有2'-O-甲基鹼基。In a preferred embodiment, the oligonucleotide is modified such that each internucleotide linkage is a phosphorothioate bond, all cytidines are 5'-methylcytidines, and all nucleotides are Has 2'-O-methyl base.
較佳的寡核苷酸之實例表徵如下: - 每個核苷酸間鍵都是硫代磷酸酯鍵,並且所有核苷酸都具有2’- O-甲基鹼基,例如:具有包含以下序列或由以下序列組成的核苷酸序列的寡核苷酸:SEQ ID NO: 12(SEQ ID NO: 1的核鹼基序列)、SEQ ID NO: 13(SEQ ID NO: 2的核鹼基序列)、SEQ ID NO: 14(SEQ ID NO: 3的核鹼基序列)、SEQ ID NO: 15(SEQ ID NO: 4的核鹼基序列)、SEQ ID NO: 16(SEQ ID NO: 5的核鹼基序列)、SEQ ID NO: 17(SEQ ID NO: 10的核鹼基序列)、SEQ ID NO: 18(SEQ ID NO: 7的核鹼基序列)、SEQ ID NO: 19(SEQ ID NO: 8的核鹼基序列)、SEQ ID NO: 20(SEQ ID NO: 9的核鹼基序列)、SEQ ID NO: 21(SEQ ID NO: 10的核鹼基序列)或SEQ ID NO: 22(SEQ ID NO: 11的核鹼基序列) - 每個核苷酸間鍵均是硫代磷酸酯鍵,所有胞苷都是5'-甲基胞苷,並且所有核苷酸都具有2’- O-甲基鹼基,例如:具有包含以下序列或由以下序列組成的核苷酸序列的寡核苷酸:SEQ ID NO: 26(SEQ ID NO: 11的核鹼基序列)、SEQ ID NO: 30(SEQ ID NO: 6的核鹼基序列)或SEQ ID NO: 34(SEQ ID NO: 7的核鹼基序列)。 Examples of preferred oligonucleotides are characterized as follows: - Each internucleotide linkage is a phosphorothioate linkage and all nucleotides have a 2'- O -methyl base, for example: having An oligonucleotide having a sequence or a nucleotide sequence consisting of: SEQ ID NO: 12 (nucleobase sequence of SEQ ID NO: 1), SEQ ID NO: 13 (nucleobase sequence of SEQ ID NO: 2 sequence), SEQ ID NO: 14 (nucleobase sequence of SEQ ID NO: 3), SEQ ID NO: 15 (nucleobase sequence of SEQ ID NO: 4), SEQ ID NO: 16 (nucleobase sequence of SEQ ID NO: 5 SEQ ID NO: 17 (the nucleobase sequence of SEQ ID NO: 10), SEQ ID NO: 18 (the nucleobase sequence of SEQ ID NO: 7), SEQ ID NO: 19 (the nucleobase sequence of SEQ ID NO: 7) ID NO: 8), SEQ ID NO: 20 (Nucleobase sequence of SEQ ID NO: 9), SEQ ID NO: 21 (SEQ ID NO: 10), or SEQ ID NO : 22 (nucleobase sequence of SEQ ID NO: 11) - Each internucleotide bond is a phosphorothioate bond, all cytidines are 5'-methylcytidines, and all nucleotides have 2'- O -methyl base, for example: an oligonucleotide having a nucleotide sequence comprising or consisting of the following sequence: SEQ ID NO: 26 (nucleobase sequence of SEQ ID NO: 11), SEQ ID NO: 30 (nucleobase sequence of SEQ ID NO: 6) or SEQ ID NO: 34 (nucleobase sequence of SEQ ID NO: 7).
因此,在較佳的實施方式中,寡核苷酸包含根據SEQ ID NO: 12至22、26、30和34中的一個的核苷酸序列。在更佳的實施方式中,寡核苷酸的核苷酸序列由根據SEQ ID NO: 12至22、26、30和34中的一個的核苷酸序列組成。在該等實施方式中,SEQ ID No: 17-19、26、30和34係較佳的;其中,SEQ ID NO: 19、26、30和34係較佳的,特別地SEQ ID NO: 19。Therefore, in a preferred embodiment, the oligonucleotide comprises a nucleotide sequence according to one of SEQ ID NO: 12 to 22, 26, 30 and 34. In a more preferred embodiment, the nucleotide sequence of the oligonucleotide consists of a nucleotide sequence according to one of SEQ ID NOs: 12 to 22, 26, 30 and 34. In these embodiments, SEQ ID NO: 17-19, 26, 30 and 34 are preferred; among them, SEQ ID NO: 19, 26, 30 and 34 are preferred, especially SEQ ID NO: 19 .
因此,在實施方式中,寡核苷酸由SEQ ID NO: 19、34、17、18、30或26組成,並表現出有吸引力的治療活性,如實驗部分所表明的(參見例如圖4、圖5、圖6)。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。Accordingly, in embodiments, the oligonucleotide consists of SEQ ID NO: 19, 34, 17, 18, 30 or 26 and exhibits attractive therapeutic activity as demonstrated in the experimental section (see e.g. Figure 4 , Figure 5, Figure 6). In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
因此,在實施方式中,寡核苷酸包含SEQ ID NO: 19或8。在實施方式中,此類寡核苷酸的長度為20至100個核苷酸。在實施方式中,寡核苷酸由SEQ ID NO: 19或8組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。Thus, in embodiments, the oligonucleotide comprises SEQ ID NO: 19 or 8. In embodiments, such oligonucleotides are 20 to 100 nucleotides in length. In embodiments, the oligonucleotide consists of SEQ ID NO: 19 or 8. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
因此,在實施方式中,寡核苷酸包含SEQ ID NO: 34或18或7。在實施方式中,此類寡核苷酸的長度為20至100個核苷酸。在實施方式中,寡核苷酸由SEQ ID NO: 34或18或7組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。Thus, in embodiments, the oligonucleotide comprises SEQ ID NO: 34 or 18 or 7. In embodiments, such oligonucleotides are 20 to 100 nucleotides in length. In embodiments, the oligonucleotide consists of SEQ ID NO: 34 or 18 or 7. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
因此,在實施方式中,寡核苷酸包含SEQ ID NO: 30或17或6。在實施方式中,此類寡核苷酸的長度為20至100個核苷酸。在實施方式中,寡核苷酸由SEQ ID NO: 30或17或6組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。Thus, in embodiments, the oligonucleotide comprises SEQ ID NO: 30 or 17 or 6. In embodiments, such oligonucleotides are 20 to 100 nucleotides in length. In embodiments, the oligonucleotide consists of SEQ ID NO: 30 or 17 or 6. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
因此,在實施方式中,寡核苷酸包含SEQ ID NO: 26或16或5。在實施方式中,此類寡核苷酸的長度為20至100個核苷酸。在實施方式中,寡核苷酸由SEQ ID NO: 26或16或5組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。Thus, in embodiments, the oligonucleotide comprises SEQ ID NO: 26 or 16 or 5. In embodiments, such oligonucleotides are 20 to 100 nucleotides in length. In embodiments, the oligonucleotide consists of SEQ ID NO: 26 or 16 or 5. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
在實施方式中,提供了寡核苷酸,該寡核苷酸包含SEQ ID NO: 12、13、14、15、16、17、18、19、20、21、22中的一個,並且長度為20至100個核苷酸,或者該寡核苷酸由SEQ ID NO: 12、13、14、15、16、17、18、19、20、21、22中的一個組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。In embodiments, an oligonucleotide is provided comprising one of SEQ ID NOs: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and having a length of 20 to 100 nucleotides, or the oligonucleotide consists of one of SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
在實施方式中,提供了寡核苷酸,該寡核苷酸包含SEQ ID NO: 34、30或26中的一個,並且長度為20至100個核苷酸,或者該寡核苷酸由SEQ ID NO: 34、30或26中的一個組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。In embodiments, an oligonucleotide is provided, the oligonucleotide comprising one of SEQ ID NO: 34, 30 or 26 and being 20 to 100 nucleotides in length, or the oligonucleotide consisting of SEQ ID NO: one of 34, 30 or 26. In this embodiment, the oligonucleotide may be further modified as defined earlier herein.
在實施方式中,提供了寡核苷酸,該寡核苷酸包含SEQ ID NO: 19或34,並且長度為20至100個核苷酸,或者該寡核苷酸由SEQ ID NO: 19或34組成。在此實施方式中,寡核苷酸可以如本文早先定義的被進一步修飾。 寡核苷酸,共軛 In embodiments, oligonucleotides are provided that comprise SEQ ID NO: 19 or 34 and are 20 to 100 nucleotides in length, or the oligonucleotide consists of SEQ ID NO: 19 or 34 composition. In this embodiment, the oligonucleotide may be further modified as defined earlier herein. Oligonucleotide, conjugated
在一些實施方式中,寡核苷酸與一或多個配體共軛。配體較佳的是能夠將寡核苷酸靶向和/或遞送至(或進入)器官、組織和/或細胞,例如腎、腎組織或腎細胞、或膀胱、膀胱組織或膀胱細胞,特別地膀胱上皮細胞。In some embodiments, an oligonucleotide is conjugated to one or more ligands. The ligand is preferably capable of targeting and/or delivering the oligonucleotide to (or into) an organ, tissue and/or cell, such as the kidney, renal tissue or kidney cells, or the bladder, bladder tissue or bladder cells, in particular bladder epithelial cells.
配體之實例係例如肽、維生素、適配體、碳水化合物或碳水化合物的混合物(Han等人, Nature Communications [自然通訊], 2016, doi:10.1038/ncomms10981;Cao等人, Mol. Ther. Nucleic Acids [分子療法核酸], 2016, doi:10.1038/mtna.2016.46)、蛋白質、小分子、抗體(或其抗原結合片段)、聚合物、藥物。碳水化合物共軛物基團配體之實例係葡萄糖、甘露糖、半乳糖、麥芽糖、果糖、N-乙醯半乳糖胺(GalNac)、葡糖胺、N-乙醯葡糖胺、葡萄糖-6-磷酸、甘露糖-6-磷酸和麥芽三糖。碳水化合物可以以多種形式存在,例如作為將碳水化合物連接到組成物組分的樹枝狀或支連結頭部分上的末端基團。碳水化合物亦可以包含在碳水化合物簇部分中,如GalNAc簇部分。碳水化合物簇部分可以包括靶向部分和視需要共軛物連接子。在一些實施方式中,碳水化合物簇部分包含1、2、3、4、5、6或更多個GalNAc基團。如本文所用,「碳水化合物簇」意指具有附接到支架或連接子基團的一或多個碳水化合物殘基的化合物,(參見,例如,Maier等人, 「Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting [與用於細胞靶向的多價碳水化合物簇共軛的反義寡核苷酸的合成],」 Bioconjugate Chem.[生物共軛物化學], 2003, (14): 18-29;Rensen等人, 「Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor [用於將脂蛋白靶向肝脫唾液酸糖蛋白受體的新型N-乙醯半乳糖胺封端的糖脂的設計與合成],」 J. Med. Chem.[藥物化學雜誌] 2004, (47): 5798-5808)。在此上下文中,「經修飾的碳水化合物」意指相對於天然存在的碳水化合物具有一或多個化學修飾的任何碳水化合物。如本文所用,「碳水化合物衍生物」意指可以使用碳水化合物作為起始材料或中間物合成的任何化合物。如本文所用,「碳水化合物」意指天然存在的碳水化合物、經修飾的碳水化合物或碳水化合物衍生物。兩種類型的賦形劑可以組合在一起形成如本文所鑒定的一種單一組成物。WO 2017/062862(浪潮生命科學公司)中描述了三價N-乙醯葡糖胺簇之實例,其亦描述了磺醯胺小分子簇。亦描述了小分子舍曲林(sertraline)的單一共軛物之實例(Ferrés-Coy等人, Mol. Psych.[分子精神病學] 2016, 21, 328),以及蛋白質結合小分子的共軛物,包括伊布洛芬(例如US 6,656,730 ISIS/伊奧尼斯製藥公司(Ionis Pharmaceutical))、精胺(例如Noir等人, J. Am. Chem Soc.[美國化學會誌] 2008, 130, 13500)、大茴香醯胺(例如Nakagawa等人, J. Am. Chem. Soc.[美國化學會誌] 2010, 132, 8848)和葉酸(例如Dohmen等人, Mol. Ther. Nucl. Acids[分子療法核酸] 2012, 1, e7)。 Examples of ligands are, for example, peptides, vitamins, aptamers, carbohydrates or mixtures of carbohydrates (Han et al., Nature Communications, 2016, doi:10.1038/ncomms10981; Cao et al., Mol. Ther. Nucleic Acids [Molecular Therapeutics Nucleic Acids], 2016, doi:10.1038/mtna.2016.46), proteins, small molecules, antibodies (or antigen-binding fragments thereof), polymers, drugs. Examples of carbohydrate conjugate group ligands are glucose, mannose, galactose, maltose, fructose, N-acetylgalactosamine (GalNac), glucosamine, N-acetylglucosamine, glucose-6 -Phosphate, mannose-6-phosphate and maltotriose. The carbohydrate may be present in a variety of forms, such as as terminal groups linking the carbohydrate to dendritic or branched head portions of the components of the composition. Carbohydrates can also be included in carbohydrate cluster moieties, such as GalNAc cluster moieties. The carbohydrate cluster moiety may include a targeting moiety and optionally a conjugate linker. In some embodiments, the carbohydrate cluster moiety contains 1, 2, 3, 4, 5, 6 or more GalNAc groups. As used herein, "carbohydrate cluster" means a compound having one or more carbohydrate residues attached to a scaffold or linker group (see, e.g., Maier et al., "Synthesis of Antisense Oligonucleotides Conjugated to a Multivalent Carbohydrate Cluster for Cellular Targeting [Synthesis of antisense oligonucleotides conjugated to multivalent carbohydrate clusters for cellular targeting],” Bioconjugate Chem. [Bioconjugate Chemistry], 2003, (14): 18-29; Rensen et al., “Design and Synthesis of Novel N-Acetylgalactosamine-Terminated Glycolipids for Targeting of Lipoproteins to the Hepatic Asiaglycoprotein Receptor Design and synthesis of galactosamine-capped glycolipids],” J. Med. Chem. [Journal of Medicinal Chemistry] 2004, (47): 5798-5808). In this context, "modified carbohydrate" means any carbohydrate that has one or more chemical modifications relative to naturally occurring carbohydrates. As used herein, "carbohydrate derivative" means any compound that can be synthesized using carbohydrates as starting materials or intermediates. As used herein, "carbohydrate" means a naturally occurring carbohydrate, a modified carbohydrate, or a carbohydrate derivative. Both types of excipients can be combined together to form a single composition as identified herein. Examples of trivalent N-acetylglucosamine clusters are described in WO 2017/062862 (Inspur Life Sciences), which also describes sulfonamide small molecule clusters. Examples of single conjugates of the small molecule sertraline are also described (Ferrés-Coy et al., Mol. Psych . [Molecular Psychiatry] 2016, 21, 328), as well as conjugates of protein-binding small molecules. , including iprofen (e.g., US 6,656,730 ISIS/Ionis Pharmaceutical), spermine (e.g., Noir et al., J. Am. Chem Soc . [Journal of the American Chemical Society] 2008, 130, 13500) , anisidamide (e.g., Nakagawa et al., J. Am. Chem. Soc . [Journal of the American Chemical Society] 2010, 132, 8848) and folic acid (e.g., Dohmen et al., Mol. Ther. Nucl. Acids [Molecular Therapy Nucleic Acids] ] 2012, 1, e7).
在實施方式中,寡核苷酸與石膽酸或二十碳五烯酸共軛。In embodiments, the oligonucleotide is conjugated to lithocholic acid or eicosapentaenoic acid.
在實施方式中,寡核苷酸(較佳的是經由其5'或3'端,更佳的是經由其3'端)與肽、維生素、適配體、碳水化合物或碳水化合物的混合物、蛋白質、小分子、抗體、聚合物、藥物、石膽酸、二十碳五烯酸或膽固醇部分共軛。In embodiments, the oligonucleotide (preferably via its 5' or 3' end, more preferably via its 3' end) is combined with a peptide, vitamin, aptamer, carbohydrate or mixture of carbohydrates, Proteins, small molecules, antibodies, polymers, drugs, lithocholic acid, eicosapentaenoic acid or cholesterol moieties are conjugated.
在較佳的實施方式中,寡核苷酸與小分子、適配體或抗體(或其抗原結合片段)共軛。較佳的抗體(或其抗原結合片段)對CD71(轉鐵蛋白受體)具有特異性(例如WO 2016/179257(CytoMx公司)中所述)或針對平衡核苷轉運蛋白(ENT),如3E10抗體,如例如Weisbart等人, Mol. Cancer Ther.[分子癌症治療學] 2012, 11, 1中所述。 In preferred embodiments, the oligonucleotide is conjugated to a small molecule, aptamer or antibody (or antigen-binding fragment thereof). Preferred antibodies (or antigen-binding fragments thereof) are specific for CD71 (transferrin receptor) (e.g. as described in WO 2016/179257 (CytoMx)) or against equilibrating nucleoside transporters (ENTs) such as 3E10 Antibodies, as described, for example, in Weisbart et al., Mol. Cancer Ther . 2012, 11, 1.
在另一個較佳的實施方式中,寡核苷酸與GalNac部分和/或膽固醇部分共軛。例如,寡核苷酸在其3'端與膽固醇部分共軛,並且在其5'端與GalNac部分共軛。In another preferred embodiment, the oligonucleotide is conjugated to a GalNac moiety and/or a cholesterol moiety. For example, the oligonucleotide is conjugated to a cholesterol moiety at its 3' end and to a GalNac moiety at its 5' end.
通常,共軛在寡核苷酸的5'或3'端,較佳的是3'端。 寡核苷酸,效應 Typically, the conjugation is at the 5' or 3' end of the oligonucleotide, preferably the 3' end. oligonucleotide, effect
在功能上,寡核苷酸(視需要具有如上所述之修飾和/或共軛)的特徵在於能夠表現出以下作用中的至少一種: 1) 調節T抗原前mRNA的剪接, 2) 減少T抗原mRNA的產生, 3) 減少VP1 mRNA,並且較佳的是VP1蛋白的產生, 4) 抑制病毒複製,較佳的是減少細胞產生的病毒顆粒數,以及 5) 限制病毒再感染的能力,亦即限制感染的細胞產生感染性病毒顆粒的能力。 Functionally, oligonucleotides (optionally modified and/or conjugated as described above) are characterized by being able to exhibit at least one of the following effects: 1) Regulate the splicing of T antigen pre-mRNA, 2) Reduce the production of T antigen mRNA, 3) Reduce VP1 mRNA and preferably VP1 protein production, 4) inhibit viral replication, preferably by reducing the number of viral particles produced by cells, and 5) Limit the ability of the virus to reinfect, that is, limit the ability of infected cells to produce infectious virus particles.
1) 可以藉由監測前mRNA的給定剪接產物的形成來評估T抗原前mRNA剪接的調節;目的係較低量的給定剪接產物,因為它表明對多瘤病毒的抑制。較低量可能意指比用寡核苷酸治療開始時相同剪接產物的量低至少5%,或低至少10%、15%、20%、30%、40%、50%、60%、70%、80%、90%或95%。可以使用PCR進行評估。1) Regulation of T antigen pre-mRNA splicing can be assessed by monitoring the formation of a given splicing product of the pre-mRNA; the aim is to target lower amounts of a given splicing product as it indicates inhibition of polyomaviruses. Lower amount may mean at least 5% lower, or at least 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70% lower than the amount of the same spliced product at the beginning of treatment with the oligonucleotide. %, 80%, 90% or 95%. This can be assessed using PCR.
2) T抗原mRNA產生的減少可為用寡核苷酸治療開始時初始產生的mRNA的至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或99%。較佳的是,T抗原mRNA不再是可偵測的。可以使用技術者已知的技術(如RT-PCR或RNA印跡法)偵測mRNA產生。2) The reduction in T antigen mRNA production can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the initially produced mRNA at the start of treatment with the oligonucleotide , 95% or 99%. Preferably, T antigen mRNA is no longer detectable. mRNA production can be detected using techniques known to those skilled in the art, such as RT-PCR or Northern blotting.
3) VP1 mRNA(並且較佳的是VP1蛋白)產生的減少可為用寡核苷酸治療開始時初始產生的mRNA(或蛋白)的至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或99%。較佳的是,VP1 mRNA(或蛋白)不再是可偵測的。可以使用技術者已知的技術(包括用於mRNA的RT-PCR或RNA印跡法和用於蛋白質的西方墨點法)偵測mRNA和蛋白質產生。3) The reduction in VP1 mRNA (and preferably VP1 protein) production can be at least 10%, 20%, 30%, 40%, 50% of the initially produced mRNA (or protein) at the start of treatment with the oligonucleotide , 60%, 70%, 80%, 90%, 95% or 99%. Preferably, VP1 mRNA (or protein) is no longer detectable. mRNA and protein production can be detected using techniques known to the skilled artisan, including RT-PCR or Northern blotting for mRNA and Western blotting for proteins.
4) 可以抑制病毒複製,使得病毒DNA的量減少用寡核苷酸治療開始時初始病毒DNA的至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或99%。在實施方式中,病毒DNA不再是可偵測的。病毒顆粒數可以減少用寡核苷酸治療開始時病毒顆粒初始數的至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或99%。在實施方式中,病毒顆粒不再是可偵測的。可以使用技術者已知的技術評估病毒複製或產生的病毒顆粒數。例如,可以使用PCR偵測病毒複製或產生的病毒顆粒數(病毒載量)。說明書的實驗部分提供了用於偵測病毒載量的示例性方法。4) Can inhibit viral replication, reducing the amount of viral DNA to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, of the initial viral DNA at the beginning of treatment with oligonucleotides 90%, 95% or 99%. In embodiments, viral DNA is no longer detectable. The number of viral particles can be reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of the initial number of viral particles at the beginning of treatment with the oligonucleotide %. In embodiments, the viral particles are no longer detectable. Viral replication or the number of viral particles produced can be assessed using techniques known to those skilled in the art. For example, PCR can be used to detect viral replication or the number of viral particles produced (viral load). The Experimental section of the instructions provides exemplary methods for detecting viral load.
5) 可以定量病毒再感染能力的限制,使得與由未用寡核苷酸處理的一個感染細胞產生的病毒所感染的細胞數相比,由用寡核苷酸處理的一個感染的細胞產生的病毒所感染的細胞數減少至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或99%5) Limitations in the reinfection capacity of viruses can be quantified such that the number of cells infected by virus produced by one infected cell treated with an oligonucleotide is compared to the number of cells infected by virus produced by an infected cell that was not treated with the oligonucleotide. The number of cells infected by the virus is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%
較佳的是,寡核苷酸至少能表現出作用1。此種對剪接的作用示於實例中。不受理論束縛,據信寡核苷酸抑制其靶向的剪接位點的使用。由此導致的大T抗原產生的減少影響衣殼蛋白的表現,從而影響病毒的產生。不受理論束縛,據信由寡核苷酸誘導的T抗原特異性剪接產物的不平衡對病毒繁殖的作用比藉由RNAi樣方法減少T抗原mRNA的作用更明顯。Preferably, the oligonucleotide exhibits at least effect 1. This effect on splicing is shown in the Examples. Without wishing to be bound by theory, it is believed that the oligonucleotide inhibits the use of the splice site it targets. The resulting reduction in large T antigen production affects the expression of capsid proteins and thus virus production. Without being bound by theory, it is believed that the imbalance of T-antigen-specific splicing products induced by oligonucleotides has a more pronounced effect on viral propagation than the reduction of T-antigen mRNA by RNAi-like methods.
在一個實施方式中,寡核苷酸至少表現出 (i) 作用1和/或作用2,以及 (ii) 作用4;或寡核苷酸至少表現出 (i) 作用1和/或作用2,以及 (ii) 作用5。較佳的是,寡核苷酸至少表現出(i) 作用1和/或作用2,(ii) 作用4以及 (iii) 作用5。In one embodiment, the oligonucleotide exhibits at least (i) Effect 1 and/or Effect 2, and (ii) Effect 4; or the oligonucleotide exhibits at least (i) Effect 1 and/or Effect 2, and (ii) role5. Preferably, the oligonucleotide exhibits at least (i) Effect 1 and/or Effect 2, (ii) Effect 4 and (iii) Effect 5.
在另一個實施方式中,寡核苷酸至少表現出 (i) 作用3以及 (ii) 作用4;或寡核苷酸至少表現出 (i) 作用3以及 (ii) 作用5。較佳的是,寡核苷酸至少表現出 (i) 作用3,(ii) 作用4以及 (iii) 作用5。In another embodiment, the oligonucleotide exhibits at least (i) Effect 3 and (ii) Effect 4; or the oligonucleotide exhibits at least (i) Effect 3 and (ii) Effect 5. Preferably, the oligonucleotide exhibits at least (i) Function 3, (ii) Function 4 and (iii) Function 5.
以下在醫療用途的上下文中定義了治療效應。 多瘤病毒 Therapeutic effects are defined below in the context of medical use. polyomavirus
在說明書的上下文中,多瘤病毒可為任何多瘤病毒。在實施方式中,多瘤病毒係人多瘤病毒,包括所有屬,像α、β和δ屬。在較佳的實施方式中,多瘤病毒係α或β病毒,較佳的是β病毒。人多瘤病毒之非限制性實例列於下表1中。在較佳的實施方式中,多瘤病毒係BK多瘤病毒(或BK病毒,本文亦稱為BKPyV或BKV)、JC多瘤病毒(或JC病毒,本文亦稱為JCV)或梅克爾細胞多瘤病毒(MC多瘤病毒、MC病毒,或本文中亦稱為MCV)。在特別較佳的實施方式中,多瘤病毒係BK病毒或JC病毒,較佳的是BK病毒。
表1:人多瘤病毒之實例
在第二態樣中,本發明係關於載體,其包含 (i) 如第一態樣所定義的寡核苷酸,(ii) 如第一態樣所定義的寡核苷酸之反向互補序列,或 (iii) 能夠被轉錄為如第一態樣所定義的寡核苷酸的DNA。In a second aspect, the invention relates to a vector comprising (i) an oligonucleotide as defined in the first aspect, (ii) the reverse complement of an oligonucleotide as defined in the first aspect sequence, or (iii) DNA capable of being transcribed into an oligonucleotide as defined in the first aspect.
在較佳的實施方式中,載體係核酸載體。因此,根據 (i) 和 (ii) 的載體較佳的為RNA載體,並且根據 (iii) 的載體較佳的為DNA載體。核酸載體包括質體載體、黏質體載體、噬菌體載體(如λ噬菌體)和病毒載體。病毒載體係較佳的,並且在一些實施方式中,病毒載體可以選自由以下組成之群組:腺病毒載體、腺相關病毒載體、反轉錄病毒載體和慢病毒載體。較佳的病毒載體係腺相關病毒載體(AAV)。實例為血清型1的AAV(AAV1)、血清型2的AAV(AAV2)、血清型3的AAV(AAV3)、血清型4的AAV(AAV4)、血清型5的AAV(AAV5)、血清型6的AAV(AAV6)、血清型7的AAV(AAV7)、血清型8的AAV(AAV8)、血清型9的AAV(AAV9)、血清型rh10的AAV(AAVrh10)、血清型rh8的AAV(AAVrh8)、血清型Cb4的AAV(AAVCb4)、血清型rh74的AAV(AAVrh74)、血清型DJ的AAV(AAVDJ)、血清型2/5的AAV(AAV2/5)、血清型2/1的AAV(AAV2/1)、血清型1/2的AAV(AAV1/2)和血清型Anc80的AAV(AAVAnc80)。AAV2係較佳的實例。In a preferred embodiment, the vector system is a nucleic acid vector. Therefore, the vector according to (i) and (ii) is preferably an RNA vector, and the vector according to (iii) is preferably a DNA vector. Nucleic acid vectors include plastid vectors, myxoid vectors, phage vectors (such as lambda phage) and viral vectors. Viral vector systems are preferred, and in some embodiments, the viral vector may be selected from the group consisting of: adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and lentiviral vectors. The preferred viral vector system is adeno-associated virus vector (AAV). Examples are AAV serotype 1 (AAV1), AAV serotype 2 (AAV2), AAV serotype 3 (AAV3), AAV serotype 4 (AAV4), AAV serotype 5 (AAV5), serotype 6 AAV of serotype 7 (AAV7), AAV of serotype 8 (AAV8), AAV of serotype 9 (AAV9), AAV of serotype rh10 (AAVrh10), AAV of serotype rh8 (AAVrh8) , AAV of serotype Cb4 (AAVCb4), AAV of serotype rh74 (AAVrh74), AAV of serotype DJ (AAVDJ), AAV of serotype 2/5 (AAV2/5), AAV of serotype 2/1 (AAV2 /1), AAV of serotype 1/2 (AAV1/2) and AAV of serotype Anc80 (AAVAnc80). AAV2 series is a better example.
然而,亦涵蓋非核酸載體,並包括例如病毒樣顆粒(VLP),或者「VLP」係指非複製的空病毒殼。VLP通常由一或多種病毒蛋白組成,如但不限於稱為衣殼蛋白、外殼蛋白、殼蛋白、表面蛋白和/或包膜蛋白的那些蛋白質。它們含有負責病毒穿透細胞的功能性病毒蛋白,從而確保有效的細胞進入。生產特定VLP的方法在本領域中係已知的。However, non-nucleic acid vectors are also encompassed, and include, for example, virus-like particles (VLPs), or "VLPs" referring to non-replicating empty viral capsids. VLPs typically consist of one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, shell proteins, surface proteins, and/or envelope proteins. They contain functional viral proteins responsible for viral penetration into cells, thus ensuring efficient cellular entry. Methods for producing specific VLPs are known in the art.
在第三態樣中,本發明關於包含如第一態樣所定義的寡核苷酸或如第二態樣所定義的載體的組成物。在較佳的實施方式中,此組成物係藥物組成物。In a third aspect, the invention relates to a composition comprising an oligonucleotide as defined in the first aspect or a vector as defined in the second aspect. In a preferred embodiment, the composition is a pharmaceutical composition.
藥物組成物較佳的是包含一或多種藥學上可接受的賦形劑,如填充劑、防腐劑、增溶劑、載劑、稀釋劑、賦形劑、鹽、輔助劑和/或溶劑,如例如Remington: The Science and Practice of Pharmacy [雷明頓:藥學的科學與實踐], 第20版. 馬里蘭州巴爾的摩: Lippincott Williams & Wilkins [利平科特•威廉斯•威爾金斯出版公司], 2000中所述之。The pharmaceutical composition preferably contains one or more pharmaceutically acceptable excipients, such as fillers, preservatives, solubilizers, carriers, diluents, excipients, salts, auxiliaries and/or solvents, such as For example, Remington: The Science and Practice of Pharmacy, 20th ed. Baltimore, MD: Lippincott Williams & Wilkins, 2000 stated.
賦形劑可以增強寡核苷酸的穩定性、溶解度、吸收、生體可用率、活性、藥物動力學、藥效學、細胞攝取和細胞內運輸,特別地它可為能夠形成複合物、奈米顆粒、微粒、奈米管、奈米凝膠、水凝膠、泊洛沙姆或普朗尼克(pluronic)、聚合物囊泡(polymersome)、膠體、微泡、囊泡、膠束、脂質體複合物(lipoplex)和/或脂質體的賦形劑。奈米顆粒之實例包括聚合物奈米顆粒、(混合)金屬奈米顆粒、碳奈米顆粒、金奈米顆粒、磁性奈米顆粒、二氧化矽奈米顆粒、脂質奈米顆粒、糖顆粒、蛋白質奈米顆粒和肽奈米顆粒。奈米顆粒和寡核苷酸組合之實例係球形核酸(SNA),如例如Barnaby等人 Cancer Treat. Res.[癌症治療研究] 2015, 166, 23中的。 Excipients can enhance the stability, solubility, absorption, bioavailability, activity, pharmacokinetics, pharmacodynamics, cellular uptake and intracellular transport of oligonucleotides. In particular, they can enhance the stability, solubility, absorption, bioavailability, activity, cellular uptake and intracellular transport of oligonucleotides. Rice particles, microparticles, nanotubes, nanogels, hydrogels, poloxamer or pluronic, polymersome, colloids, microvesicles, vesicles, micelles, lipids excipients for lipoplexes and/or liposomes. Examples of nanoparticles include polymer nanoparticles, (hybrid) metal nanoparticles, carbon nanoparticles, gold nanoparticles, magnetic nanoparticles, silica nanoparticles, lipid nanoparticles, sugar particles, Protein nanoparticles and peptide nanoparticles. An example of a combination of nanoparticles and oligonucleotides is a spherical nucleic acid (SNA), as for example in Barnaby et al. Cancer Treat. Res . 2015, 166, 23.
較佳的賦形劑係靶向賦形劑,其能夠將寡核苷酸靶向和/或遞送至(或進入)器官、組織和/或細胞,例如腎、腎組織或腎細胞、或膀胱、膀胱組織或膀胱細胞,特別地膀胱上皮細胞。該等賦形劑中的許多係本領域已知的(例如參見Bruno, K.等人, (2011), Adv. Drug. Deliv. Rev [先進藥物遞送綜述], 63(13): 210-1226),實例包括聚合物(例如聚乙烯亞胺(PEI)、聚丙烯亞胺(PPI)、葡聚糖衍生物、氰基丙烯酸丁酯(PBCA)、氰基丙烯酸己酯(PHCA)、聚(乳酸-共-乙醇酸)(PLGA)、多胺(例如精胺、亞精胺、腐胺、屍胺)、殼聚糖、聚(醯胺基胺)(PAMAM)、聚(酯胺)、聚乙烯醚、聚乙烯吡咯啶酮(PVP)、聚乙二醇(PEG)環糊精、透明質酸、多聚乙醯神經胺酸(colominic acid)及其衍生物)、樹枝狀聚合物(例如聚(醯胺基胺))、脂質(例如1,2-二油醯基-3-二甲基銨丙烷(DODAP)、二油醯基二甲基氯化銨(DODAC)、磷脂醯膽鹼衍生物[例如1,2-二硬脂醯基-sn-甘油-3-磷酸膽鹼(DSPC)]、溶血磷脂醯膽鹼衍生物[例如1-硬脂醯基-2-溶血-sn-甘油-3-磷酸膽鹼(S-LysoPC)]、鞘磷脂、2-{3-[雙-(3-胺基-丙基)-胺基]-丙基胺基}-N-雙十四烷基胺基甲醯基甲基乙醯胺(RPR209120)、磷酸甘油衍生物[例如1,2-二棕櫚醯基-sn-甘油-3-磷酸甘油鈉鹽(DPPG-Na)]、磷脂酸衍生物[1,2-二硬脂醯基-sn-甘油-3-磷脂酸鈉鹽(DSPA)]、磷脂醯乙醇胺衍生物[例如二油醯基-L-R-磷脂醯乙醇胺(DOPE)、1,2-二硬脂醯基-sn-甘油-3-磷酸乙醇胺(DSPE)、2-二植烷醯基-sn-甘油-3-磷酸乙醇胺(DPhyPE)]、N-[1-(2,3-二油醯基氧基)丙基]-N,N,N-三甲基銨(DOTAP)、N-[1-(2,3-二油醯基氧基)丙基]-N,N,N-三甲基銨(DOTMA)、1,3-二油醯基氧基-2-(6-羧基-精胺基)-丙醯胺(DOSPER)、1,2-二肉豆蔻基氧基丙基-3-二甲基羥乙基銨(DMRIE)、N1-膽固醇基氧基羰基-3,7-二氮雜壬烷-1,9-二胺(CDAN)、二甲基雙十八烷基溴化銨(DDAB)、1-棕櫚醯基-2-油醯基-sn-甘油-3-磷酸膽鹼(POPC)、b-L-精胺醯基-2,3-L-二胺基丙酸-N-棕櫚基-N-油醯基-醯胺三鹽酸鹽(AtuFECT01)、N,N-二甲基-3-胺基丙烷衍生物[例如1,2-二硬脂醯氧基-N,N-二甲基-3-胺基丙烷(DSDMA)、1,2-二油烯基氧基-N,N-二甲基-3-胺基丙烷(DoDMA)、1,2-二亞油氧基-N,N-3-二甲基胺基丙烷(DLinDMA)、2,2-二亞油基-4-二甲基胺基甲基[1,3]-二氧戊環(DLin-K-DMA)]、磷脂醯絲胺酸衍生物[1,2-二油烯基-sn-甘油-3-磷酸-L-絲胺酸鈉鹽(DOPS)])、蛋白質(例如白蛋白、明膠、缺端膠原(atellocollagen))和線性或環狀肽(例如魚精蛋白、PepFects、NickFects、聚精胺酸、聚離胺酸、CADY、MPG、細胞穿透肽(CPP)、靶向肽、細胞轉位肽、內體逃逸肽)。已經描述了此類肽的實例,例如肌肉靶向肽(例如Jirka等人, Nucl. Acid Ther.[核酸療法] 2014, 24, 25)、CPP(例如Pip系列,包括WO 2013/030569和寡聚精胺酸系列,例如US 9,161,948(薩雷普塔公司(Sarepta))、WO 2016/187425(薩雷普塔公司)和M12肽,例如Gao等人, Mol. Ther.[分子療法] 2014, 22, 1333中的)、或血腦屏障(BBB)穿越肽(crossing peptide),如(支鏈)ApoE衍生物(Shabanpoor等人, Nucl. Acids Ther.[核酸療法] 2017, 27, 130)。如上所述關於寡核苷酸共軛的碳水化合物和碳水化合物簇亦適合用作賦形劑。Preferred excipients are targeting excipients capable of targeting and/or delivering the oligonucleotide to (or into) organs, tissues and/or cells, such as the kidney, renal tissue or kidney cells, or the bladder. , bladder tissue or bladder cells, especially bladder epithelial cells. Many of these excipients are known in the art (see, for example, Bruno, K. et al., (2011), Adv. Drug. Deliv. Rev, 63(13): 210-1226 ), examples include polymers (such as polyethyleneimine (PEI), polypropyleneimine (PPI), dextran derivatives, butyl cyanoacrylate (PBCA), hexyl cyanoacrylate (PHCA), poly( Lactic-co-glycolic acid) (PLGA), polyamines (e.g. spermine, spermidine, putrescine, cadaverine), chitosan, poly(amidoamine) (PAMAM), poly(esteramine), Polyvinyl ether, polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) cyclodextrin, hyaluronic acid, polyacetylneuraminic acid (colominic acid and its derivatives), dendrimers ( For example, poly(amide amine)), lipids (such as 1,2-dioleyl-3-dimethylammonium propane (DODAP), dioleyl dimethyl ammonium chloride (DODAC), phospholipids Base derivatives [e.g. 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC)], lysophosphatidylcholine derivatives [e.g. 1-stearyl-2-hemolys-sn -glycerol-3-phosphocholine (S-LysoPC)], sphingomyelin, 2-{3-[bis-(3-amino-propyl)-amino]-propylamino}-N-bis-deca Tetraalkylaminoformylmethylacetamide (RPR209120), glycerol phosphate derivatives [such as 1,2-dipalmitoyl-sn-glycerol-3-glycerol phosphate sodium salt (DPPG-Na)], phospholipids Acid derivatives [1,2-distearyl-sn-glycerol-3-phosphatidic acid sodium salt (DSPA)], phospholipidylethanolamine derivatives [such as dioleyl-L-R-phosphatidylethanolamine (DOPE), 1,2-distearyl-sn-glycerol-3-phosphoethanolamine (DSPE), 2-distearyl-sn-glycerol-3-phosphoethanolamine (DPhyPE)], N-[1-(2 ,3-dioleyloxy)propyl]-N,N,N-trimethylammonium (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N ,N,N-trimethylammonium (DOTMA), 1,3-dioleyloxy-2-(6-carboxy-sperminyl)-propanamide (DOSPER), 1,2-dimyristin Oxypropyl-3-dimethylhydroxyethylammonium (DMRIE), N1-cholesteryloxycarbonyl-3,7-diazanonane-1,9-diamine (CDAN), dimethyl Dioctadecyl ammonium bromide (DDAB), 1-palmitoyl-2-oleyl-sn-glyceryl-3-phosphocholine (POPC), b-L-sperminyl-2,3-L- Diaminopropionic acid-N-palmityl-N-oleyl-amide trihydrochloride (AtuFECT01), N,N-dimethyl-3-aminopropane derivatives [such as 1,2-disulfide Fattyoxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,N-dimethyl-3-aminopropane (DoDMA), 1,2-dilinoleoxy-N,N-3-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]- Dioxolane (DLin-K-DMA)], phospholipid serine derivative [1,2-diolelenyl-sn-glycerol-3-phosphate-L-serine sodium salt (DOPS)]) , proteins (e.g. albumin, gelatin, atellocollagen) and linear or cyclic peptides (e.g. protamine, PepFects, NickFects, polyarginine, polylysine, CADY, MPG, cell-penetrating peptides (CPP), targeting peptide, cell translocation peptide, endosomal escape peptide). Examples of such peptides have been described, such as muscle-targeting peptides (e.g. Jirka et al., Nucl. Acid Ther. 2014, 24, 25), CPPs (e.g. the Pip series, including WO 2013/030569 and Oligo Arginine series, such as US 9,161,948 (Sarepta), WO 2016/187425 (Sarepta) and M12 peptides, such as Gao et al., Mol. Ther. [Molecular Therapy] 2014, 22 , 1333), or blood-brain barrier (BBB) crossing peptides such as (branched) ApoE derivatives (Shabanpoor et al., Nucl. Acids Ther. 2017, 27, 130). Carbohydrates and carbohydrate clusters, as described above with respect to oligonucleotide conjugation, are also suitable for use as excipients.
將藥物組成物配製成包含藥學上有效量的寡核苷酸。它可以配製用於經由局部、全身和/或腸胃外途徑投與,例如靜脈內、皮下、腹膜內、鞘內、肌內、眼部、經鼻、泌尿生殖、皮內、皮膚、腸道、玻璃體內、海綿竇內、腦內、鞘內、硬膜外或口服途徑。The pharmaceutical composition is formulated to contain a pharmaceutically effective amount of the oligonucleotide. It may be formulated for administration via local, systemic and/or parenteral routes, such as intravenous, subcutaneous, intraperitoneal, intrathecal, intramuscular, ocular, nasal, genitourinary, intradermal, cutaneous, enteral, Intravitreal, intracavernous sinus, intracerebral, intrathecal, epidural, or oral routes.
較佳的是,將藥物組成物配製成用於口服遞送的乳劑、混懸液、丸劑、片劑、膠囊或軟凝膠,或用於遞送到呼吸道和肺的氣溶膠或乾粉形式。Preferably, the pharmaceutical compositions are formulated as emulsions, suspensions, pills, tablets, capsules or soft gels for oral delivery, or as aerosols or dry powders for delivery to the respiratory tract and lungs.
在實施方式中,藥物組成物進一步包含用於治療多瘤病毒感染的另一種活性成分。In embodiments, the pharmaceutical composition further comprises another active ingredient for the treatment of polyomavirus infection.
在第四態樣中,本發明關於如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物,用於作為藥品使用,特別地用於在治療受試者的多瘤病毒感染中使用。In a fourth aspect, the present invention relates to an oligonucleotide as defined in the first aspect, a carrier as defined in the second aspect or a pharmaceutical composition as defined in the third aspect, for use as a medicine , specifically for use in the treatment of polyomavirus infection in a subject.
為簡潔起見,寡核苷酸、載體和藥物組成物在下文中統稱為「藥品」。For the sake of brevity, oligonucleotides, vectors, and pharmaceutical compositions are collectively referred to below as "drugs."
因此,雖然該藥品通常用於治療受試者的多瘤病毒感染,但其較佳的是亦用於治療與多瘤病毒相關的疾病。多瘤病毒的含義如上關於第一態樣所定義。「與多瘤病毒相關的疾病」包括膀胱炎(特別地出血性膀胱炎,例如骨髓移植的接受者中的)、輸尿管炎、間質性腎炎(亦稱為腎病;特別地移植腎病)和進行性多部腦白質病(特別地在免疫功能低下的受試者中)。Therefore, although the drug is typically used to treat polyomavirus infections in subjects, it is preferably also used to treat polyomavirus-related diseases. Polyomavirus has the meaning as defined above with respect to the first aspect. "Polyomavirus-related diseases" include cystitis (especially hemorrhagic cystitis, such as in recipients of bone marrow transplants), ureteritis, interstitial nephritis (also called nephropathy; especially transplant nephropathy), and Sexual multipartite leukoencephalopathy (especially in immunocompromised subjects).
如本文所用,術語「感染」係指病毒感染,亦即病毒進入宿主的至少一個細胞並在該至少一個細胞內複製。感染可能是急性的(亦即活躍的)或者亦可能是潛伏的(亦即不活躍的、隱藏的、休眠的)(如例如在多瘤病毒的情況下)。在急性感染中,病毒正在複製,感染細胞並可能引起症狀,而在潛伏感染中,病毒不會獨立於宿主細胞基因組進行複製並感染更多細胞,而是「隱藏」在細胞中。潛伏感染可以被急性感染打斷,其中隱藏的病毒開始複製並感染另外的細胞。在預先存在的潛伏感染的情況下,治療感染的用途較佳的是涉及藉由防止隱藏的病毒感染另外的細胞(亦即擴散)來預防急性感染。換言之,此可以描述為潛伏感染的治療,其中治療不一定是治癒性的(但可以控制病毒)。換言之,用藥品治療的受試者可能是無症狀的。在實施方式中,用藥品治療的受試者係無症狀且血清反應陽性的。在另一個實施方式中,用藥品治療的受試者係無症狀且血清反應陰性的。As used herein, the term "infection" refers to a viral infection in which a virus enters and replicates within at least one cell of a host. The infection may be acute (i.e. active) or latent (i.e. inactive, latent, dormant) (as in the case of polyomaviruses, for example). In an acute infection, the virus is replicating, infecting cells and possibly causing symptoms, whereas in a latent infection, the virus does not replicate independently of the host cell genome and infect more cells, but "hide" within the cell. Latent infection can be interrupted by acute infection, in which the hidden virus begins to replicate and infect additional cells. In the case of a pre-existing latent infection, the use of treating the infection preferably involves preventing the acute infection by preventing the hidden virus from infecting other cells (ie, spreading). In other words, this can be described as treatment of latent infection, where the treatment is not necessarily curative (but can control the virus). In other words, subjects treated with the drug may be asymptomatic. In embodiments, the subject treated with the drug is asymptomatic and seropositive. In another embodiment, the subject treated with the drug is asymptomatic and seronegative.
以治療有效量投與藥品。可以在體外(亦即無細胞或在細胞中)或在體內(亦即在動物(如動物模型)或患者中)評估此類治療效應的誘導。它可以在分子水平和/或細胞水平上進行評估。Administer the drug in a therapeutically effective amount. The induction of such therapeutic effects can be assessed in vitro (ie, cell-free or in cells) or in vivo (ie, in animals (eg, animal models) or in patients). It can be assessed at the molecular level and/or the cellular level.
「治療效應」特別係指關於以上本發明之第一態樣定義的作用1) 至5),亦即該等作用亦分別稱為治療效應1) 至5)。進一步的治療效應係: 6) 預防與感染相關的細胞效應, 7) 減輕與感染相關的細胞效應, 8) 預防感染的受試者的疾病,和/或 9) 減慢,較佳的是停止,更佳的是逆轉疾病進展。 "Therapeutic effect" particularly refers to the effects 1) to 5) defined above in relation to the first aspect of the present invention, that is, these effects are also referred to as therapeutic effects 1) to 5) respectively. Further therapeutic effects include: 6) Prevent infection-related cellular effects, 7) Alleviating the cellular effects associated with infection, 8) Prevent disease in infected subjects, and/or 9) Slow, preferably stop, and better yet reverse disease progression.
6) 和7):與感染相關的細胞效應可能是細胞死亡(例如藉由細胞凋亡或細胞裂解),其緩解將是細胞死亡的減少。與治療開始時的細胞死亡水平相比,此類細胞死亡的減少可能為至少5%。較佳的是,細胞死亡的減少意指至少10%,甚至更佳的是至少20%、至少30%、至少40%、至少50%、至少70%、至少80%、至少90%或100%。細胞死亡可以藉由直接偵測細胞死亡或藉由偵測細胞存活來確定,例如藉由收穫細胞群體和(免疫)染色細胞週期標誌物,藉由FACS分析或藉由PCR(用於細胞週期RNA的表現水平)用適當的標誌物來確定一或多個細胞週期階段。6) and 7): The cellular effect associated with the infection may be cell death (e.g. by apoptosis or cell lysis) and its mitigation would be a reduction in cell death. The reduction in such cell death may be at least 5% compared to the level of cell death at the start of treatment. Preferably, reduction in cell death means at least 10%, even more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 80%, at least 90% or 100% . Cell death can be determined by directly detecting cell death or by detecting cell survival, for example by harvesting cell populations and (immuno)staining for cell cycle markers, by FACS analysis or by PCR (for cell cycle RNA level of performance) to determine one or more cell cycle phases using appropriate markers.
8) 和9):疾病是否被預防或進展是否減慢、停止或逆轉可以藉由評估與多瘤病毒感染相關的疾病的症狀或參數來確定。此類症狀係本領域已知的。例如,為了評估腎病的進展,可以確定參數(如腎小球濾過率或肌酐水平)。8) and 9): Whether disease is prevented or whether progression is slowed, stopped, or reversed can be determined by assessing symptoms or parameters of the disease associated with polyomavirus infection. Such symptoms are known in the art. For example, to assess the progression of kidney disease, parameters such as glomerular filtration rate or creatinine levels can be determined.
可在8) 或9) 下評估為疾病或病症的存在或狀態或進展的(分子)標誌物的另外的參數包括:細胞週期蛋白E2(CCNE2)、細胞分裂週期蛋白6(CDC6)、細胞週期蛋白E2(CCNA2)、E2F轉錄因子8(E2F8)、存活蛋白(BIRC5)、RAD51相關蛋白-1(RAD51AP1)、BRCA1相互作用蛋白C-末端解旋酶1(BRIP1)和脂蛋白元B mRNA編輯酶3B(APOBEC3B)。Additional parameters that may be assessed under 8) or 9) as (molecular) markers of the presence or status or progression of a disease or disorder include: Cyclin E2 (CCNE2), Cell Division Cyclin 6 (CDC6), Cell Cycle Protein E2 (CCNA2), E2F transcription factor 8 (E2F8), survivin (BIRC5), RAD51-associated protein-1 (RAD51AP1), BRCA1-interacting protein C-terminal helicase 1 (BRIP1) and lipoprotein B mRNA editing Enzyme 3B (APOBEC3B).
如本文所定義的用寡核苷酸治療可以增加該等基因中至少一個的下調或降低(Abend, J.等人 (2010) Global effects of BKV infection of gene expression in human primary epithelial cells [BKV感染對人原代上皮細胞中基因表現的整體影響]; 397 (1): 73)。在實施方式中,在BKV感染或再感染細胞後誘導下調或降低。Treatment with oligonucleotides, as defined herein, can increase the downregulation or decrease of at least one of these genes (Abend, J. et al. (2010) Global effects of BKV infection of gene expression in human primary epithelial cells [BKV infection on Global impact of gene expression in human primary epithelial cells]; 397 (1): 73). In embodiments, downregulation or reduction is induced upon infection or reinfection of cells with BKV.
在實施方式中,下調或降低為在用寡核苷酸治療開始時所述基因的初始表現水平的至少10%、20%、30%、40%、50%、60%、70%、80%、90%、95%、99%。在實施方式中,表現不再是可偵測的。可以使用技術者已知的技術評估表現。在實施方式中,使用RNA印跡法或PCR評估表現。In embodiments, the downregulation or reduction is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% of the initial expression level of the gene at the beginning of treatment with the oligonucleotide , 90%, 95%, 99%. In embodiments, the manifestation is no longer detectable. Performance can be evaluated using techniques known to those skilled in the art. In embodiments, performance is assessed using Northern blotting or PCR.
因此,在實施方式中,如本文所定義的寡核苷酸、載體或藥物組成物均可用於作為藥品使用,較佳的是用於治療受試者的多瘤病毒感染,其中所述寡核苷酸、載體或藥物組成物能夠表現出以下作用中的至少一種: 1) 調節T抗原前mRNA的剪接, 2) 減少T抗原mRNA的產生, 3) 減少VP1 mRNA,並且較佳的是VP1蛋白的產生, 4) 抑制病毒複製, 5) 限制病毒再感染的能力,亦即限制感染的細胞產生感染性病毒顆粒的能力, 6) 預防與感染相關的細胞效應, 7) 減輕與感染相關的細胞效應, 8) 預防感染的受試者的疾病,和 9) 減慢,較佳的是停止,更佳的是逆轉疾病進展。 Accordingly, in embodiments, an oligonucleotide, vector or pharmaceutical composition as defined herein may be used as a pharmaceutical, preferably for the treatment of polyomavirus infection in a subject, wherein the oligonucleotide The glycolic acid, carrier or pharmaceutical composition can exhibit at least one of the following effects: 1) Regulate the splicing of T antigen pre-mRNA, 2) Reduce the production of T antigen mRNA, 3) Reduce VP1 mRNA and preferably VP1 protein production, 4) Inhibit virus replication, 5) Limit the ability of the virus to reinfect, that is, limit the ability of infected cells to produce infectious virus particles, 6) Prevent infection-related cellular effects, 7) Alleviating the cellular effects associated with infection, 8) prevent disease in infected subjects, and 9) Slow, preferably stop, and better yet reverse disease progression.
在實施方式中,如本文所定義的寡核苷酸、載體或藥物組成物用於作為藥品使用,較佳的是用於治療受試者的多瘤病毒感染,其中當與所述多瘤病毒感染相關的疾病的至少一個參數已經降低時,該疾病已被預防(8)或進展減慢、停止或逆轉(9),所述參數選自:腎小球濾過率、肌酐水平、細胞週期蛋白E2(CCNE2)、細胞分裂週期蛋白6(CDC6)、細胞週期蛋白E2(CCNA2)、E2F轉錄因子8(E2F8)、存活蛋白(BIRC5)、RAD51相關蛋白-1(RAD51AP1)、BRCA1相互作用蛋白C-末端解旋酶1(BRIP1)和脂蛋白元B mRNA編輯酶3B(APOBEC3B)。In embodiments, the oligonucleotide, vector or pharmaceutical composition as defined herein is for use as a medicament, preferably for the treatment of a polyomavirus infection in a subject, wherein when combined with said polyomavirus Infection-related disease has been prevented (8) or its progression has been slowed, stopped or reversed (9) when at least one parameter of the disease has been reduced, selected from: glomerular filtration rate, creatinine level, cell cycle proteins E2 (CCNE2), cell division cyclin 6 (CDC6), cyclin E2 (CCNA2), E2F transcription factor 8 (E2F8), survivin (BIRC5), RAD51-associated protein-1 (RAD51AP1), BRCA1-interacting protein C -Terminal helicase 1 (BRIP1) and lipoprotein B mRNA editing enzyme 3B (APOBEC3B).
設想治療效應包括如以上關於第一態樣所定義的治療效應1) 至5) 中的至少一種,並且較佳的是亦包括治療效應6) 至9) 中的至少一種。It is envisaged that the therapeutic effect includes at least one of therapeutic effects 1) to 5) as defined above with respect to the first aspect, and preferably also includes at least one of therapeutic effects 6) to 9).
藉由與治療開始時的狀態進行比較來評估效應。Effects are assessed by comparison with the status at the start of treatment.
受試者較佳的是人。通常,多瘤病毒在允許其的宿主(亦即允許病毒繞過其防禦並複製的宿主,如免疫功能低下的宿主)中感染並複製。此亦可以用術語免疫不全來描述,此係一種免疫系統對抗傳染病和癌症的能力受損或完全喪失的狀態。此狀態可能是暫時的,亦可能是永久的。在一個實施方式中,免疫不全係獲得性的(「繼發性的」),通常是由於影響患者免疫系統的外在因素所致。該等外在因素之實例包括HIV感染、(極端)年齡和環境因素,如營養不良。免疫不全亦可由藥物誘導,如糖皮質素、細胞抑制劑、抗體和作用於免疫親和蛋白的化合物(如鈣調磷酸酶抑制劑、貝拉西普(belatacept)(一種具有CTLA-4的細胞外結構域的免疫球蛋白樣分子)和類似分子)。此可能是期望的作用,如在器官移植手術中作為抗排斥措施,以及在患有過度活躍免疫系統的受試者中,如在自體免疫性疾病中。然而,有時此種期望的作用具有降低受試者抵抗多瘤病毒感染的能力的另外的作用。患有任何類型的免疫不全的受試者都被稱為免疫功能低下。在另一個實施方式中,免疫不全係遺傳的。例如,其可能是影響B細胞或T細胞淋巴球發育、吞噬細胞、補體、細胞介素或其受體、抗體或先天或適應性免疫系統的其他組分的遺傳缺陷(通常是隱性的)。Preferably, the test subject is a human. Typically, polyomaviruses infect and replicate in permissive hosts (that is, hosts that allow the virus to bypass its defenses and replicate, such as immunocompromised hosts). This is also described by the term immunodeficiency, a state in which the immune system's ability to fight infectious diseases and cancer is impaired or completely lost. This state may be temporary or permanent. In one embodiment, the immunodeficiency is acquired ("secondary"), usually due to extrinsic factors that affect the patient's immune system. Examples of such external factors include HIV infection, (extreme) age and environmental factors such as malnutrition. Immunosufficiency can also be induced by drugs such as glucocorticoids, cytostatics, antibodies, and compounds that act on immunoaffinity proteins (eg, calcineurin inhibitors, belatacept (an extracellular drug with CTLA-4 immunoglobulin-like molecules) and similar molecules). This may be a desired effect, such as in organ transplant surgeries as an anti-rejection measure, and in subjects with overactive immune systems, such as in autoimmune diseases. However, sometimes this desired effect has the additional effect of reducing the subject's ability to resist polyomavirus infection. Subjects suffering from any type of immunodeficiency are said to be immunocompromised. In another embodiment, the immunodeficiency is inherited. For example, it may be a genetic defect (often recessive) that affects B-cell or T-cell lymphocyte development, phagocytes, complement, interleukins or their receptors, antibodies, or other components of the innate or adaptive immune system. .
因此,在實施方式中,受試者免疫功能低下,例如由於以上因素之一和/或另一種感染、癌症、或使用投與於受試者以治療另一種病症或疾病的藥物。在一個實施方式中,其他病症係器官、組織或細胞(特別地腎)移植。因此,在一個實施方式中,受試者係器官、組織或細胞移植(本文中亦統稱為「移植」)的接受者。Thus, in embodiments, the subject is immunocompromised, for example due to one of the above factors and/or another infection, cancer, or use of a drug administered to the subject to treat another condition or disease. In one embodiment, the other condition is organ, tissue or cell (especially kidney) transplantation. Accordingly, in one embodiment, the subject is a recipient of an organ, tissue or cell transplant (also collectively referred to herein as a "transplant").
在第五態樣中,本發明關於離體方法,該等方法包括抑制細胞中多瘤病毒複製的離體方法和產生移植的離體方法。In a fifth aspect, the invention relates to ex vivo methods, including ex vivo methods for inhibiting polyomavirus replication in cells and ex vivo methods for generating transplants.
抑制細胞中多瘤病毒複製的離體方法包括 (i) 提供用多瘤病毒感染的細胞,並使該細胞與如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物接觸;或 (ii) 提供包含如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物的細胞,並使該細胞與多瘤病毒接觸。產生移植物的離體方法包括提供一或多種供體器官、組織或細胞(較佳的是包含腎細胞),以及使該一或多種供體器官、組織或細胞的細胞與如第一態樣所定義的寡核苷酸、如第二態樣所定義的載體或如第三態樣所定義的藥物組成物接觸。 一般定義 An ex vivo method of inhibiting polyomavirus replication in a cell includes (i) providing a cell infected with a polyomavirus and contacting the cell with an oligonucleotide as defined in the first aspect, an oligonucleotide as defined in the second aspect contacting a carrier or a pharmaceutical composition as defined in the third aspect; or (ii) providing an oligonucleotide comprising an oligonucleotide as defined in the first aspect, a carrier as defined in the second aspect or a pharmaceutical composition as defined in the third aspect cells of the defined pharmaceutical composition and brought into contact with the polyomavirus. Ex vivo methods of producing a graft include providing one or more donor organs, tissues or cells (preferably comprising kidney cells), and allowing cells of the one or more donor organs, tissues or cells to react in a first aspect The oligonucleotide as defined, the carrier as defined in the second aspect or the pharmaceutical composition as defined in the third aspect is contacted. general definition
除非另外說明,否則本文所用的所有技術和科學術語具有與本發明所屬領域的普通技術者常見和通常理解的相同的含義,並且基於本揭露進行閱讀。Unless otherwise stated, all technical and scientific terms used herein have the same meanings as commonly and commonly understood by one of ordinary skill in the art to which this invention belongs and are read in light of this disclosure.
「核鹼基」,有時稱為鹼基,通常是腺嘌呤、胞嘧啶、鳥嘌呤、胸腺嘧啶或尿嘧啶或其衍生物。胞嘧啶、胸腺嘧啶和尿嘧啶係嘧啶鹼基,並且通常經由其1-氮與支架連接。腺嘌呤和鳥嘌呤係嘌呤鹼基,並且通常經由其9-氮與支架連接。如本文所指的RNA核鹼基係腺嘌呤、胞嘧啶、鳥嘌呤和尿嘧啶。A "nucleobase", sometimes called a base, is usually adenine, cytosine, guanine, thymine or uracil or their derivatives. Cytosine, thymine and uracil are pyrimidine bases and are usually linked to the scaffold via their 1-nitrogen. Adenine and guanine are purine bases and are usually linked to the scaffold via their 9-nitrogen. The RNA nucleobases as referred to herein are adenine, cytosine, guanine and uracil.
除非另外指示,否則本文所指的「核苷酸」代表RNA核苷酸,較佳的是天然存在的RNA核苷酸。RNA中最常見的天然存在的核苷酸係單磷酸腺苷、單磷酸胞苷、單磷酸鳥苷和單磷酸尿苷。它們由戊糖核糖、經由磷酸酯連接的5'-連接的磷酸基團和1'-連接的鹼基組成。糖連接鹼基和磷酸,並且因此通常被稱為核苷酸的支架。因此,戊糖中的修飾通常被稱為支架修飾。因此,糖修飾可稱為支架修飾。對於大的修飾(severe modification),原始戊糖可能會被另一個類似地連接鹼基和磷酸的部分完全替換。因此可以理解,雖然戊糖通常是支架,但支架不一定是戊糖。核苷酸通常經由其5'-磷酸部分與相鄰核苷酸單體的3'-羥基部分的縮合而與相鄰核苷酸連接。類似地,其3'-羥基部分通常與相鄰核苷酸單體的5'-磷酸連接。此形成磷酸二酯鍵。磷酸二酯和支架形成交替共聚物。鹼基被接枝到此共聚物上,亦即接枝到支架部分上。由於此特徵,由寡核苷酸的連接單體形成的交替共聚物通常被稱為寡核苷酸的骨架。由於磷酸二酯鍵將相鄰的單體連接在一起,因此它們通常被稱為骨架鍵。應當理解,當磷酸基團被修飾以使其成為類似部分(如硫代磷酸酯)時,此類部分仍被稱為單體的骨架鍵。此被稱為骨架鍵修飾。因此,通常,寡核苷酸的骨架由交替的支架和骨架鍵組成。Unless otherwise indicated, "nucleotides" as referred to herein represent RNA nucleotides, preferably naturally occurring RNA nucleotides. The most common naturally occurring nucleotides in RNA are adenosine monophosphate, cytidine monophosphate, guanosine monophosphate, and uridine monophosphate. They consist of the pentose ribose sugar, a 5'-linked phosphate group and a 1'-linked base linked via a phosphate ester. Sugars link bases and phosphates, and are therefore often referred to as the scaffolding of nucleotides. Therefore, modifications in pentose sugars are often called scaffold modifications. Therefore, sugar modification can be called scaffold modification. For severe modifications, the original pentose sugar may be completely replaced by another moiety that similarly connects the base and phosphate. It can therefore be understood that although a pentose sugar is often the scaffold, the scaffold is not necessarily a pentose sugar. Nucleotides are typically linked to adjacent nucleotides via condensation of their 5'-phosphate moiety with the 3'-hydroxyl moiety of the adjacent nucleotide monomer. Similarly, its 3'-hydroxyl moiety is usually linked to the 5'-phosphate of the adjacent nucleotide monomer. This forms a phosphodiester bond. The phosphodiester and scaffold form alternating copolymers. The bases are grafted onto this copolymer, that is, onto the scaffold portion. Because of this characteristic, the alternating copolymer formed by the linking monomers of an oligonucleotide is often referred to as the backbone of the oligonucleotide. Because phosphodiester bonds link adjacent monomers together, they are often called backbone bonds. It should be understood that when the phosphate group is modified so that it becomes a similar moiety (such as a phosphorothioate), such moieties are still referred to as the backbone bonds of the monomer. This is called skeleton bond modification. Therefore, typically, the backbone of an oligonucleotide consists of alternating scaffolds and backbone bonds.
在寡核苷酸之上下文中,術語「特異性結合」意指寡核苷酸能夠與其靶標退火。術語「結合」可替換為「雜交」、「靶向」、「針對」、「反義於」或「互補於」。可以使用EMSA(電泳遷移率變動分析)使用寡核苷酸並將其與多瘤病毒RNA孵育來評估寡核苷酸與其靶標前mRNA的結合。In the context of an oligonucleotide, the term "specifically binds" means that the oligonucleotide is capable of annealing to its target. The term "binding" may be replaced by "hybridizing", "targeting", "against", "antisense to" or "complementary to". Binding of the oligonucleotide to its target pre-mRNA can be assessed using EMSA (Electrophoretic Mobility Shift Assay) using the oligonucleotide and incubating it with polyomavirus RNA.
當用於寡核苷酸時,術語「退火」應理解為寡核苷酸與至少基本上互補的序列(對於涉及氫鍵的鹼基配對)的鍵合,該鹼基配對可為沃森-克裡克(Watson-Crick)、胡斯坦(Hoogsteen)或反向胡斯坦(reversed Hoogsteen)鹼基配對。嚴格雜交條件涉及在68℃下在5x SSC/5x Denhardt溶液/1.0% SDS中雜交,以及在室溫下在0.2x SSC/0.1% SDS中洗滌,或涉及其本領域公認的等同條件(例如,在60℃下在2.5 x SSC緩衝液中進行雜交,然後在37℃下以低緩衝液濃度進行幾個洗滌步驟,並保持穩定的條件)。中等條件涉及在42℃下在3x SSC中洗滌,或其本領域公認的等同條件。鹽濃度和溫度的參數可以改變,以實現探針和靶核酸之間同一性的最佳水平。有關此類條件的指導可在本領域獲得,例如,Sambrook等人, 1989, Molecular Cloning, A Laboratory Manual [分子選殖,實驗室手冊], 冷泉港出版社, 紐約;以及Ausubel等人(編輯), 1995, Current Protocols in Molecular Biology [分子生物學當前方案], (John Wiley & Sons, N.Y.[約翰威利父子出版社], 紐約) 第2.10單元。When applied to oligonucleotides, the term "annealing" is understood to mean the bonding of the oligonucleotide to an at least substantially complementary sequence (for base pairing involving hydrogen bonding), which base pairing may be Watson- Watson-Crick, Hoogsteen or reversed Hoogsteen base pairing. Stringent hybridization conditions involve hybridization in 5x SSC/5x Denhardt's solution/1.0% SDS at 68°C and washes in 0.2x SSC/0.1% SDS at room temperature, or their art-recognized equivalents (e.g., Hybridize in 2.5 x SSC buffer at 60 °C, followed by several wash steps at low buffer concentration at 37 °C and maintain stable conditions). Moderate conditions involve washing in 3x SSC at 42°C, or its art-recognized equivalent. The parameters of salt concentration and temperature can be varied to achieve optimal levels of identity between probe and target nucleic acid. Guidance on such conditions is available in the art, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, New York; and Ausubel et al. (eds.) , 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y., New York) Unit 2.10.
互補股的雜交典型地隨著序列的長度而改善。兩條股的特異性雜交係用12、13、14、15、16、17、18、19或20(較佳的是18、19或20)或更多個互補核鹼基的連續片段實現的。寡核苷酸的序列可以(但不一定)與其要雜交的靶序列100%互補。此外,寡核苷酸可以與一或多個區段雜交,使得在雜交事件中不涉及間插或相鄰區段。例如,寡核苷酸包含與前mRNA內的靶區域的至少70%、75%、80%、85%、90%、95%、96%、97%、98%或99%的序列互補性。例如,在寡核苷酸的20個核鹼基中有18個與靶區域互補並且因此特異性雜交的寡核苷酸將代表90%互補性。當18個核苷酸的寡核苷酸的序列係多瘤病毒大T抗原前mRNA的至少12個核鹼基的連續片段之反向互補序列時,其餘6個互補核鹼基可能與該12個成簇或與該12個不連續。使用本領域已知的BLAST程式(基本局部比對搜索工具)和PowerBLAST程式(Altschul等人, J. Mol. Biol.[分子生物學雜誌], 1990, 215, 403 -410;Zhang和Madden,Genome Res.[基因組研究], 1997, 7,649-656)可以常規地確定寡核苷酸與靶前mRNA區域的互補性百分比。Hybridization of complementary strands typically improves with sequence length. Specific hybridization of the two strands is achieved using a contiguous segment of 12, 13, 14, 15, 16, 17, 18, 19 or 20 (preferably 18, 19 or 20) or more complementary nucleobases . The sequence of an oligonucleotide can, but does not have to, be 100% complementary to the target sequence to which it hybridizes. Additionally, oligonucleotides can hybridize to one or more segments such that intervening or adjacent segments are not involved in the hybridization event. For example, the oligonucleotide contains at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence complementarity to the target region within the pre-mRNA. For example, an oligonucleotide in which 18 of the 20 nucleobases of the oligonucleotide are complementary to the target region and therefore hybridize specifically will represent 90% complementarity. When the sequence of the 18-nucleotide oligonucleotide is the reverse complement of a contiguous segment of at least 12 nucleobases of the polyomavirus large T antigen pre-mRNA, the remaining 6 complementary nucleobases may be identical to the 12-nucleobase sequence. clustered or discontinuous with the 12. Use the BLAST program (Basic Local Alignment Search Tool) and the PowerBLAST program known in the art (Altschul et al., J. Mol. Biol. [Journal of Molecular Biology], 1990, 215, 403-410; Zhang and Madden, Genome Res. [Genome Research], 1997, 7, 649-656) The percent complementarity of an oligonucleotide to the target pre-mRNA region can be routinely determined.
較佳的是,如果序列的至少12個核鹼基的連續片段與靶標至少基本上互補,則寡核苷酸能夠與靶標退火。「基本上互補」意指與靶序列的反向互補序列基本上相同。「基本上相同」意指寡核苷酸不需要與參考序列100%相同,可以包含如本文所定義的誤配和/或間隔子。較佳的是,基本上相同的寡核苷酸(如果不是100%相同)包含1至3個,亦即1、2或3個誤配和/或間隔子,較佳的是每個寡核苷酸一個誤配或間隔子,使得預期的退火不會因誤配和/或間隔子而失敗。為了能退火(儘管存在誤配和/或間隔子),較佳的是,寡核苷酸每10個核苷酸不包含多於1個誤配(如果第一個小數為5或更高,則向上入,否則向下舍)。如本文所用,術語「間隔子」係指非核苷酸間隔子分子,當連接兩個核苷酸時,其將兩個核苷酸之間的距離增加到約一個核苷酸的距離(亦即如果兩個核苷酸由第三個核苷酸連接,它們將分開的距離)。間隔子之非限制性實例係肌苷、d-尿嘧啶、鹵代鹼基、胺基-dT、C3、C12、Spacer 9、Spacer 18和dSpacer。Preferably, the oligonucleotide is capable of annealing to the target if a contiguous stretch of at least 12 nucleobases of the sequence is at least substantially complementary to the target. "Substantially complementary" means substantially identical to the reverse complement of the target sequence. "Substantially identical" means that the oligonucleotide need not be 100% identical to the reference sequence and may contain mismatches and/or spacers as defined herein. Preferably, substantially identical oligonucleotides (if not 100% identical) contain from 1 to 3, i.e. 1, 2 or 3 mismatches and/or spacers, preferably per oligonucleotide nucleotide a mismatch or spacer so that the intended annealing does not fail due to mismatches and/or spacers. In order to anneal (despite the presence of mismatches and/or spacers), it is preferred that the oligonucleotide does not contain more than 1 mismatch per 10 nucleotides (if the first decimal is 5 or higher, Then enter it upward, otherwise round it down). As used herein, the term "spacer" refers to a non-nucleotide spacer molecule that, when connecting two nucleotides, increases the distance between the two nucleotides to about one nucleotide distance (i.e. The distance that two nucleotides would separate if they were joined by a third nucleotide). Non-limiting examples of spacers are inosine, d-uracil, halobase, amine-dT, C3, C12, Spacer 9, Spacer 18 and dSpacer.
寡核苷酸可以包含3'和/或5'突出端,亦即核鹼基連續片段,其基本上不與靶區域互補。此類突出端不會阻止寡核苷酸與其靶區域特異性結合。Oligonucleotides may contain 3' and/or 5' overhangs, ie, contiguous stretches of nucleobases that are not substantially complementary to the target region. Such overhangs do not prevent the oligonucleotide from binding specifically to its target region.
術語「互補序列」和「反向互補序列」的使用類似,因為靶核酸的互補序列具有靶序列反向互補序列的序列。其中,「反向」反映了通常從核酸的5'端開始敘述序列。The terms "complementary sequence" and "reverse complement" are used similarly because the complementary sequence of a target nucleic acid has the sequence of the reverse complement of the target sequence. Among them, "reverse" reflects that the sequence is usually described starting from the 5' end of the nucleic acid.
「前mRNA」或「先質mRNA」係信使核糖核酸(mRNA)的未成熟單股。多瘤病毒T抗原前mRNA係藉由轉錄從細胞核中的多瘤病毒DNA模板合成的。前mRNA含有一或多個內含子,該等內含子在前mRNA成熟為mRNA的過程中被剪接。剪接過程從轉錄物中去除內含子並將外顯子連接在一起。內含子的兩側典型地為供體位點(內含子的5'端)和受體位點(內含子的3'端)。剪接位點係剪接所必需的,並且典型地包括內含子5'端的幾乎不變的序列GU和內含子3'端的通常具有不變的AG序列的剪接受體位點。GU和AG序列以及插入序列從前mRNA中剪接出來。多瘤病毒T抗原前mRNA的一個特徵係它可以被可變剪接或不剪接,從而導致產生至少兩個,通常是3、4或5個不同的剪接mRNA。病毒繁殖取決於病毒基因組的可用性、病毒蛋白的存在、細胞機制以及特別地各個階段和組分之間微妙的相互作用。病毒RNA的剪接過程係調節病毒繁殖過程的重要方法,並且影響細胞中形成的某些產物的水平和可能的時間。術語「剪接位點」係指定義剪接基因座的序列(「剪接序列」)。"Pre-mRNA" or "pre-mRNA" is the immature single strand of messenger ribonucleic acid (mRNA). Polyomavirus T antigen pre-mRNA is synthesized by transcription from a polyomavirus DNA template in the nucleus. Pre-mRNA contains one or more introns, which are spliced during the maturation of pre-mRNA into mRNA. The splicing process removes introns from the transcript and joins the exons together. Introns are typically flanked by donor sites (5' end of the intron) and acceptor sites (3' end of the intron). Splice sites are required for splicing and typically include a nearly invariant sequence GU at the 5' end of the intron and a splice acceptor site with an often invariant AG sequence at the 3' end of the intron. GU and AG sequences as well as insert sequences are spliced out of the pre-mRNA. A characteristic feature of the polyomavirus T antigen pre-mRNA is that it can be alternatively spliced or unspliced, resulting in the production of at least two, and usually 3, 4, or 5 different spliced mRNAs. Viral propagation depends on the availability of viral genomes, the presence of viral proteins, cellular machinery and, in particular, subtle interactions between various stages and components. The splicing process of viral RNA is an important way to regulate the viral reproduction process and affects the levels and possibly the timing of certain products formed in the cell. The term "splice site" refers to the sequence that defines the splicing locus (the "splice sequence").
如本文所用,「能夠被轉錄」意指DNA核鹼基序列與RNA核鹼基序列相同,不同之處在於DNA在RNA的所有尿嘧啶位置都具有胸腺嘧啶。As used herein, "capable of being transcribed" means that the DNA nucleobase sequence is the same as the RNA nucleobase sequence, except that DNA has thymine at all uracil positions of RNA.
關於寡核苷酸的作用,本文提及的「用寡核苷酸治療開始」意指包括使感染的細胞與寡核苷酸接觸的治療。治療可為體外(例如細胞培養)或體內(非人動物或人)治療。With regard to the effect of oligonucleotides, reference herein to "initiation of treatment with oligonucleotides" means a treatment involving contacting infected cells with oligonucleotides. Treatment can be in vitro (eg, cell culture) or in vivo (in non-human animals or humans).
在最廣泛的意義上,「離體」意指體外。在較佳的實施方式中,其指的是在自然條件變化最小的外部環境中在生物體的組織內或組織上進行的實驗或量測。 實例 實例 1結果 ASO nanowalk 和候選修飾 In its broadest sense, "ex vivo" means outside the body. In a preferred embodiment, it refers to experiments or measurements performed in or on the tissues of an organism in an external environment with minimal changes in natural conditions. Example Example 1 Results ASO nanowalk and candidate modifications
此前,我們開發了反義寡核苷酸(ASO),該反義寡核苷酸靶向BKV早期編碼區前mRNA的外顯子-內含子接點,其導致產生大T抗原、小t抗原和截短的T抗原mRNA(WO 2019/168402)。該等研究產生了幾種ASO,該等ASO顯示出減弱以下表現的能力:1) 大T抗原和VP1 mRNA;2) VP1蛋白;以及3) 病毒DNA產生。在BKV早期編碼區前mRNA中,大多數5' ASO在大T抗原序列的內含子區僅具有1個核鹼基,而大多數3' ASO在大T抗原外顯子1的外顯子部分僅具有1個核鹼基。如圖2所示,ASO的定位以5'至3'的方向持續移動SEQ ID NO: 1下游的2個核苷酸,從而允許整個外顯子1-內含子接點與本文所述之SEQ ID NO: 1至11(RNA序列)和相應的2'-OMe修飾的ASO(SEQ ID NO: 12至22)有效拼接。因此,對於該等研究,生成了11個另外的ASO(SEQ ID NO: 12至22)並篩選其在人PTEC中的活性。Previously, we developed antisense oligonucleotides (ASOs) that target the exon-intron junction of the pre-mRNA of the early coding region of BKV, which results in the generation of large T antigen, small T antigen Antigen and truncated T-antigen mRNA (WO 2019/168402). These studies yielded several ASOs that demonstrated the ability to attenuate: 1) large T antigen and VP1 mRNA; 2) VP1 protein; and 3) viral DNA production. In the BKV early coding region pre-mRNA, most 5' ASOs have only 1 nucleobase in the intronic region of the large T antigen sequence, while most 3' ASOs have only one nucleobase in the exon 1 of the large T antigen. Part has only 1 nucleobase. As shown in Figure 2, the positioning of the ASO continuously shifts 2 nucleotides downstream of SEQ ID NO: 1 in the 5' to 3' direction, allowing the entire exon 1-intron junction to be aligned with that described herein. SEQ ID NO: 1 to 11 (RNA sequence) and the corresponding 2'-OMe modified ASO (SEQ ID NO: 12 to 22) were efficiently spliced. Therefore, for these studies, 11 additional ASOs (SEQ ID NO: 12 to 22) were generated and screened for activity in human PTEC.
令人驚訝的是,該等新2'-OMe修飾的ASO(圖2)中的幾個對大T抗原外顯子1和/或內含子供體位點表現出優越的靶向活性(圖3至圖4)。Surprisingly, several of these new 2'-OMe modified ASOs (Figure 2) showed superior targeting activity to the large T antigen exon 1 and/or intron donor sites (Figure 3 to Figure 4).
該一見解係藉由對人近端小管上皮細胞(hPTEC)進行篩選研究獲得的,其導致對1) TAg和VP1 mRNA;2) VP1蛋白;和3) 病毒顆粒產生的有效功效,如圖3所示。隨後,我們藉由用5-甲基胞苷(SEQ ID NO: 26、30、34和38)和不同百分比的2'-甲氧基-乙基(MOE)修飾的寡核苷酸(SEQ ID NO: 23、24、25、27、28、29、31、32、33、35、36、37)替換2'OMe-胞嘧啶來對該等主要候選物的各種修飾進行廣泛測試。該等研究一致表明,具有2'-O-甲基修飾的ASO使VP1蛋白和BKV DNA複製的減少最多(圖4)。該等資料,以及我們先前的研究(其中我們改變了摻入ASO骨架中的硫代磷酸酯的量)促使我們繼續使用完全2'-OMe和硫代磷酸化的ASO。 靶向 BKV 的 ASO 限制「再感染」 This insight was gained through screening studies on human proximal tubule epithelial cells (hPTEC), which resulted in effective efficacy on 1) TAg and VP1 mRNA; 2) VP1 protein; and 3) viral particle production, as shown in Figure 3 shown. Subsequently, we synthesized oligonucleotides modified with 5-methylcytidine (SEQ ID NO: 26, 30, 34, and 38) and different percentages of 2'-methoxy-ethyl (MOE) (SEQ ID NO: 23, 24, 25, 27, 28, 29, 31, 32, 33, 35, 36, 37) Various modifications of these leading candidates were extensively tested by replacing 2'OMe-cytosine. These studies consistently showed that ASOs with 2'-O-methyl modification caused the greatest reduction in VP1 protein and BKV DNA replication (Fig. 4). This information, as well as our previous studies in which we varied the amount of phosphorothioate incorporated into the ASO backbone, prompted us to proceed with the use of fully 2'-OMe and phosphorothioated ASOs. ASO targeting BKV limits “re-infection”
根據觀察到的回應於用我們的靶向BKV的ASO治療產生的病毒DNA減少,我們選擇進行「再感染」研究。為此,我們用SEQ ID NO: 26、SEQ ID NO: 17、SEQ ID NO: 34或SEQ ID NO: 19處理hPTEC,並在16 h後用BKV感染該等hPTEC。將細胞在培養基中孵育7天,然後收穫含有活性和非活性病毒顆粒的條件培養基。隨後將條件培養基稀釋10倍並置於未處理的hPTEC上。72 h後,將細胞固定並針對大T抗原陽性進行免疫染色(圖5),表明相對於對照細胞(左小圖),用SEQ ID NO: 19(和HYB_03(SEQ ID NO: 68))處理後大T抗原陽性細胞的數量明顯減少。此外,用SEQ ID NO: 19(和HYB_03(SEQ ID NO: 68))處理後的陽性程度低於對照細胞(以及SEQ ID NO: 26和SEQ ID NO: 17處理的細胞)觀察到的陽性程度。如圖6所示,TAg和VP1 mRNA水平的評估表明,用SEQ ID NO: 19預處理後降低程度最大,VP1蛋白的評估亦是如此。用SEQ ID NO: 19處理後,病毒DNA水平和再感染細胞的數目明顯降低。 靶向 BKV 的 ASO 誘導 BKV 早期編碼區前 mRNA 的異常剪接 Based on the observed reduction in viral DNA in response to treatment with our BKV-targeting ASO, we elected to conduct a "reinfection" study. To this end, we treated hPTEC with SEQ ID NO: 26, SEQ ID NO: 17, SEQ ID NO: 34 or SEQ ID NO: 19 and infected the hPTEC with BKV 16 h later. Cells were incubated in culture medium for 7 days and then conditioned medium containing active and inactive viral particles was harvested. Conditioned medium was then diluted 10-fold and plated onto untreated hPTEC. After 72 h, cells were fixed and immunostained for large T antigen positivity (Fig. 5), showing treatment with SEQ ID NO: 19 (and HYB_03 (SEQ ID NO: 68)) relative to control cells (left panel) The number of large T antigen-positive cells was significantly reduced. Furthermore, the degree of positivity after treatment with SEQ ID NO: 19 (and HYB_03 (SEQ ID NO: 68)) was lower than that observed with control cells (and cells treated with SEQ ID NO: 26 and SEQ ID NO: 17) . As shown in Figure 6, assessment of TAg and VP1 mRNA levels showed the greatest reduction after pretreatment with SEQ ID NO: 19, as was assessment of VP1 protein. After treatment with SEQ ID NO: 19, viral DNA levels and the number of reinfected cells were significantly reduced. ASO targeting BKV induces abnormal splicing of BKV early coding region pre - mRNA
隨後,我們在pRPc細胞中研究了我們的靶向BKV的ASO破壞BKV早期編碼區前mRNA的適當剪接的能力。為此,我們將我們的ASO投與於pRPc細胞,並在轉染後24小時收穫RNA。如圖7所示,該等研究表明,lipofectamine和加擾對照ASO處理的細胞沒有顯示改變的剪接特徵(泳道1-3)。相比之下,HYB_03(SEQ ID NO: 68)和SEQ ID NO: 19均導致大T抗原mRNA水平(產物1)降低,並向略微增加的小t抗原mRNA(產物3,泳道4-5)轉移。同時,明確的證據表明,產物1和3之間存在異常剪接產物,以及小於產物1的mRNA產物。此外,我們將誤配核鹼基引入SEQ ID NO: 19的位置10(SEQ ID NO: 39),位置5和10(SEQ ID NO: 40)以及位置5、10和15(SEQ ID NO:41)中,導致剪接效應逐漸滴定(gradual titration)(圖7,泳道6-8)。該等研究清楚地表明,SEQ ID NO: 19以雜交依賴性方式介導大T抗原mRNA的消除。接下來,我們類似地評估了設計為gapmer(RNA-DNA-RNA雜交反義寡核苷酸)的SEQ ID NO: 19是否可以以RNA酶H依賴性方式導致相似或更高水平的大T抗原(前)mRNA降解。如圖8(泳道3)所示,SEQ ID NO: 19 GapmeR(SEQ ID NO: 42)並未產生大T抗原mRNA水平(產物1)的有效降低,而靶向早期編碼區前mRNA的外顯子1的另外的GapmeR(SEQ ID NO: 42至45)類似地沒有導致大T或小t抗原mRNA表現水平的顯著降低。 PBMC 暴露於靶向 BKV 的 ASO 不會影響活力 Subsequently, we studied the ability of our BKV-targeting ASO to disrupt proper splicing of BKV early coding region pre-mRNA in pRPc cells. To do this, we administered our ASO to pRPc cells and harvested RNA 24 hours post-transfection. As shown in Figure 7, these studies showed that cells treated with lipofectamine and scrambled control ASO did not display altered splicing characteristics (lanes 1-3). In contrast, both HYB_03 (SEQ ID NO: 68) and SEQ ID NO: 19 resulted in a decrease in large T antigen mRNA levels (product 1) and a slight increase in small t antigen mRNA (product 3, lanes 4-5) transfer. At the same time, clear evidence shows that there is an abnormal splicing product between products 1 and 3, as well as an mRNA product smaller than product 1. In addition, we introduced mismatched nucleobases into SEQ ID NO: 19 at position 10 (SEQ ID NO: 39), positions 5 and 10 (SEQ ID NO: 40) and positions 5, 10 and 15 (SEQ ID NO: 41 ), resulting in a gradual titration of the splicing effect (Fig. 7, lanes 6-8). These studies clearly demonstrate that SEQ ID NO: 19 mediates the elimination of large T antigen mRNA in a hybridization-dependent manner. Next, we similarly evaluated whether SEQ ID NO: 19, designed as a gapmer (RNA-DNA-RNA hybrid antisense oligonucleotide), could lead to similar or higher levels of large T antigen in an RNase H-dependent manner (Pre)mRNA degradation. As shown in Figure 8 (lane 3), SEQ ID NO: 19 GapmeR (SEQ ID NO: 42) did not produce an efficient reduction in large T antigen mRNA levels (product 1), whereas targeting the exoexpression of early coding region pre-mRNA Additional GapmeRs of subunit 1 (SEQ ID NOs: 42 to 45) similarly did not result in significant reductions in large T or small t antigen mRNA expression levels. Exposure of PBMC to BKV- targeting ASOs does not affect viability
接下來,我們評估了暴露於增加水平的靶向BKV的ASO是否會影響細胞活力,由此將周邊血來源的單核細胞(PBMC)暴露於1或10 μM ASO 48 h。如圖9所示,引入了細胞活力的多種對照,包括在培養48 h之前暴露於65˚C 30分鐘(引發細胞活力降低)以及用R848(TLR3促效劑)處理以增強PBMC活力(Doyle S.L.,等人 (2007), J. Biol Chem.[生物化學雜誌] 282 (51): 36953-36960)。暴露於SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19(不增加其濃度)似乎不影響PBMC活力。 PBMC 暴露於靶向 BKV 的 ASO 導致細胞介素產生的輕度激活 We next evaluated whether exposure to increasing levels of BKV-targeting ASO affects cell viability, whereby peripheral blood-derived mononuclear cells (PBMC) were exposed to 1 or 10 μM ASO for 48 h. As shown in Figure 9 , multiple controls for cell viability were introduced, including exposure to 65˚C for 30 min before culture for 48 h (triggering a decrease in cell viability) and treatment with R848 (TLR3 agonist) to enhance PBMC viability (Doyle SL , et al. (2007), J. Biol Chem. [Journal of Biological Chemistry] 282 (51): 36953-36960). Exposure to SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34, or SEQ ID NO: 19 (without increasing their concentration) does not appear to affect PBMC viability. Exposure of PBMC to BKV - targeting ASOs results in mild activation of interleukin production
為了控制單核細胞和巨噬細胞回應於暴露於增加水平的靶向BKV的ASO的促炎細胞介素表現的潛在激活,我們用1或10 µM ASO處理PBMC 48 h,並收穫培養基用於細胞介素產生的多重ELISA分析。如圖10和圖11所示,暴露於SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19未顯示促炎或抗炎細胞介素產生的顯著增加。 凝血時間不受靶向 BKV 的 ASO 的影響 To control for the potential activation of pro-inflammatory cytokine expression of monocytes and macrophages in response to exposure to increasing levels of BKV-targeting ASO, we treated PBMC with 1 or 10 µM ASO for 48 h and harvested the culture medium for use in the cells Multiplex ELISA analysis of interleukin production. As shown in Figures 10 and 11, exposure to SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34, or SEQ ID NO: 19 did not show a significant increase in pro- or anti-inflammatory cytokine production. Clotting times are not affected by BKV - targeting ASOs
有時,ASO被描述為影響血液生理凝固所需的時間。此對於接受基於寡核苷酸的療法(包括腎移植後靶向BKV的ASO)的患者可能是有害的。因此,我們選擇藉由確定活化部分凝血激酶時間(aPTT)來評估血液暴露於增加濃度的我們的靶向BKV的ASO是否會增加凝血時間。為此,收集人血漿並將其暴露於濃度為1 µM和10 µM的SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19。如圖12所示,暴露於較高濃度的靶向BKV的ASO時,凝血時間略有增加, 靶向 BKV 的 ASO 顯示在腎中的優異生物分佈 ASO is sometimes described as affecting the time it takes for blood to physiologically clot. This may be detrimental in patients receiving oligonucleotide-based therapies, including BKV-targeting ASOs after kidney transplantation. Therefore, we chose to evaluate whether exposure of blood to increasing concentrations of our BKV-targeting ASO would increase clotting time by determining activated partial thromboplastin time (aPTT). For this purpose, human plasma is collected and exposed to SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34 or SEQ ID NO: 19 at concentrations of 1 µM and 10 µM. As shown in Figure 12, clotting times increased slightly upon exposure to higher concentrations of BKV-targeting ASOs, and BKV - targeting ASOs showed excellent biodistribution in the kidney.
由於BKV主要存在於腎的小管上皮細胞(和膀胱上皮)中,因此我們用五種候選ASO進行了四次靜脈內給藥研究(第0天、第3天、第7天和第10天濃度為40 mg/kg,第14天處死,圖13左小圖),以檢查我們的靶向BKV的ASO在腎(並且特別地近端和遠端小管上皮細胞)中的生物分佈。如圖13(左側圖像和中間圖像)所示,具有SEQ ID NO: 19的寡核苷酸清楚地顯示在近端小管中的攝取,其中在腎片段的遠端隔室中觀察到弱染色(圖13,右側圖像)。我們開發了一種雜交ELISA(hELISA)探針,用於以定量方式確定各種器官中的SEQ ID NO: 19水平。如圖14所示,該等研究表明,腎係SEQ ID NO: 19攝取的主要器官(以每個器官每克計),其攝取量為肝的大約2.75倍,脾的4倍。該等腎外器官被廣泛認為是ASO攝取的優異儲存庫,清楚地揭示了ASO在腎中的優越分佈。 靶向 BKV 的 ASO 治療方案不影響腎或肝功能 Since BKV is primarily found in the tubular epithelium of the kidney (and bladder epithelium), we conducted four intravenous dosing studies (day 0, day 3, day 7, and day 10 concentrations) with five candidate ASOs. at 40 mg/kg and sacrificed on day 14, Figure 13 left panel) to examine the biodistribution of our BKV-targeting ASO in the kidney (and specifically in proximal and distal tubular epithelial cells). As shown in Figure 13 (left and middle images), the oligonucleotide with SEQ ID NO: 19 clearly shows uptake in the proximal tubule, where weak uptake was observed in the distal compartment of the renal segment. Staining (Fig. 13, right image). We developed a hybrid ELISA (hELISA) probe for determining SEQ ID NO: 19 levels in various organs in a quantitative manner. As shown in Figure 14, these studies show that the kidney is the main organ ingested by SEQ ID NO: 19 (on a per gram basis per organ), and its uptake is approximately 2.75 times that of the liver and 4 times that of the spleen. These extrarenal organs are widely considered to be excellent reservoirs for ASO uptake, clearly revealing the superior distribution of ASO in the kidney. BKV - targeting ASO regimen does not affect kidney or liver function
在我們的四次靜脈內給藥研究(第0天、第3天、第7天和第10天的濃度為40 mg/kg,第14天處死)的背景下,我們沒有觀察到任何明顯的腎或肝毒性/損傷跡象。如圖15所示,用SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19處理後,肌酐和血尿素氮的血清水平以及尿白蛋白水平完全在正常範圍內。類似地,在4次投與SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19後,天冬胺酸胺基轉移酶和丙胺酸胺基轉移酶的血清水平及其比率沒有產生任何肝毒性證據。 靶向 BKV 的 ASO 治療方案不導致明顯的腎損傷 We did not observe any significant Signs of renal or hepatotoxicity/injury. As shown in Figure 15, after treatment with SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34 or SEQ ID NO: 19, the serum levels of creatinine and blood urea nitrogen and the urinary albumin level were completely within the normal range. within. Similarly, serum aspartate aminotransferase and alanine aminotransferase after 4 administrations of SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34, or SEQ ID NO: 19 The levels and their ratios did not produce any evidence of hepatotoxicity. BKV - targeting ASO regimen does not cause significant renal injury
四次投與SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19後的免疫組織化學染色(針對腎損傷的證據)未顯示明顯/急性腎損傷的跡象。KIM-1和天狼猩紅(picrosirius red)染色的初步評估未明確指出腎損傷或膠原沈積(見圖16)。 材料與方法 用於系統發育分析的登錄號 Immunohistochemical staining (for evidence of kidney injury) after four administrations of SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34, or SEQ ID NO: 19 showed no evidence of overt/acute kidney injury. Initial evaluation with KIM-1 and picrosirius red staining did not clearly indicate renal injury or collagen deposition (see Figure 16). Materials and Methods Accession numbers used for phylogenetic analysis
BK多瘤病毒分離株的完整基因組序列係從公開的NCBI資料庫下載的。從該等記錄中,只有報告完整基因組的分離株用於保存大T抗原中的剪接位點。Dunlop菌株用作參考基因組。分離株「MM」和「FNL-9」分別由於內含子中的大量缺失或與受體剪接位點重疊的重複而被去除。以下提供了245個獨特基因組序列的登錄號: AB211369.1;AB211370.1;AB211371.1;AB211372.1;AB211373.1;AB211374.1;AB211375.1;AB211376.1;AB211377.1;AB211378.1;AB211379.1;AB211381.1;AB211382.1;AB211383.1;AB211384.1;AB211385.1;AB211386.1;AB211387.1;AB211388.1;AB211389.1;AB211390.1;AB211391.1;AB213487.1;AB217917.1;AB217918.1;AB217919.1;AB217920.1;AB217921.1;AB260028.1;AB260029.1;AB260030.1;AB260031.1;AB260032.1;AB260033.1;AB263912.1;AB263913.1;AB263914.1;AB263915.1;AB263916.1;AB263917.1;AB263918.1;AB263919.1;AB263920.1;AB263921.1;AB263922.1;AB263923.1;AB263924.1;AB263925.1;AB263926.1;AB263927.1;AB263928.1;AB263929.1;AB263930.1;AB263931.1;AB263932.1;AB263934.1;AB263935.1;AB263936.1;AB263938.1;AB269825.1;AB269826.1;AB269827.1;AB269828.1;AB269829.1;AB269830.1;AB269831.1;AB269832.1;AB269834.1;AB269836.1;AB269837.1;AB269838.1;AB269840.1;AB269841.1;AB269842.1;AB269843.1;AB269844.1;AB269845.1;AB269846.1;AB269847.1;AB269848.1;AB269849.1;AB269850.1;AB269851.1;AB269852.1;AB269853.1;AB269854.1;AB269855.1;AB269856.1;AB269857.1;AB269858.1;AB269859.1;AB269860.1;AB269861.1;AB269862.1;AB269863.1;AB269864.1;AB269865.1;AB269866.1;AB269867.1;AB269868.1;AB269869.1;AB298941.1;AB298942.1;AB298945.1;AB298946.1;AB298947.1;AB301086.1;AB301087.1;AB301089.1;AB301090.1;AB301091.1;AB301092.1;AB301093.1;AB301094.1;AB301095.1;AB301096.1;AB301097.1;AB301099.1;AB301100.1;AB301101.1;AB365130.1;AB365132.1;AB365133.1;AB365134.1;AB365136.1;AB365137.1;AB365138.1;AB365139.1;AB365140.1;AB365141.1;AB365142.1;AB365144.1;AB365145.1;AB365146.1;AB365148.1;AB365149.1;AB365150.1;AB365151.1;AB365153.1;AB365154.1;AB365156.1;AB365157.1;AB365158.1;AB365159.1;AB365160.1;AB365162.1;AB365164.1;AB365165.1;AB365166.1;AB365167.1;AB365168.1;AB365170.1;AB365173.1;AB365174.1;AB365175.1;AB365176.1;AB365178.1;AB369087.1;AB369088.1;AB369089.1;AB369090.1;AB369092.1;AB369093.1;AB369094.1;AB369095.1;AB369096.1;AB369097.1;AB369098.1;AB369099.1;AB369101.1;AB464953.1;AB464954.1;AB464956.1;AB464957.1;AB464958.1;AB464960.1;AB464961.1;AB464962.1;AB485695.1;AB485696.1;AB485697.1;AB485698.1;AB485699.1;AB485700.1;AB485701.1;AB485703.1;AB485704.1;AB485707.1;AB485709.1;AB485710.1;AB485711.1;AB485712.1;AY628224.1;AY628225.1;AY628226.1;AY628227.1;AY628228.1;AY628229.1;AY628230.1;AY628231.1;AY628232.1;AY628233.1;AY628234.1;AY628235.1;AY628236.1;AY628237.1;AY628238.1;DQ305492.1;EF376992.1;FR720308.1;FR720309.1;FR720310.1;FR720311.1;FR720312.1;FR720313.1;FR720315.1;FR720317.1;FR720318.1;FR720320.1;FR720321.1;JF894228.1;JN192431.1;JN192432.1;JN192433.1;JN192435.1;JN192437.1;JN192438.1;JN192439.1;JN192440.1;JQ713822.1;KF055891.1;KF055892.1;KF055893.1;KP412983.1;KP984526.1;KY114802.1;KY114803.1;KY132094.1;KY487998.1;LC029413.1;LC309239.1;LC309240.1;LT960370.1;M23122.1;V01108.1。 The complete genome sequences of BK polyomavirus isolates were downloaded from the publicly available NCBI database. From these records, only isolates reporting complete genomes were used to preserve splice sites in the large T antigen. The Dunlop strain was used as the reference genome. Isolates “MM” and “FNL-9” were removed due to large deletions in introns or duplications overlapping the receptor splice site, respectively. Accession numbers for 245 unique genome sequences are provided below: AB211369.1; AB211370.1; AB211371.1; AB211372.1; AB211373.1; AB211374.1; AB211375.1; AB211376.1; 382. 1;AB211383.1;AB211384.1;AB211385.1;AB211386.1;AB211387.1;AB211388.1;AB211389.1;AB211390.1;AB211391.1;AB213487.1;AB217917.1;AB217918.1; AB217919.1; AB217920.1; AB217921.1; AB260028.1; AB260029.1; AB260030.1; AB260031.1; AB260032.1; 915. 1;AB263916.1;AB263917.1;AB263918.1;AB263919.1;AB263920.1;AB263921.1;AB263922.1;AB263923.1;AB263924.1;AB263925.1;AB263926.1;AB263927.1; AB263928.1; AB263929.1; AB263930.1; AB263931.1; AB263932.1; AB263934.1; AB263935.1; AB263936.1; 828. 1; AB269829.1; AB269830.1; AB269831.1; AB269832.1; AB269834.1; AB269836.1; AB269837.1; AB269838.1; AB269840.1; AB269841.1; AB269844.1; AB269845.1; AB269846.1; AB269847.1; AB269848.1; AB269849.1; AB269850.1; AB269851.1; 856. 1;AB269857.1;AB269858.1;AB269859.1;AB269860.1;AB269861.1;AB269862.1;AB269863.1;AB269864.1;AB269865.1;AB269866.1;AB269867.1;AB269868.1; AB269869.1; AB298941.1; AB298942.1; AB298945.1; AB298946.1; AB298947.1; AB301086.1; AB301087.1; 093. 1; AB301094.1; AB301095.1; AB301096.1; AB301097.1; AB301099.1; AB301100.1; AB301101.1; AB365130.1; AB365132.1; AB365133.1; AB365137.1; AB365138.1; AB365139.1; AB365140.1; AB365141.1; AB365142.1; AB365144.1; 151. 1; AB365153.1; AB365154.1; AB365156.1; AB365157.1; AB365158.1; AB365159.1; AB365160.1; AB365162.1; AB365164.1; AB365165.1; AB365168.1; AB365170.1; AB365173.1; AB365174.1; AB365175.1; AB365176.1; AB365178.1; 093. 1; AB369094.1; AB369095.1; AB369096.1; AB369097.1; AB369098.1; AB369099.1; AB369101.1; AB464960.1; AB464961.1; AB464962.1; AB485695.1; AB485696.1; AB485697.1; AB485698.1; AB485699.1; 707. 1; AB485709.1; AB485710.1; AB485711.1; AB485712.1; AY628224.1; AY628225.1; AY628226.1; AY628227.1; AY628232.1;AY628233.1;AY628234.1;AY628235.1;AY628236.1;AY628237.1;AY628238.1;DQ305492.1;EF376992.1;FR720308.1;FR720309.1;FR720310.1;FR7 20311. 1; FR720312.1; FR720313.1; FR720315.1; FR720317.1; FR720318.1; FR720320.1; FR720321.1; JF894228.1; JN192431.1; JN192432.1; JN192433.1; JN19243 5.1; JN192437.1; JN192438.1; JN192439.1; JN192440.1; JQ713822.1; KF055891.1; KF055892.1; 3.1; KY132094. 1; KY487998.1; LC029413.1; LC309239.1; LC309240.1; LT960370.1; M23122.1; V01108.1.
類似地,下載了13種不同原型人多瘤病毒的完整基因組序列。登錄號如下: NC_001538;NC_001699;NC_009238;NC_009539;NC_010277;NC_014406;NC_014407;NC_014361;NC_015150;NC_018102;NC_020106;NC_020890;NC_024118。 大 T 抗原剪接位點的保守性 Similarly, the complete genome sequences of 13 different prototype human polyomaviruses were downloaded. The accession numbers are as follows: NC_001538; NC_001699; NC_009238; NC_009539; NC_010277; NC_014406; NC_014407; NC_014361; Conservation of the large T antigen splice site
來自所有參考人多瘤病毒的全基因組核苷酸序列在2018年2月20日從NCBI網站(https://www.ncbi.nlm.nih.gov/nuccore)下載,並使用默認設置用WebPrank(線上提供:https://www.ebi.ac.uk/goldman-srv/webprank/)比對。構建了系統發育UPGMA樹,並為每個剪接位點創建序列標識,以顯示不同人多瘤病毒之間的保守性。下面提供了所有下載的refseq登錄號:Whole-genome nucleotide sequences from all reference human polyomaviruses were downloaded from the NCBI website (https://www.ncbi.nlm.nih.gov/nuccore) on February 20, 2018, and analyzed with WebPrank using default settings ( Available online: https://www.ebi.ac.uk/goldman-srv/webprank/) comparison. A phylogenetic UPGMA tree was constructed and sequence identities were created for each splice site to show conservation among different human polyomaviruses. All downloaded refseq accession numbers are provided below:
參考序列:NC_001538、NC_001699、NC_009238、NC_009539、NC_010277、NC_014406、NC_014407、NC_014361、NC_015150、NC_018102、NC_020106、NC_020890、NC_024118Reference sequences: NC_001538, NC_001699, NC_009238, NC_009539, NC_010277, NC_014406, NC_014407, NC_014361, NC_015150, NC_018102, NC_020106, NC_020890, NC_024118
所有人多瘤病毒分離株的全基因組核苷酸序列在2018年2月20日從NCBI網站下載。僅從獨特基因組序列中檢索大T抗原的全基因序列,並使用默認設置用WebPrank比對。為大T抗原中的每個剪接位點創建序列標識,以顯示不同人多瘤病毒內部和之間的保守性。The complete genome nucleotide sequences of human polyomavirus isolates were downloaded from the NCBI website on February 20, 2018. Full gene sequences of the large T antigen were retrieved from unique genomic sequences only and aligned with WebPrank using default settings. Sequence identifiers were created for each splice site in the large T antigen to show conservation within and between different human polyomaviruses.
所有下載的登錄號如下所示: BKPyV:AB211369.1、AB211370.1、AB211371.1、AB211372.1、AB211373.1、AB211374.1、AB211375.1、AB211376.1、AB211377.1、AB211378.1、AB211379.1、AB211380.1、AB211381.1、AB211382.1、AB211383.1、AB211384.1、AB211385.1、AB211386.1、AB211387.1、AB211388.1、AB211389.1、AB211390.1、AB211391.1、AB213487.1、AB217917.1、AB217918.1、AB217919.1、AB217920.1、AB217921.1、AB260028.1、AB260029.1、AB260030.1、AB260031.1、AB260032.1、AB260033.1、AB260034.1、AB263912.1、AB263913.1、AB263914.1、AB263915.1、AB263916.1、AB263917.1、AB263918.1、AB263919.1、AB263920.1、AB263921.1、AB263922.1、AB263923.1、AB263924.1、AB263925.1、AB263926.1、AB263927.1、AB263928.1、AB263929.1、AB263930.1、AB263931.1、AB263932.1、AB263933.1、AB263934.1、AB263935.1、AB263936.1、AB263937.1、AB263938.1、AB269822.1、AB269823.1、AB269824.1、AB269825.1、AB269826.1、AB269827.1、AB269828.1、AB269829.1、AB269830.1、AB269831.1、AB269832.1、AB269833.1、AB269834.1、AB269835.1、AB269836.1、AB269837.1、AB269838.1、AB269839.1、AB269840.1、AB269841.1、AB269842.1、AB269843.1、AB269844.1、AB269845.1、AB269846.1、AB269847.1、AB269848.1、AB269849.1、AB269850.1、AB269851.1、AB269852.1、AB269853.1、AB269854.1、AB269855.1、AB269856.1、AB269857.1、AB269858.1、AB269859.1、AB269860.1、AB269861.1、AB269862.1、AB269863.1、AB269864.1、AB269865.1、AB269866.1、AB269867.1、AB269868.1、AB269869.1、AB298940.1、AB298941.1、AB298942.1、AB298943.1、AB298944.1、AB298945.1、AB298946.1、AB298947.1、AB301086.1、AB301087.1、AB301088.1、AB301089.1、AB301090.1、AB301091.1、AB301092.1、AB301093.1、AB301094.1、AB301095.1、AB301096.1、AB301097.1、AB301098.1、AB301099.1、AB301100.1、AB301101.1、AB301102.1、AB301103.1、AB365130.1、AB365131.1、AB365132.1、AB365133.1、AB365134.1、AB365135.1、AB365136.1、AB365137.1、AB365138.1、AB365139.1、AB365140.1、AB365141.1、AB365142.1、AB365143.1、AB365144.1、AB365145.1、AB365146.1、AB365147.1、AB365148.1、AB365149.1、AB365150.1、AB365151.1、AB365152.1、AB365153.1、AB365154.1、AB365155.1、AB365156.1、AB365157.1、AB365158.1、AB365159.1、AB365160.1、AB365161.1、AB365162.1、AB365163.1、AB365164.1、AB365165.1、AB365166.1、AB365167.1、AB365168.1、AB365169.1、AB365170.1、AB365171.1、AB365172.1、AB365173.1、AB365174.1、AB365175.1、AB365176.1、AB365177.1、AB365178.1、AB369087.1、AB369088.1、AB369089.1、AB369090.1、AB369091.1、AB369092.1、AB369093.1、AB369094.1、AB369095.1、AB369096.1、AB369097.1、AB369098.1、AB369099.1、AB369100.1、AB369101.1、AB464953.1、AB464954.1、AB464955.1、AB464956.1、AB464957.1、AB464958.1、AB464959.1、AB464960.1、AB464961.1、AB464962.1、AB464963.1、AB485694.1、AB485695.1、AB485696.1、AB485697.1、AB485698.1、AB485699.1、AB485700.1、AB485701.1、AB485702.1、AB485703.1、AB485704.1、AB485705.1、AB485706.1、AB485707.1、AB485708.1、AB485709.1、AB485710.1、AB485711.1、AB485712.1、AY628224.1、AY628225.1、AY628226.1、AY628227.1、AY628228.1、AY628229.1、AY628230.1、AY628231.1、AY628232.1、AY628233.1、AY628234.1、AY628235.1、AY628236.1、AY628237.1、AY628238.1、DQ305492.1、EF376992.1、FR720308.1、FR720309.1、FR720310.1、FR720311.1、FR720312.1、FR720313.1、FR720314.1、FR720315.1、FR720316.1、FR720317.1、FR720318.1、FR720319.1、FR720320.1、FR720321.1、FR720322.1、FR720323.1、JF894228.1、JN192431.1、JN192432.1、JN192433.1、JN192434.1、JN192435.1、JN192436.1、JN192437.1、JN192438.1、JN192439.1、JN192440.1、JN192441.1、JQ713822.1、KF055891.1、KF055892.1、KF055893.1、KP412983.1、KP984526.1、KY114802.1、KY114803.1、KY132094.1、KY487998.1、LC029411.1、LC029412.1、LC029413.1、LC029414.1、LC309239.1、LC309240.1、LT934539.1、LT960370.1、M23122.1、MF627830.1、MF627831.1、V01108.1、V01109.1 剪接位點保守性和親緣關係樹 The accession numbers for all downloads are as follows: BKPyV: AB211369.1, AB211370.1, AB211371.1, AB211372.1, AB211373.1, AB211374.1, AB211375.1, AB211376.1, AB211377.1, AB211378.1 , AB211379.1, AB211380.1, AB211381.1, AB211382.1, AB211383.1, AB211384.1, AB211385.1, AB211386.1, AB211387.1, AB211388.1, AB211389.1, AB211390.1, AB2 11391 .1, AB213487.1, AB217917.1, AB217918.1, AB217919.1, AB217920.1, AB217921.1, AB260028.1, AB260029.1, AB260030.1, AB260031.1, AB260032.1, AB260033.1 , AB260034.1, AB263912.1, AB263913.1, AB263914.1, AB263915.1, AB263916.1, AB263917.1, AB263918.1, AB263919.1, AB263920.1, AB263921.1, AB263922.1, AB2 63923 .1, AB263924.1, AB263925.1, AB263926.1, AB263927.1, AB263928.1, AB263929.1, AB263930.1, AB263931.1, AB263932.1, AB263933.1, AB263934.1, AB263935.1 , AB263936.1, AB263937.1, AB263938.1, AB269822.1, AB269823.1, AB269824.1, AB269825.1, AB269826.1, AB269827.1, AB269828.1, AB269829.1, AB269830.1, AB2 69831 .1, AB269832.1, AB269833.1, AB269834.1, AB269835.1, AB269836.1, AB269837.1, AB269838.1, AB269839.1, AB269840.1, AB269841.1, AB269842.1, AB269843.1 , AB269844.1, AB269845.1, AB269846.1, AB269847.1, AB269848.1, AB269849.1, AB269850.1, AB269851.1, AB269852.1, AB269853.1, AB269854.1, AB269855.1, AB2 69856 .1, AB269857.1, AB269858.1, AB269859.1, AB269860.1, AB269861.1, AB269862.1, AB269863.1, AB269864.1, AB269865.1, AB269866.1, AB269867.1, AB269868.1 , AB269869.1, AB298940.1, AB298941.1, AB298942.1, AB298943.1, AB298944.1, AB298945.1, AB298946.1, AB298947.1, AB301086.1, AB301087.1, AB301088.1, AB3 01089 .1, AB301090.1, AB301091.1, AB301092.1, AB301093.1, AB301094.1, AB301095.1, AB301096.1, AB301097.1, AB301098.1, AB301099.1, AB301100.1, AB301101.1 , AB301102.1, AB301103.1, AB365130.1, AB365131.1, AB365132.1, AB365133.1, AB365134.1, AB365135.1, AB365136.1, AB365137.1, AB365138.1, AB365139.1, AB3 65140 .1, AB365141.1, AB365142.1, AB365143.1, AB365144.1, AB365145.1, AB365146.1, AB365147.1, AB365148.1, AB365149.1, AB365150.1, AB365151.1, AB365152.1 , AB365153.1, AB365154.1, AB365155.1, AB365156.1, AB365157.1, AB365158.1, AB365159.1, AB365160.1, AB365161.1, AB365162.1, AB365163.1, AB365164.1, AB3 65165 .1, AB365166.1, AB365167.1, AB365168.1, AB365169.1, AB365170.1, AB365171.1, AB365172.1, AB365173.1, AB365174.1, AB365175.1, AB365176.1, AB365177.1 , AB365178.1, AB369087.1, AB369088.1, AB369089.1, AB369090.1, AB369091.1, AB369092.1, AB369093.1, AB369094.1, AB369095.1, AB369096.1, AB369097.1, AB3 69098 .1, AB369099.1, AB369100.1, AB369101.1, AB464953.1, AB464954.1, AB464955.1, AB464956.1, AB464957.1, AB464958.1, AB464959.1, AB464960.1, AB464961.1 , AB464962.1, AB464963.1, AB485694.1, AB485695.1, AB485696.1, AB485697.1, AB485698.1, AB485699.1, AB485700.1, AB485701.1, AB485702.1, AB485703.1, AB4 85704 .1, AB485705.1, AB485706.1, AB485707.1, AB485708.1, AB485709.1, AB485710.1, AB485711.1, AB485712.1, AY628224.1, AY628225.1, AY628226.1, AY628227.1 , AY628228.1, AY628229.1, AY628230.1, AY628231.1, AY628232.1, AY628233.1, AY628234.1, AY628235.1, AY628236.1, AY628237.1, AY628238.1, DQ305492.1, EF 376992 .1, FR720308.1, FR720309.1, FR720310.1, FR720311.1, FR720312.1, FR720313.1, FR720314.1, FR720315.1, FR720316.1, FR720317.1, FR720318.1, FR720319.1 , FR720320.1, FR720321.1, FR720322.1, FR720323.1, JF894228.1, JN192431.1, JN192432.1, JN192433.1, JN192434.1, JN192435.1, JN192436.1, JN192 437.1, JN192438 .1, JN192439.1, JN192440.1, JN192441.1, JQ713822.1, KF055891.1, KF055892.1, KF055893.1, KP412983.1, KP984526.1, KY114802.1, KY114803.1, KY13 2094.1 , KY487998.1, LC029411.1, LC029412.1, LC029413.1, LC029414.1, LC309239.1, LC309240.1, LT934539.1, LT960370.1, M23122.1, MF627830.1, MF627831.1, V01 108 .1. V01109.1 splice site conservation and genetic relationship tree
使用clustalW(R中的「msa」包)比對13種不同的多瘤病毒參考序列和所有獨特BK多瘤病毒分離株的大T抗原的全基因序列(包括內含子序列)。使用UPGMA方法(R中的「phangorn」和「ggtree」包)構建了親緣關係樹。為受體和供體剪接位點構建序列標記,以顯示亞型之間的核苷酸特異性保守性(R中的「msa」包)。 ASO 設計 The full gene sequences (including intronic sequences) of the large T antigen of 13 different polyomavirus reference sequences and all unique BK polyomavirus isolates were aligned using clustalW (the “msa” package in R). Kinship trees were constructed using the UPGMA method ("phangorn" and "ggtree" packages in R). Construct sequence markers for acceptor and donor splice sites to show nucleotide-specific conservation between isoforms (‘msa’ package in R). ASO design
將反義寡核苷酸(ASO)設計為靶向BK病毒大T抗原的供體剪接位點(SEQ ID NO: 1至10)。將另外的ASO設計為衍生自SEQ ID NO: 1至10並具有改進的化學性質。它們含有2′-O-甲基鹼基,長度為20個核苷酸,並用全硫代磷酸酯骨架(*)和視需要用5甲基胞苷(指示的位置)進一步修飾。使用RNA結構預測ASO的二級結構和結合能。所有ASO序列如下所示:
表 3 :具有經修飾的RNA化學性質的寡核苷酸
從ATCC獲得永生化近端小管腎上皮HK2細胞(ATCC ® CRL-2190™),並將其保持在37℃,5% CO 2,杜氏改良Eagle培養基(Dulbecco’s Modified Eagle’s medium)-F12 1 : 1混合物(具有15 mM Hepes、2.5 mM L-麩醯胺酸(龍沙公司(Lonza)),並補充有三碘甲腺原胺酸、表皮生長因子(EGF)、胰島素-轉鐵蛋白-硒-乙醇胺(ITS-X)、氫化可的松和100 U/mL青黴素-鏈黴素)中。將人腎近端小管上皮細胞(PTEpiC)(Sciencell公司,#4100)維持在由500 ml基礎培養基、2%胎牛血清和1X上皮細胞生長補充劑組成的完全上皮細胞培養基(Sciencell公司,#4101)中。在第4代和第6代之間進行hPTEC實驗。pRPc細胞(一種用BKV的早期編碼區轉化的小鼠細胞系)(Negrini, M.等人, Cancer Research [癌症研究], 1992)組成型表現BKV大T抗原。將pRPc細胞維持在補充有10% FCS的杜氏改良Eagle培養基中。將所有細胞在37℃和5% CO 2下,在100 U/mL青黴素、100 μg/mL鏈黴素溶液(英傑公司(Invitrogen),布雷達,荷蘭)存在下培養。 Immortalized proximal tubule renal epithelial HK2 cells (ATCC® CRL-2190™) were obtained from ATCC and maintained at 37°C, 5% CO2 , Dulbecco's Modified Eagle's medium-F12 1:1 mixture (With 15 mM Hepes, 2.5 mM L-Glutamine (Lonza), supplemented with Triiodothyronine, Epidermal Growth Factor (EGF), Insulin-Transferrin-Se-Ethanolamine ( ITS-X), hydrocortisone, and 100 U/mL penicillin-streptomycin). Human renal proximal tubule epithelial cells (PTEpiC) (Sciencell, #4100) were maintained in complete epithelial medium (Sciencell, #4101) consisting of 500 ml of basal medium, 2% fetal calf serum, and 1X Epithelial Cell Growth Supplement. )middle. hPTEC experiments were performed between passages 4 and 6. pRPc cells, a mouse cell line transformed with the early coding region of BKV (Negrini, M. et al., Cancer Research, 1992), constitutively express the BKV large T antigen. pRPc cells were maintained in Dulbecco's modified Eagle medium supplemented with 10% FCS. All cells were cultured at 37°C and 5% CO in the presence of 100 U/mL penicillin, 100 μg/mL streptomycin solution (Invitrogen, Breda, The Netherlands).
從ATCC獲得BK多瘤病毒(ATCC® VR-837™),並將其在完全HK2培養基中稀釋以減少感染負荷。對於處理實驗,將細胞以32,000個細胞/cm 2的密度接種在6孔或12孔板(康寧公司(Corning))中並生長過夜。藉由將細胞與lipofectamine 3000(賽默飛世爾公司(Thermo Fisher))以50 nM的ASO濃度孵育5 h來進行ASO處理,然後洗掉lipofectamine。藉由將細胞與含有BK多瘤病毒的培養基孵育2 h來在洗滌細胞後24 h進行BK多瘤病毒感染,然後洗滌細胞3次以去除過量的病毒顆粒。在洗滌該等初始洗滌液後以及在感染後第3天、第5天和第7天收集上清液,以使用PCR確定病毒顆粒的產生。在感染前收集病毒載量樣本以確定感染負荷。在第7天收穫RNA和蛋白質以確定大T抗原和VP1的表現。 病毒載量確定 BK polyomavirus (ATCC® VR-837™) was obtained from ATCC and diluted in complete HK2 medium to reduce infection load. For treatment experiments, cells were seeded in 6- or 12-well plates (Corning) at a density of 32,000 cells/ cm and grown overnight. ASO treatment was performed by incubating cells with lipofectamine 3000 (Thermo Fisher) at an ASO concentration of 50 nM for 5 h, and then the lipofectamine was washed away. BK polyomavirus infection was performed 24 h after washing the cells by incubating the cells with BK polyomavirus-containing medium for 2 h, and then washing the cells three times to remove excess viral particles. Supernatants were collected after these initial washes and at days 3, 5, and 7 post-infection to determine viral particle production using PCR. Viral load samples are collected prior to infection to determine infectious load. RNA and protein were harvested on day 7 to determine the expression of large T antigen and VP1. Viral load determination
為了確定培養上清液中的病毒載量,在每個時間點從每個孔中收集200 µL。將Pierce通用核酸酶(Pierce Universal Nuclease)添加到每個樣本中以在室溫下降解未包裝的DNA 15分鐘,並且然後用5 mM EDTA滅活。使用DNA迷你套組(kit)(凱傑公司(Qiagen))從上清液中分離病毒DNA,並使用Taqman PCR確定病毒載量,如下所述( Wunderink, H.F.,等人, J. Clin. Virol.[臨床病毒學雜誌], 2017)。 To determine the viral load in the culture supernatant, 200 µL was collected from each well at each time point. Pierce Universal Nuclease was added to each sample to degrade unpackaged DNA for 15 min at room temperature and then inactivated with 5 mM EDTA. Viral DNA was isolated from the supernatant using a DNA minikit (Qiagen), and viral load was determined using Taqman PCR as described below ( Wunderink, HF , et al., J. Clin. Virol .[Journal of Clinical Virology], 2017).
為了監測DNA提取的品質和潛在的PCR抑制,我們向裂解緩衝液中添加低濃度的海豹皰疹病毒(phocine herpesvirus)。在最終體積為100 μL的洗脫緩衝液中洗脫DNA,其中10 μL用作即時定量PCR(qPCR)的輸入。使用引物440BKVs 5′-GAAAAGGAGAGTGTCCAGGG-3′(SEQ ID NO: 46)和441BKVas 5′-GAACTTCTACTCCTCCTTTTATTAGT-3′(SEQ ID NO: 47)及Taqman探針576BKV-TQ-FAM FAM 5′-CCAAAAAGCCAAAGGAACCC-3′-BHQ1(SEQ ID NO: 48),擴增BKPyV VP1基因內的90-bp片段(序列由LUMC醫學微生物學系(LUMC Department of Medical Microbiology)提供)。BKPyV qPCR和海豹皰疹病毒PCR被雙重用於DNA品質和潛在的PCR抑制監測。此外,BKPyV qPCR經驗證可偵測BKPyV基因型I-IV。To monitor the quality of DNA extraction and potential PCR inhibition, we added low concentrations of phocine herpesvirus to the lysis buffer. Elute DNA in a final volume of 100 μL of elution buffer, of which 10 μL is used as input for real-time quantitative PCR (qPCR). Use primers 440BKVs 5′-GAAAAGGAGAGTGTCCAGGG-3′ (SEQ ID NO: 46) and 441BKVas 5′-GAACTTCTACTCCTCCTTTATTAGT-3′ (SEQ ID NO: 47) and Taqman probe 576BKV-TQ-FAM FAM 5′-CCAAAAAGCCAAAGGAACCC-3′ -BHQ1 (SEQ ID NO: 48), amplifies a 90-bp fragment within the BKPyV VP1 gene (sequence provided by LUMC Department of Medical Microbiology). BKPyV qPCR and seal herpesvirus PCR were used dually to monitor DNA quality and potential PCR inhibition. In addition, BKPyV qPCR was validated to detect BKPyV genotypes I-IV.
在50 μL的總體積中進行定量PCR反應,其中含有25 μL HotStarTaq預混液(凱傑公司,希爾登,德國)、0.5 μmol/L每種引物、0.35 μmol/L BKPyV探針和3.5 mmol/L MgCl 2。使用CFX96即時偵測系統(伯樂公司(Bio-Rad),赫拉克勒斯,加利福尼亞州,美國)在以下循環條件下進行反應:在95℃下15 min,然後進行45次擴增循環(95℃下30 s;55℃下30 s;72℃下30 s)。為了定量,使用定量的BKPyV陽性尿樣的標準品。BKPyV qPCR的分析靈敏度為約10個拷貝/mL。在每個板上,包括3個陰性對照;該等對照在所有PCR測定中均測試為陰性。循環閾值 ≥ 40的PCR結果被認為是陰性的。 抗體和西方墨點法 Quantitative PCR reactions were performed in a total volume of 50 μL containing 25 μL HotStarTaq Master Mix (Qiagen, Hilden, Germany), 0.5 μmol/L of each primer, 0.35 μmol/L BKPyV probe, and 3.5 mmol/ L MgCl 2 . Reactions were performed using the CFX96 Instant Detection System (Bio-Rad, Hercules, CA, USA) under the following cycling conditions: 15 min at 95°C, followed by 45 cycles of amplification (95°C 30 s at 55℃; 30 s at 72℃). For quantification, quantitative standards of BKPyV-positive urine samples were used. The analytical sensitivity of BKPyV qPCR is approximately 10 copies/mL. On each plate, include 3 negative controls; these controls tested negative in all PCR assays. PCR results with a cycle threshold ≥ 40 were considered negative. Antibodies and Western blotting
使用BCA方法確定蛋白質濃度。在4%-15% TGX凝膠上跑樣,並將其轉移到硝化纖維素或PVDF膜上。所用的抗體係:兔多株抗肌動蛋白HRP(上樣對照)、兔多株抗SV40 VP1(ab53977,艾博抗公司(Abcam))、小鼠單株抗SV40 T抗原[PAb416](ab16879,艾博抗公司)、小鼠單株抗SV40 T抗原(PAb108,賽默飛世爾公司)、兔多株抗SV40 VP1(艾博抗公司,ab53977)、生物素化蓮花豆凝集素(Lotus Tetragonolobus Lectin)(LTL;載體實驗室(Vector Laboratories),B-1325)、綿羊多株抗裂隙素(AF4269,R&D系統公司(R&D Systems))、純化的小鼠抗E-鈣黏著蛋白(貝迪公司(Becton Dickinson),610181)、兔多株抗GAPDH(細胞傳訊公司(Cell Signalling),D16H11)和兔多株抗硫代磷酸酯(由麻塞諸塞大學醫學院(University of Massachusetts Medical School)Jonathan Watts博士友情提供)。將大T抗原和VP1的一抗在4℃下孵育過夜,並將肌動蛋白的一抗在室溫下孵育30分鐘。用於大T抗原和VP1的二抗分別為山羊多株抗小鼠HRP(P044701-2,安捷倫公司(Agilent))和山羊多株抗兔HRP(P044801-2,安捷倫公司)。此外,山羊抗兔488(生命技術公司(Life Technologies),A-11008)、驢抗兔488(英傑公司,A21206)、驢抗大鼠647(英傑公司,ab150155)、驢抗羊647(英傑公司,A21448)、驢抗小鼠IgG2a 647(英傑公司,A31571)、鏈黴親和素568(英傑公司,S11226)和同種型對照兔IgG(Dako公司,X0936)。將膜與SuperSignal™ West Femto最高靈敏度底物(賽默飛世爾公司)一起孵育,並使用ChemiDoc MP成像系統(伯樂公司)視覺化蛋白質條帶。將細胞核用Hoechst複染。 免疫組織化學 Determine protein concentration using the BCA method. Run samples on a 4%-15% TGX gel and transfer to nitrocellulose or PVDF membrane. Antibody systems used: rabbit polyclonal anti-actin HRP (loading control), rabbit polyclonal anti-SV40 VP1 (ab53977, Abcam), mouse monoclonal anti-SV40 T antigen [PAb416] (ab16879 , Abcam Company), mouse monoclonal anti-SV40 T antigen (PAb108, Thermo Fisher Company), rabbit multi-strain anti-SV40 VP1 (Abcam Company, ab53977), biotinylated lotus bean lectin (Lotus Tetragonolobus Lectin (LTL; Vector Laboratories, B-1325), sheep polystrain anti-slittin (AF4269, R&D Systems), purified mouse anti-E-cadherin (Beidi (Becton Dickinson, 610181), rabbit polyclonal anti-GAPDH (Cell Signalling, D16H11), and rabbit polyclonal anti-phosphorothioate (by Jonathan Watts, University of Massachusetts Medical School Courtesy of Dr.). Primary antibodies for large T antigen and VP1 were incubated overnight at 4°C, and primary antibodies for actin were incubated for 30 min at room temperature. The secondary antibodies used for large T antigen and VP1 were goat polyclonal anti-mouse HRP (P044701-2, Agilent) and goat polyclonal anti-rabbit HRP (P044801-2, Agilent), respectively. In addition, goat anti-rabbit 488 (Life Technologies, A-11008), donkey anti-rabbit 488 (Invitrogen, A21206), donkey anti-rat 647 (Invitrogen, ab150155), donkey anti-sheep 647 (Invitrogen) , A21448), donkey anti-mouse IgG2a 647 (Invitrogen, A31571), streptavidin 568 (Invitrogen, S11226), and isotype control rabbit IgG (Dako, X0936). The membrane was incubated with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific) and protein bands were visualized using the ChemiDoc MP Imaging System (Bio-Rad). Nuclei were counterstained with Hoechst. Immunohistochemistry
在處死小鼠後,切除小鼠腎、肝、肺、脾、膀胱和心臟並用PBS灌注並固定在10%緩衝福馬林和石蠟包埋中。將腎組織切成4 μm厚切片並放置在載玻片上。將新鮮切片的切片在二甲苯中脫蠟10分鐘,然後將切片在分級乙醇系列中再水化並置於PBS中。隨後,對切片進行ASO染色(使用偵測硫代磷酸酯骨架的抗體或藉由與基於RNA的探針的螢光原位雜交),對於腎損傷,藉由腎小管的腎損傷標誌物-1(KIM-1)的染色,或對於間質膠原,藉由天狼星紅染色。 即時 qPCR After the mice were sacrificed, the kidneys, liver, lungs, spleen, bladder, and heart were excised and perfused with PBS and fixed in 10% buffered formalin and embedded in paraffin. Kidney tissue was cut into 4 μm thick sections and placed on glass slides. Freshly sectioned sections were deparaffinized in xylene for 10 min, and then the sections were rehydrated in a graded ethanol series and placed in PBS. Subsequently, sections were stained for ASO (using antibodies that detect phosphorothioate scaffolds or by fluorescent in situ hybridization with RNA-based probes) and, for renal injury, by renal tubular renal injury marker-1. (KIM-1) staining, or for interstitial collagen, by Sirius red staining. real-time qPCR
將ASO處理和BKV感染的細胞在Trizol中裂解,並使用RNeasy套組(凱傑公司)分離RNA。在分離過程中添加DNA酶I(凱傑公司)處理以去除多餘的DNA,並使用Promega反轉錄酶、DTT、dNTP和隨機引物合成cDNA。在CFX384 Touch™即時PCR偵測系統(伯樂公司)上用SYBR™ Select預混液(賽默飛世爾公司)和以下引物進行即時PCR:
表 4 :引物
將磁性球添加到StartMax比色皿(思塔高診斷股份有限公司(Diagnostica Stago))中。用Owren-Koller稀釋劑(思塔高診斷股份有限公司)稀釋血漿,然後每個比色皿中添加50 μL aPTT試劑(TriniClot)。隨後,將含有5 uL ASO溶液的50 μL稀釋血漿添加到aPTT試劑中,並在37℃下孵育170秒。接下來,將比色皿置於磁場中,將磁場激活以激活凝血。在180秒時,使用連續移液器(repeater pipette)在每個比色皿中添加50 μL 25 mM CaCl 2,並且每次移液操作都會自動記錄凝血時間量測值。 周邊血單核細胞細胞介素產生測定 Magnetic beads were added to StartMax cuvettes (Diagnostica Stago). Plasma was diluted with Owren-Koller diluent (Stage Diagnostics, Inc.), and then 50 μL of aPTT reagent (TriniClot) was added to each cuvette. Subsequently, 50 μL of diluted plasma containing 5 uL of ASO solution was added to the aPTT reagent and incubated at 37°C for 170 seconds. Next, the cuvette is placed in a magnetic field, which is activated to activate clotting. At 180 seconds, 50 μL of 25 mM CaCl 2 was added to each cuvette using a repeater pipette, and clotting time measurements were automatically recorded with each pipetting operation. Peripheral blood mononuclear cell interleukin production assay
將PBMC解凍並添加至培養基中5分鐘,並藉由離心收集。將細胞重懸,並藉由台盼藍拒染法評估初始活力,然後將細胞稀釋至1.67×10 6個細胞/mL。隨後,在培養基(R848(Invivogen公司,美國))中製備陰性對照(培養基)和陽性對照。藉由以下來評估回應於暴露於1 μM或10 μM ASO的細胞介素產生:向圓底孔中添加20 μL陰性或陽性對照或適當的ASO濃度,然後添加180 μL含有PBMC的培養基並在37℃(5% CO 2)下孵育48 h。隨後,將培養基移液到Eppendorf管中,以1200 rpm離心6分鐘,並將上清液轉移到定製的多重elisa板中,以偵測GM-CSF、IFN-γ、IL-6、IL-12(p70亞基)、MIP-1b、TNF-α、G-CSF、IFN-α2、IL-1b、IL-2、IL-10和IL-17。 PBMC were thawed and added to the culture medium for 5 minutes and collected by centrifugation. The cells were resuspended and the initial viability was assessed by trypan blue exclusion method, and then the cells were diluted to 1.67 × 10 6 cells/mL. Subsequently, negative control (medium) and positive control were prepared in culture medium (R848 (Invivogen Inc., USA)). Assess interleukin production in response to exposure to 1 μM or 10 μM ASO by adding 20 μL of negative or positive control or appropriate ASO concentration to round-bottomed wells, then adding 180 μL of medium containing PBMC and incubating at 37 Incubate at ℃ (5% CO 2 ) for 48 h. Subsequently, the culture medium was pipetted into Eppendorf tubes, centrifuged at 1200 rpm for 6 min, and the supernatant was transferred to a custom-made multiplex elisa plate to detect GM-CSF, IFN-γ, IL-6, IL- 12 (p70 subunit), MIP-1b, TNF-α, G-CSF, IFN-α2, IL-1b, IL-2, IL-10 and IL-17.
藉由以下來評估細胞活力:在平底孔和190 μL含有PBMC的培養基中添加陰性或陽性對照材料或ASO,並在37℃(5% CO 2)下孵育48 h。隨後,每孔中添加CellTiter-Blue試劑,將孔混合4 h並孵育4 h,然後在555/585 nm下量測螢光。 雜交 ELISA ( hELISA ) Cell viability was assessed by adding negative or positive control material or ASO to flat-bottomed wells and 190 μL of medium containing PBMC, and incubating at 37°C (5% CO 2 ) for 48 h. Subsequently, CellTiter-Blue reagent was added to each well, the wells were mixed for 4 h and incubated for 4 h, and then the fluorescence was measured at 555/585 nm. Hybrid ELISA ( hELISA )
將從小鼠收穫的組織置於裂解緩衝液中並稀釋至非飽和濃度。隨後,藉由將1 µL標準曲線或組織樣本添加到96孔板的單個孔中之49 µL樣本緩衝液中,將標準曲線和組織樣本在樣本緩衝液中以1 : 50稀釋。接下來,每孔添加50 μL探針混合物(由20 nM捕獲探針和20 nM偵測探針組成),然後用光折射密封件覆蓋96孔板。接下來,將探針在熱循環儀中在95℃ 5分鐘,40℃ 30分鐘,最後保持在12℃來雜交。接下來,藉由用KPL洗滌緩衝液洗滌來製備MSD Gold板(Mesoscale公司),然後將雜交樣本從96孔板轉移到MSD Gold板中(一式兩孔)。用光折射密封件覆蓋MSD Gold板,並將其在室溫下以650 rpm在定軌振盪器上孵育30分鐘。接下來,將每個孔用KPL緩衝液洗滌3次,然後將0.5 µg/mL SULFO標籤抗地高辛抗體(在1%阻斷劑A緩衝液中)添加到每個孔中。用光折射條密封板,並將其在定軌振盪器上在650 rpm孵育60分鐘。隨後將板用KPL緩衝液洗滌3次,然後將MSD Gold緩衝液添加到每個孔中,並藉由分光光度法讀取板。 動物 Tissue harvested from mice was placed in lysis buffer and diluted to non-saturating concentrations. Subsequently, dilute the standard curve and tissue sample 1:50 in sample buffer by adding 1 µL of standard curve or tissue sample to 49 µL of sample buffer in a single well of a 96-well plate. Next, add 50 μL of probe mix (consisting of 20 nM capture probe and 20 nM detection probe) per well, and then cover the 96-well plate with a photorefractive seal. Next, hybridize the probe in a thermal cycler at 95°C for 5 minutes, 40°C for 30 minutes, and finally at 12°C. Next, MSD Gold plates (Mesoscale) were prepared by washing with KPL wash buffer, and hybridization samples were transferred from the 96-well plate to MSD Gold plates (in duplicate). Cover the MSD Gold plate with the photorefractive seal and incubate it on an orbital shaker at 650 rpm for 30 min at room temperature. Next, each well was washed 3 times with KPL buffer, and 0.5 µg/mL SULFO-tagged anti-digoxigenin antibody (in 1% Blocker A buffer) was added to each well. Seal the plate with a photorefractive strip and incubate it on an orbital shaker at 650 rpm for 60 min. The plate was then washed 3 times with KPL buffer, then MSD Gold buffer was added to each well and the plate was read spectrophotometrically. animal
將C57Bl6野生型小鼠安置在萊頓大學醫學中心(Leiden University Medical Center animal facility)的動物設施中。小鼠隨意接受食物和水。對於生物分佈和初步安全性研究,在第0天、第3天、第7天和第10天經由尾靜脈靜脈內投與ASO(40 mg/kg)。處死後(第14天),從每隻小鼠收集血液(和尿液),並將其在室溫下靜置30分鐘,然後在4℃下以6,000 rpm離心以收集上層血清。在LUMC的臨床化學實驗室確定肌酐、血尿素氮(BUN或尿素)、白蛋白、天冬胺酸轉胺酶(AST)和丙胺酸轉胺酶(ALT)。 實例 2結果 根據本發明的 ASO 具有改進的抗病毒活性 C57Bl6 wild-type mice were housed in the Leiden University Medical Center animal facility. Mice received food and water ad libitum. For biodistribution and preliminary safety studies, ASO (40 mg/kg) was administered intravenously via the tail vein on days 0, 3, 7, and 10. After sacrifice (day 14), blood (and urine) was collected from each mouse and allowed to stand at room temperature for 30 minutes, then centrifuged at 6,000 rpm at 4°C to collect the upper serum. Creatinine, blood urea nitrogen (BUN or urea), albumin, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were determined in LUMC's clinical chemistry laboratory. Example 2 Results ASO according to the invention has improved antiviral activity
為了舉例說明根據本發明的ASO的抗病毒活性,將根據SEQ ID NO: 8的ASO(ASO8)的抗病毒活性分別與根據WO 2019/168402的SEQ ID NO 23和24(ASO23和ASO24)的結構相似的ASO的抗病毒活性進行比較。ASO23和ASO24與ASO8的不同之處僅在於,它們在多瘤病毒大T抗原前mRNA中的20個核苷酸靶區域從上游或下游一個核苷酸開始。To illustrate the antiviral activity of the ASO according to the present invention, the antiviral activity of the ASO according to SEQ ID NO: 8 (ASO8) was compared with the structures of SEQ ID NOs 23 and 24 (ASO23 and ASO24) according to WO 2019/168402, respectively. The antiviral activities of similar ASOs were compared. ASO23 and ASO24 differ from ASO8 only in that their 20-nucleotide target region in the polyomavirus large T antigen pre-mRNA begins one nucleotide upstream or downstream.
在用BKV感染前24 h,用50 nM ASO處理人腎近端上皮細胞(PTEC),並在感染後第5天定量BKV RNA和蛋白質表現。在此體外模型中,相對於未處理的對照,用ASO8處理導致T-Ag和VP1 mRNA分別減少93.8%和96.5%。該等減少優於用ASO23(T-Ag:91.9%和VP1:92.0%)和ASO24(T-Ag:92.4%和VP1:90.4%)實現的減少。參見圖17A。ASO8提供的改進藉由下游VP1蛋白表現的測定證實,ASO8(92.5%)處理後更有效地抑制VP1蛋白表現,而ASO23和ASO24分別為80.7%和89.4%。參見圖17B。總體而言,該等結果表明ASO8在抑制BKV複製態樣係有效的,並且與結構相似的ASO相比有所改進。鑒於結構相似以及因此幾乎相同的靶區域,此種改進完全出乎意料。 材料與方法 細胞的轉染和感染 Human proximal kidney epithelial cells (PTEC) were treated with 50 nM ASO 24 h before infection with BKV, and BKV RNA and protein expression were quantified on day 5 post-infection. In this in vitro model, treatment with ASO8 resulted in a 93.8% and 96.5% reduction in T-Ag and VP1 mRNA, respectively, relative to untreated controls. These reductions are superior to those achieved with ASO23 (T-Ag: 91.9% and VP1: 92.0%) and ASO24 (T-Ag: 92.4% and VP1: 90.4%). See Figure 17A. The improvement provided by ASO8 was confirmed by the measurement of downstream VP1 protein expression. ASO8 (92.5%) was more effective in inhibiting VP1 protein expression after treatment, compared with 80.7% and 89.4% for ASO23 and ASO24 respectively. See Figure 17B. Overall, these results indicate that ASO8 is effective in inhibiting BKV replication and is improved compared to structurally similar ASOs. This improvement was completely unexpected given the similar structures and therefore almost identical target areas. Materials and Methods Transfection and Infection of Cells
將人腎近端上皮細胞(PTECS,Sciencell公司,目錄號:4100)維持在37℃和5% CO 2下在REBM基礎培養基(龍沙公司,目錄號:CC-3191)(用REGMTM SingleQuotsTM補充劑(龍沙公司,目錄號:CC-4127,在本文稱為REGM)補充到0.5% FCS)中。在轉染之前,將PTEC以21,000個細胞/cm 2的密度接種在12孔板(康寧公司目錄號:3512)中過夜。按照製造商的說明,使用Lipofectamine 3000(英傑公司,目錄號:L3000075)實現50 nM ASO的轉染。孵育5 h後,將所有REGM替換為新鮮的REGM,並將細胞維持過夜,之後將細胞用BKV(ATCC,目錄號:VR-837;從原始原液以1 : 3000稀釋)感染2 h。吸出含有病毒的REGM,並在添加新鮮的REGM之前將所有孔洗滌3次。感染後將細胞維持在培養基中5天,在感染後第3天補充REGM。將未處理的細胞作為對照。 定量即時 PCR Human kidney proximal epithelial cells (PTECS, Sciencell Inc., catalog number: 4100) were maintained at 37°C and 5% CO in REBM basal medium (Lonza, catalog number: CC-3191) (supplemented with REGMTM SingleQuotsTM (Lonza, catalog number: CC-4127, referred to herein as REGM) supplemented to 0.5% FCS). Prior to transfection, PTEC were seeded in 12-well plates (Corning Incorporated Cat. No.: 3512) at a density of 21,000 cells/cm overnight . Transfection of 50 nM ASO was achieved using Lipofectamine 3000 (Invitrogen, catalog number: L3000075) according to the manufacturer's instructions. After 5 h of incubation, all REGM was replaced with fresh REGM and the cells were maintained overnight, after which the cells were infected with BKV (ATCC, catalog number: VR-837; diluted 1:3000 from the original stock solution) for 2 h. Aspirate the virus-containing REGM and wash all wells 3 times before adding fresh REGM. Cells were maintained in culture medium for 5 days post-infection and REGM was supplemented on day 3 post-infection. Untreated cells served as controls. Quantitative real-time PCR
使用RNeasy套組(凱傑公司,目錄號:74106)按照製造商的說明,用RLT緩衝液和1% 2-巰基乙醇(西格瑪奧德里奇公司(Sigma-Aldrich),目錄號:M3148-100 mL)和DNA酶I處理(凱傑公司,目錄號:1010395)分離RNA。RNA洗脫後包括另外的DNA去除步驟,以使用TURBO DNA-free™套組(賽默飛世爾科技公司(Thermo Fisher Scientific),目錄號:AM1907)去除任何多餘的BKV DNA。使用M-MLV反轉錄酶(目錄號:M1708)在M-MLV RT 5X緩衝液(目錄號:M531A)、10 mM dNTP混合物(目錄號:U1518)、RNasin®核糖核酸酶抑制劑(目錄號:N2518)、寡聚(dT)15引物(目錄號:C110A)和0.1 M分子級DTT(所有普洛麥格公司(Promega),目錄號:Y00147)中進行cDNA的合成。根據下表,使用SYBR Select預混液(賽默飛世爾科技公司,目錄號:4472908)和以下引物在CFX Opus即時PCR系統(伯樂公司)上進行BKV RNA表現的定量即時PCR分析:T-Ag:GAGGAGGATGTAAAGGTAGCTCA(正向,SEQ ID NO: 70)和ACTGGCAAACATATCTTCATGGC(反向,SEQ ID NO: 71);VP1:TGCAGGGTCACAAAAAGTGC(正向,SEQ ID NO: 72)和AGCACTCCCTGCATTTCCAA(反向,SEQ ID NO: 73);GAPDH:ACAACTTTGGTATCGTGGAAGG(正向,SEQ ID NO: 74)和GCCATCACGCCACAGTTTC(反向,SEQ ID NO: 75)。使用ΔΔCt方法確定mRNA表現的變化。
感染後第5天,在RIPA裂解和提取緩衝液(賽默飛世爾科技公司,目錄號:89901)中用1 : 100 Pierce蛋白酶和磷酸酶抑制劑微型片劑(賽默飛世爾科技公司,目錄號:A32959)裂解細胞,並使用Pierce™ BCA蛋白質測定套組(賽默飛世爾科技公司,目錄號:23225)確定蛋白質濃度。使用Jess Simple Western系統(生物技術公司(Biotechne))定量VP1蛋白表現。簡而言之,使用12-230 kDa分離模組(ProteinSimple公司,目錄號:SM-W004-1)和抗兔偵測模組(ProteinSimple公司,目錄號:DM-001)分析0.5 mg/mL蛋白質裂解物樣本。需要時,將抗體在抗體稀釋劑中稀釋。使用1 : 20抗SV40 VP1抗體(艾博抗公司,目錄號:ab53977)和1 : 20山羊抗兔免疫球蛋白/HRP(安捷倫公司,目錄號:P044801-2)偵測VP1蛋白。將β-肌動蛋白表現用作上樣對照,使用1 : 20 β-肌動蛋白小鼠單株抗體(細胞傳訊技術公司(Cell Signaling Technology),目錄號:#3700S)和1 : 20山羊抗小鼠免疫球蛋白/HRP(安捷倫公司,目錄號:P044701-2)用作二抗。該測定由375 V電壓下30分鐘分離時間,隨後是5 min抗體稀釋時間,60分鐘一抗時間和30分鐘二抗時間組成。使用高動態範圍化學發光信號的垂直線(dropped line)進行峰面積計算。在逐個毛細管的基礎上調整峰發現閾值和寬度,以確保信號的適當擬合。On day 5 post-infection, cells were treated with 1:100 Pierce Protease and Phosphatase Inhibitor Minitablets (Thermo Fisher Scientific, Cat. No. 89901) in RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific, Cat. No. 89901). No.: A32959), the cells were lysed, and the protein concentration was determined using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Cat. No.: 23225). VP1 protein expression was quantified using the Jess Simple Western system (Biotechne). Briefly, 0.5 mg/mL protein was analyzed using the 12-230 kDa separation module (ProteinSimple, catalog number: SM-W004-1) and the anti-rabbit detection module (ProteinSimple, catalog number: DM-001) Lysate sample. If necessary, dilute the antibody in antibody diluent. VP1 protein was detected using 1:20 anti-SV40 VP1 antibody (Abcam, catalog number: ab53977) and 1:20 goat anti-rabbit immunoglobulin/HRP (Agilent, catalog number: P044801-2). β-Actin expression was used as a loading control using 1:20 β-actin mouse monoclonal antibody (Cell Signaling Technology, Cat. #3700S) and 1:20 goat anti- Mouse immunoglobulin/HRP (Agilent, catalog number: P044701-2) was used as the secondary antibody. The assay consists of a 30-minute separation time at 375 V, followed by a 5-minute antibody dilution time, a 60-minute primary antibody time, and a 30-minute secondary antibody time. Peak area calculations were performed using the dropped line of the high dynamic range chemiluminescence signal. Adjust the peak finding threshold and width on a capillary-by-capillary basis to ensure appropriate fit of the signal.
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圖 1 : BKV T-Ag 剪接位點在基因型之間係保守的,允許「一體適用( one-size-fits-all )」的 ASO 方法。下載BKV分離株的公開可用的完整基因組序列,並且比對全基因T-Ag序列並研究基因型之間的保守性。使用文獻中描述的參考分離株鑒定基因型。發現表明,T-Ag中的供體剪接位點在基因型之間主要是保守的,而受體剪接位點在非編碼內含子區中的變化更大。基於該等發現,我們鑒定了一段與供體剪接位點重疊的核苷酸,該供體剪接位點顯示出基因型之間的100%序列保守性。簡明BKV T-Ag親緣關係樹(左)與描繪每個基因型的剪接位點保守性的序列標識(右)。字母(核苷酸)的大小表示每個基因型在該位置上存在的核苷酸的相對發生率。缺失被描述為序列標識中的空白區或空位。序列在表1中給出。
圖2 :設計多個ASO 以靶向BK 病毒大T 抗原的外顯子1 - 內含子供體位點。所靶向的大T抗原供體剪接位點被定義為SEQ ID NO: 1至10,並使用內部開發的設計演算法進行設計。簡而言之,該演算法考慮了反義寡核苷酸(ASO)GC含量(40%-60%)、解鏈溫度(>48℃)和CpG模體的缺失。為每個潛在的ASO分配一個分數,當不滿足要求時,該分數會增加。通常,分數低的ASO優於分數高的ASO,但亦考慮了靶RNA和ASO的預測二級結構,以及與靶RNA中的剪接(RESCUE)強化子和/或緘默子的結合。重要的是,分數考慮了是否滿足設計要求,但不能預測功效。最後,可靶向的序列需要含有至少1個外顯子或內含子核苷酸。淺色框(light box)定義衍生自SEQ ID NO: 1至10中的每一個的2’-OMe-完全修飾的ASO(SEQ ID NO: 12至21),並且深色框(dark box)定義靶向外顯子 - 內含子接點(SEQ ID NO: 42)或外顯子1序列SEQ ID NO: 43至45的GapmeR寡核苷酸。截短的ASO由SEQ ID NO: 11表示。由SEQ ID NO: 11表示的ASO包含SEQ ID NO: 5的一部分。由SEQ ID NO: 11表示的ASO已被進一步修飾為2’-OMe完全修飾的ASO(所有核苷酸都具有2’-O-甲基鹼基),並且每個核苷酸間鍵都是硫代磷酸酯鍵。此經進一步修飾的ASO由SEQ ID NO: 22表示。 Figure 2 : Design of multiple ASOs to target the exon 1- intron donor site of the BK virus large T antigen. The large T antigen donor splice sites targeted were defined as SEQ ID NO: 1 to 10 and designed using an in-house developed design algorithm. Briefly, this algorithm takes into account antisense oligonucleotide (ASO) GC content (40%-60%), melting temperature (>48°C), and absence of CpG motifs. Each potential ASO is assigned a score, which increases when requirements are not met. Generally, ASOs with low scores are preferred over those with high scores, but the predicted secondary structure of the target RNA and ASO, as well as binding to splicing (RESCUE) enhancers and/or silencers in the target RNA, are also considered. Importantly, the score takes into account whether design requirements are met but does not predict efficacy. Finally, targetable sequences need to contain at least 1 exon or intron nucleotide. The light box definition is a 2'-OMe-fully modified ASO (SEQ ID NO: 12 to 21) derived from each of SEQ ID NOs: 1 to 10, and the dark box definition GapmeR oligonucleotides targeting the exon-intron junction (SEQ ID NO: 42) or the exon 1 sequence SEQ ID NO: 43 to 45. The truncated ASO is represented by SEQ ID NO: 11. The ASO represented by SEQ ID NO: 11 contains a portion of SEQ ID NO: 5. The ASO represented by SEQ ID NO: 11 has been further modified into a 2'-OMe fully modified ASO (all nucleotides have 2'-O-methyl bases), and each internucleotide bond is Phosphorothioate bond. This further modified ASO is represented by SEQ ID NO: 22.
圖3 :靶向BKV 大T 抗原外顯子1 - 內含子接點的經化學修飾的ASO 介導10 至100 倍的BKV 參數抑制。將人原代腎小管上皮細胞(PTEC)用新的候選物2'-OMe修飾的ASO(SEQ ID NO: 12至21)處理,並隨後用BKV感染。使用PCR或西方墨點法研究BKV mRNA和蛋白質表現以及上清液中的病毒顆粒釋放(n=3個重複)。圖顯示每個單獨的ASO在以下各種結果量測上相對於加擾對照的倍數變化:T-Ag mRNA、VP1 mRNA、VP1蛋白和病毒產生。深灰色粗線表示觀察到的HYB_03(20-mer,2’O-甲基核苷酸和硫代磷酸酯骨架,由SEQ ID NO: 68表示)的平均總體減少,其用作參考。SEQ ID NO: 67係衍生SEQ ID NO: 68的天然RNA序列。SEQ ID NO: 67由5’-CAGCACAAACCUCUGAGCUA-3’表示。 Figure 3 : Chemically modified ASOs targeting the BKV large T antigen exon 1- intron junction mediate 10- to 100 -fold inhibition of BKV parameters. Human primary renal tubular epithelial cells (PTEC) were treated with the novel candidate 2'-OMe modified ASO (SEQ ID NO: 12 to 21) and subsequently infected with BKV. BKV mRNA and protein expression and viral particle release in supernatants were studied using PCR or Western blotting (n = 3 replicates). The graph shows the fold change of each individual ASO relative to the scrambled control on various outcome measures: T-Ag mRNA, VP1 mRNA, VP1 protein, and viral production. The thick dark gray line represents the average overall reduction observed for HYB_03 (20-mer, 2'O-methyl nucleotide and phosphorothioate backbone, represented by SEQ ID NO: 68), which was used as a reference. SEQ ID NO: 67 is the native RNA sequence from which SEQ ID NO: 68 is derived. SEQ ID NO: 67 is represented by 5'-CAGCACAAACCUCUGAGCUA-3'.
SEQ ID NO: 12活性的可變性係由於不同重複的細胞的較差的核定位和活力造成的。選擇的新的候選序列(SEQ ID NO: 16至19)因其抑制BKV mRNA和蛋白質表現以及病毒產生的高效力而脫穎而出。通常,與2’-OMe修飾的加擾對照相比,用該等新的2'-OMe修飾的ASO候選物處理後,病毒顆粒的mRNA表現和產生被抑制至少10倍,而蛋白質表現減少100倍。Variability in SEQ ID NO: 12 activity is due to poor nuclear localization and viability of cells from different replicates. The new candidate sequences selected (SEQ ID NO: 16 to 19) stood out for their high potency in inhibiting BKV mRNA and protein expression and virus production. Typically, treatment with these new 2'-OMe-modified ASO candidates resulted in at least a 10-fold inhibition of viral particle mRNA expression and production and a 100-fold reduction in protein expression compared to 2'-OMe-modified scrambled controls. times.
圖4 :2’-OMe 化學在靶向外顯子1 - 內含子接點時產生比具有2’-MOE 化學的ASO 更大的BKV 參數降低。將人原代腎小管上皮細胞(PTEC)用新的候選物ASO(SEQ ID NO: 16至19)的化學變體處理,並隨後用BKV感染。使用PCR或西方墨點法研究BKV蛋白表現和上清液中的病毒顆粒釋放。將FAM標記的加擾對照、具有FAM共軛物的HYB_03(亦即SEQ ID NO: 68(+FAM))以及未標記的HYB_03(-FAM)(亦即SEQ ID NO: 68(-FAM))作為參考或對照(n=3個重複)。圖顯示在各種結果量測(VP1蛋白和病毒產生)上,每個單獨的ASO相對於FAM標記的加擾對照的倍數變化。有趣的是,含有MOE-PS的ASO(SEQ ID NO: 23、24、25、27、28、29、31、32、33、35、36、37、38或69)在抑制BKV方面表現出相當低的活性,而OMe-PS變體(SEQ ID NO: 17、18、19、22、26、30、34、38和68,HYB_03)顯示出對蛋白質表現和病毒產生的強烈抑制。5'-甲基胞苷的存在似乎不會干擾ASO的功效。除HYB_03(SEQ ID NO: 68)外,4種最有效的ASO被選為最有希望的候選物,亦即SEQ ID NO: 30、18、34和19。 Figure 4 : 2'-OMe chemistry produces greater reductions in BKV parameters when targeting exon 1- intron junctions than ASOs with 2'-MOE chemistry . Human primary renal tubular epithelial cells (PTEC) were treated with chemical variants of the novel candidate ASO (SEQ ID NO: 16 to 19) and subsequently infected with BKV. Study BKV protein expression and viral particle release in supernatants using PCR or Western blotting. FAM labeled scrambling control, HYB_03 with FAM conjugate (i.e. SEQ ID NO: 68 (+FAM)) and unlabeled HYB_03 (-FAM) (i.e. SEQ ID NO: 68 (-FAM)) As reference or control (n = 3 replicates). The graph shows the fold change of each individual ASO relative to the FAM-labeled scrambled control on various outcome measures (VP1 protein and virus production). Interestingly, ASOs containing MOE-PS (SEQ ID NO: 23, 24, 25, 27, 28, 29, 31, 32, 33, 35, 36, 37, 38 or 69) showed comparable efficacy in inhibiting BKV low activity, while the OMe-PS variant (SEQ ID NO: 17, 18, 19, 22, 26, 30, 34, 38 and 68, HYB_03) showed strong inhibition of protein expression and virus production. The presence of 5'-methylcytidine does not appear to interfere with ASO efficacy. In addition to HYB_03 (SEQ ID NO: 68), the 4 most potent ASOs were selected as the most promising candidates, namely SEQ ID NO: 30, 18, 34 and 19.
圖 5 :具有 SEQ ID NO: 19 的寡核苷酸在「再感染」後產生 BKV 感染細胞的有效減少。進行再感染測定以確定ASO是否限制活性病毒的產生,由此將人近端小管上皮細胞用加擾對照和表現出抗BKV活性的幾種ASO(HYB_03(SEQ ID NO: 68)、SEQ ID NO: 26、SEQ ID NO: 17、SEQ ID NO: 34、SEQ ID NO: 19)處理。將人PTEC用ASO預處理(感染前24小時)並用BKV感染,之後將細胞孵育7天的時間段。在第7天收穫培養上清液,並將其以10倍稀釋形式置於新鮮鋪板的PTEC上3天。針對細胞核(DAPI)和大T抗原蛋白的表現來染色細胞。 Figure 5 : Oligonucleotide with SEQ ID NO: 19 produces an efficient reduction of BKV- infected cells after "re-infection". Reinfection assays were performed to determine whether ASOs limit the production of active virus, whereby human proximal tubule epithelial cells were treated with a scramble control and several ASOs exhibiting anti-BKV activity (HYB_03 (SEQ ID NO: 68), SEQ ID NO. : 26, SEQ ID NO: 17, SEQ ID NO: 34, SEQ ID NO: 19) processing. Human PTEC were pretreated with ASO (24 h before infection) and infected with BKV, after which the cells were incubated for a period of 7 days. Culture supernatant was harvested on day 7 and plated at a 10-fold dilution onto freshly plated PTEC for 3 days. Cells were stained for expression of nuclei (DAPI) and large T antigen protein.
圖 6 :具有 SEQ ID NO: 19 的寡核苷酸在「再感染」後產生 BKV 衍生的 RNA 、蛋白質和病毒 DNA 的有效減少。進行再感染測定以確定ASO是否限制活性病毒的產生,由此將人近端小管上皮細胞用加擾對照和表現出抗BKV活性的幾種ASO(HYB_03(SEQ ID NO: 68)、SEQ ID NO: 26、SEQ ID NO: 17、SEQ ID NO: 34、SEQ ID NO: 19)處理。將人PTEC用ASO預處理(感染前24小時)並用BKV感染,之後將細胞孵育7天的時間段。在第7天收穫培養上清液,並將其以10倍稀釋形式置於新鮮鋪板的PTEC上3天,此時我們評估了大T mRNA、VP1 mRNA以及蛋白質和病毒DNA產生的水平。 Figure 6 : Oligonucleotide with SEQ ID NO: 19 produces efficient reduction of BKV- derived RNA , protein and viral DNA after "re-infection". Reinfection assays were performed to determine whether ASOs limit the production of active virus, whereby human proximal tubule epithelial cells were treated with a scramble control and several ASOs exhibiting anti-BKV activity (HYB_03 (SEQ ID NO: 68), SEQ ID NO. : 26, SEQ ID NO: 17, SEQ ID NO: 34, SEQ ID NO: 19) processing. Human PTEC were pretreated with ASO (24 h before infection) and infected with BKV, after which the cells were incubated for a period of 7 days. Culture supernatants were harvested on day 7 and plated at 10-fold dilutions on freshly plated PTEC for 3 days, at which time we assessed the levels of large T mRNA, VP1 mRNA, and protein and viral DNA production.
圖7 :靶向剪接的ASO 誘導早期編碼區前mRNA 的異常剪接,同時從大T 抗原轉變為小t 抗原。用含有Gardner BK病毒株的早期編碼區的pRPc載體轉化小鼠Balb/c細胞(Negrini, M.等人, Cancer Research [癌症研究], 1992)。此驅動了BKV的早期編碼區在該等細胞中的組成型表現,如藉由大T抗原mRNA(產物1)和小t抗原mRNA(產物3)的大量表現所證明的。藉由lipofectamine以25 nM的濃度投與加擾對照、HYB_03(SEQ ID NO: 68)和SEQ ID NO: 19,並在處理後24小時收穫RNA。藉由lipofectamine以25 nM的濃度投與SEQ ID NO: 19的誤配對照(核鹼基10處的單個誤配(SEQ ID NO: 39)、核鹼基5和10處的雙誤配(SEQ ID NO: 40)和核鹼基5、10和15處的三誤配(SEQ ID NO: 41)),並在處理後24 h收穫RNA。注釋:該等細胞由Massimo Negrini教授(費拉拉大學(University of Ferrara),義大利)友情提供。用HYB_03和SEQ ID NO: 19處理顯著影響早期編碼區前mRNA的剪接和表現水平,導致從作為主要產物的大T抗原(帶1)明顯轉變為各種RNA產物,包括小t抗原(帶3)的增加。引入單個誤配導致正常剪接的部分恢復,而2-3個誤配幾乎完全恢復作為主要RNA種類的大T抗原的剪接。該等資料提供了靶標接合和特異性的明確證據。 Figure 7 : ASO targeting splicing induces abnormal splicing of pre-mRNA in the early coding region and simultaneously switches from large T antigen to small T antigen. Mouse Balb/c cells were transformed with the pRPc vector containing the early coding region of the Gardner BK strain (Negrini, M. et al., Cancer Research, 1992). This drives the constitutive expression of the early coding region of BKV in these cells, as evidenced by the abundant expression of large T-antigen mRNA (product 1) and small t-antigen mRNA (product 3). Scrambling control, HYB_03 (SEQ ID NO: 68) and SEQ ID NO: 19 were administered by lipofectamine at a concentration of 25 nM, and RNA was harvested 24 hours after treatment. Mismatch controls of SEQ ID NO: 19 (single mismatch at nucleobase 10 (SEQ ID NO: 39), double mismatch at nucleobases 5 and 10 (SEQ ID NO: 39)) were administered by lipofectamine at a concentration of 25 nM. ID NO: 40) and three mismatches at nucleobases 5, 10, and 15 (SEQ ID NO: 41)), and RNA was harvested 24 h after treatment. Note: These cells were kindly provided by Professor Massimo Negrini (University of Ferrara, Italy). Treatment with HYB_03 and SEQ ID NO: 19 significantly affects the splicing and expression levels of early coding region pre-mRNA, resulting in a clear shift from the large T antigen as the main product (band 1) to various RNA products, including the small t antigen (band 3) increase. Introduction of a single mismatch resulted in partial restoration of normal splicing, whereas 2-3 mismatches almost completely restored splicing of the large T antigen as the predominant RNA species. These data provide clear evidence of target engagement and specificity.
圖8 :靶向大T 抗原剪接的GapmeR 不誘導異常剪接,並且不介導大T 抗原mRNA 的意義不大的減少。用含有Gardner BK病毒株的早期編碼區的pRPc載體轉化小鼠Balb/c細胞( Negrini, M.等人, Cancer Research[癌症研究], 1992)。此驅動了BKV的早期編碼區在該等細胞中的組成型表現,如藉由大T抗原mRNA(產物1)和小t抗原mRNA(產物3)的大量表現所證明的。與使用立體阻礙來調節剪接的反義寡核苷酸相反,GapmeR被設計為源自SEQ ID NO: 19的序列,並藉由lipofectamine以25 nM的濃度投與,並在處理後24小時收穫RNA。SEQ ID NO: 19的GapmeR版本藉由lipofectamine以25 nM的濃度投與,並在處理後24 h收穫RNA(SEQ ID NO: 42),但其對於大T或小t抗原RNA表現水平沒有表現出任何顯著的活性。靶向早期編碼區(前)mRNA的外顯子1的另外的GapmeR(SEQ ID NO: 43至45)不介導早期編碼區mRNA的顯著減少。因此,GapmeR對T抗原剪接的影響不如先前用靶向剪接位點的ASO HYB_03(SEQ ID NO: 68)和SEQ ID NO: 19觀察到的那麼大。該等發現表明,儘管GapmeR能夠降解靶mRNA,但靶mRNA的剪接基本上不受影響。注釋:該等細胞由Massimo Negrini教授(費拉拉大學,義大利)友情提供。 Figure 8 : GapmeR targeting large T antigen splicing does not induce aberrant splicing and does not mediate a nonsignificant reduction in large T antigen mRNA . Mouse Balb/c cells were transformed with the pRPc vector containing the early coding region of the Gardner BK strain ( Negrini, M. et al., Cancer Research [Cancer Research], 1992 ). This drives the constitutive expression of the early coding region of BKV in these cells, as evidenced by the abundant expression of large T-antigen mRNA (product 1) and small t-antigen mRNA (product 3). In contrast to antisense oligonucleotides that use steric hindrance to modulate splicing, GapmeR was designed to be derived from the sequence of SEQ ID NO: 19 and administered by lipofectamine at a concentration of 25 nM, with RNA harvested 24 hours after treatment . The GapmeR version of SEQ ID NO: 19 was administered with lipofectamine at a concentration of 25 nM and RNA was harvested 24 h after treatment (SEQ ID NO: 42), but it did not exhibit expression levels for large T or small t antigen RNA. Any significant activity. Additional GapmeR (SEQ ID NO: 43 to 45) targeting exon 1 of early coding region (pre)mRNA did not mediate significant reduction of early coding region mRNA. Therefore, the effect of GapmeR on T-antigen splicing is not as great as previously observed with the splice site-targeting ASOs HYB_03 (SEQ ID NO: 68) and SEQ ID NO: 19. These findings indicate that although GapmeR can degrade target mRNA, the splicing of target mRNA is essentially unaffected. Note: These cells were kindly provided by Professor Massimo Negrini (University of Ferrara, Italy).
圖 9 : PBMC 活力不受用 SEQ ID NO: 30 、 SEQ ID NO: 18 、 SEQ ID NO: 34 和 SEQ ID NO: 19 處理的影響。從4個不同供體的膚色血球層中分離健康人周邊血單核細胞(PBMC),並在濃度遞增的ASO存在下將其孵育48小時。孵育後,使用Cell titer Blue活力測定法量測細胞活力。將PBMC在65℃下孵育10分鐘以作為陽性對照。作為另外的對照,使用1 µM R848(TLR7/8促效劑)激活PBMC,並將所有條件與鹽水處理的對照進行比較。細胞活力表示為相對於鹽水處理的對照的百分比。如圖所示,在48小時後,在65℃孵育的陽性對照中未偵測到活細胞,而在ASO處理的PBMC中未觀察到對細胞活力的顯著影響。 Figure 9 : PBMC viability is not affected by treatment with SEQ ID NO: 30 , SEQ ID NO: 18 , SEQ ID NO: 34 and SEQ ID NO: 19 . Healthy human peripheral blood mononuclear cells (PBMC) were isolated from the skin-colored hemocyte layers of 4 different donors and incubated in the presence of increasing concentrations of ASO for 48 hours. After incubation, cell viability was measured using the Cell titer Blue viability assay. PBMC were incubated at 65°C for 10 minutes as a positive control. As an additional control, PBMC were activated with 1 µM R848 (TLR7/8 agonist) and all conditions were compared to saline-treated controls. Cell viability was expressed as percentage relative to saline-treated control. As shown, after 48 hours, no viable cells were detected in the positive control incubated at 65°C, while no significant effect on cell viability was observed in ASO-treated PBMC.
圖 10 :用 SEQ ID NO: 30 、 SEQ ID NO: 18 、 SEQ ID NO: 34 和 SEQ ID NO: 19 處理介導 PBMC 中促炎細胞介素回應的最小激活。從4個不同供體的膚色血球層中分離健康人周邊血單核細胞(PBMC),並在濃度遞增的ASO存在下將其孵育48小時。孵育後,收穫上清液以量測細胞介素產生。作為陽性對照,使用1 µM R848(TLR7/8促效劑,資料未顯示)激活PBMC,並將所有條件與鹽水處理的對照進行比較。回應於暴露於SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34和SEQ ID NO: 19的6種細胞介素的相對產生和釋放如左圖所示。觀察到回應於ASO處理的一些細胞介素水平的輕微瞬時增加,但從未達到與R848處理的對照相同的程度。資料表示所有4個供體中偵測限內的細胞介素。 Figure 10 : Treatment with SEQ ID NO: 30 , SEQ ID NO: 18 , SEQ ID NO: 34 and SEQ ID NO: 19 mediates minimal activation of pro-inflammatory cytokine responses in PBMC . Healthy human peripheral blood mononuclear cells (PBMC) were isolated from the skin-colored hemocyte layers of 4 different donors and incubated in the presence of increasing concentrations of ASO for 48 hours. After incubation, the supernatant was harvested to measure interleukin production. As a positive control, PBMC were activated with 1 µM R848 (TLR7/8 agonist, data not shown) and all conditions were compared to saline-treated controls. The relative production and release of 6 interleukins in response to exposure to SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 19 is shown on the left. A slight transient increase in some interleukin levels was observed in response to ASO treatment, but never to the same extent as in R848-treated controls. Data represent interleukins within detection limits for all 4 donors.
圖 11 :用 SEQ ID NO: 30 、 SEQ ID NO: 18 、 SEQ ID NO: 34 和 SEQ ID NO: 19 處理介導 PBMC 中促炎和抗炎細胞介素的最小激活。從4個不同供體的膚色血球層中分離健康的人周邊血單核細胞(PBMC),並在濃度遞增的SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34和SEQ ID NO: 19存在下將其孵育48小時。孵育後,收穫上清液以量測細胞介素產生。作為陽性對照,使用1 µM R848(TLR7/8促效劑,資料未顯示)激活PBMC,並將所有條件與鹽水處理的對照進行比較。回應於用SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34和SEQ ID NO: 19處理的6種細胞介素的相對產生和釋放如左圖所示。觀察到回應於ASO處理的一些細胞介素水平的輕微瞬時增加,但從未達到與R848處理的對照相同的程度。資料表示4個供體中的2個的在偵測限內的細胞介素。 Figure 11 : Treatment with SEQ ID NO: 30 , SEQ ID NO: 18 , SEQ ID NO: 34 and SEQ ID NO: 19 mediates minimal activation of pro- and anti-inflammatory cytokines in PBMC . Healthy human peripheral blood mononuclear cells (PBMC) were isolated from skin-colored hemocytes of 4 different donors and prepared at increasing concentrations of SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34, and SEQ ID NO : Incubate it for 48 hours in the presence of 19. After incubation, the supernatant was harvested to measure interleukin production. As a positive control, PBMC were activated with 1 µM R848 (TLR7/8 agonist, data not shown) and all conditions were compared to saline-treated controls. The relative production and release of 6 interleukins in response to treatment with SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 19 is shown on the left. A slight transient increase in some interleukin levels was observed in response to ASO treatment, but never to the same extent as in R848-treated controls. Data represent interleukins within detection limits for 2 of 4 donors.
圖 12 :人血漿暴露於具有 SEQ ID NO: 19 、 SEQ ID NO 30 、 SEQ ID NO: 18 或 SEQ ID NO: 34 的寡核苷酸對血漿凝固時間的影響最小。將濃度遞增的ASO添加到正常人血漿中,之後使用活化部分凝血激酶時間(aPTT)測試激活凝血級聯的內在途徑。將ASO條件與鹽水處理的對照血漿進行比較。鹽水處理的對照血漿中內在凝血級聯的激活導致凝血時間為33.8秒。向血漿中添加SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34和SEQ ID NO: 19導致凝血時間的劑量依賴性延長。資料表示n = 1。 Figure 12 : Exposure of human plasma to oligonucleotides having SEQ ID NO: 19 , SEQ ID NO 30 , SEQ ID NO: 18 or SEQ ID NO: 34 has minimal effect on plasma clotting time. Increasing concentrations of ASO were added to normal human plasma, and the intrinsic pathways that activate the coagulation cascade were tested using activated partial thromboplastin time (aPTT). ASO conditions were compared to saline-treated control plasma. Activation of the intrinsic coagulation cascade in saline-treated control plasma resulted in a clotting time of 33.8 seconds. Addition of SEQ ID NO: 30, SEQ ID NO: 18, SEQ ID NO: 34 and SEQ ID NO: 19 to plasma resulted in a dose-dependent prolongation of clotting time. Data indicate n = 1.
圖13 :靶向BKV 的ASO 明顯被小鼠腎皮質的近端和遠端小管吸收。A)9週齡雄性C57BL6小鼠在第0天、第3天、第7天、第10天接受40 mg/kg HYB_03(SEQ ID NO: 68)、SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19的靜脈內注射。將該等小鼠與鹽水處理的動物進行比較。群組大小為每個時間點每群組5隻小鼠。 B)如藉由使用特定的Cy5標記的FISH探針(SEQ ID NO: 19)染色證明的,高水平的SEQ ID NO: 19分佈到腎,並且特別地近端小管上皮細胞的視覺證據。將腎單位的單個片段定義為腎小球(glom)、近端小管上皮細胞(prox)和遠端小管上皮細胞(dist)、以及集合管上皮(「coll」),其基於裂隙素(「glom」)、LTL(「prox」)、E-鈣黏著蛋白(「dist」)和水孔蛋白-2(「coll」)的免疫組織化學染色。B) (右側圖像),在腎的髓質部分偵測到痕量水平的SEQ ID NO: 19(見箭頭)。 Figure 13 : ASO targeting BKV was significantly absorbed by proximal and distal tubules of mouse renal cortex. A) 9-week-old male C57BL6 mice received 40 mg/kg HYB_03 (SEQ ID NO: 68), SEQ ID NO: 30, and SEQ ID NO: 18 on days 0, 3, 7, and 10 , SEQ ID NO: 34 or SEQ ID NO: 19 for intravenous injection. The mice were compared to saline-treated animals. Group size was 5 mice per group per time point. B) Visual evidence of high levels of SEQ ID NO: 19 distributed to the kidney, and specifically proximal tubule epithelial cells, as demonstrated by staining with a specific Cy5-labeled FISH probe (SEQ ID NO: 19). The individual segments of the nephron are defined as the glomerulus (glom), the proximal tubular epithelium (prox) and the distal tubular epithelium (dist), and the collecting duct epithelium ("coll") based on the schizotypyrin ("glom") ”), LTL (“prox”), E-cadherin (“dist”), and aquaporin-2 (“coll”). B) (right image), trace levels of SEQ ID NO: 19 were detected in the medullary portion of the kidney (see arrow).
圖 14 :腎和高血流量器官的 SEQ ID NO: 19 攝取的雜交 ELISA 分析。9週齡雄性C57BL6小鼠在第0天、第3天、第7天、第10天接受40 mg/kg HYB_03(SEQ ID NO: 68)、SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19的靜脈內注射,並與鹽水處理的動物進行比較(每群組n = 5隻小鼠)。顯示了用SEQ ID NO: 19進行的實驗的結果。左小圖:顯示了兩個腎(左和右)、肝、脾和肺中SEQ ID NO: 19水平的定量,表明每克腎組織的(相對)攝取量係每克肝組織的大約2.5倍。類似地,與肺和脾相比,觀察到腎的相對攝取量更大。右小圖:描繪了單個小鼠組織水平,表明體內組織分佈特徵一致。 Figure 14 : Hybrid ELISA analysis of SEQ ID NO: 19 uptake in kidneys and high blood flow organs . 9-week-old male C57BL6 mice received 40 mg/kg HYB_03 (SEQ ID NO: 68), SEQ ID NO: 30, SEQ ID NO: 18, SEQ on days 0, 3, 7, and 10 ID NO: 34 or SEQ ID NO: 19 and compared to saline-treated animals (n = 5 mice per group). Results of experiments performed with SEQ ID NO: 19 are shown. Left panel: shows quantification of SEQ ID NO: 19 levels in two kidneys (left and right), liver, spleen and lung, indicating that the (relative) uptake per gram of kidney tissue is approximately 2.5 times that per gram of liver tissue . Similarly, greater relative uptake was observed in the kidney compared with the lung and spleen. Right panel: depicts the level of individual mouse tissues, demonstrating consistent tissue distribution characteristics in vivo.
圖 15 :在 2 週內多次給藥 SEQ ID NO: 30 、 SEQ ID NO: 18 、 SEQ ID NO: 34 或 SEQ ID NO: 19 不會導致小鼠血清中腎或肝損傷的跡象。9週齡雄性C57BL6小鼠在第0天、第3天、第7天、第10天接受40 mg/kg HYB_03(SEQ ID NO: 68)、SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19的靜脈內注射,並與鹽水處理的動物進行比較。在第一次投與後14天研究腎和肝功能標誌物的血清水平(每群組n=5隻小鼠)。上小圖:腎(肌酐、尿素和白蛋白)和肝(AST和ALT)生物標誌物的血清水平,表明器官功能和/或損傷。雖然在一些群組之間偵測到一些微小差異,但所有水平都很好地在正常範圍內,因此沒有觀察到腎或肝損傷的跡象。 Figure 15 : Multiple administration of SEQ ID NO: 30 , SEQ ID NO: 18 , SEQ ID NO: 34 or SEQ ID NO: 19 over 2 weeks did not result in signs of renal or liver damage in the serum of mice. 9-week-old male C57BL6 mice received 40 mg/kg HYB_03 (SEQ ID NO: 68), SEQ ID NO: 30, SEQ ID NO: 18, SEQ on days 0, 3, 7, and 10 ID NO: 34 or SEQ ID NO: 19 and compared to saline treated animals. Serum levels of renal and liver function markers were studied 14 days after the first dose (n=5 mice per cohort). Upper panel: Serum levels of renal (creatinine, urea, and albumin) and hepatic (AST and ALT) biomarkers indicating organ function and/or injury. Although some minor differences were detected between some groups, all levels were well within the normal range and therefore no signs of renal or liver damage were observed.
圖 16 :在 2 週內重複給藥 SEQ ID NO: 30 、 SEQ ID NO: 18 、 SEQ ID NO: 34 或 SEQ ID NO: 19 不會導致腎損傷的跡象。9週齡雄性C57BL6小鼠在第0天、第3天、第7天、第10天接受40 mg/kg HYB_03(SEQ ID NO: 68)、SEQ ID NO: 30、SEQ ID NO: 18、SEQ ID NO: 34或SEQ ID NO: 19的靜脈內注射,並與鹽水處理的動物進行比較(每群組n = 5隻小鼠)。處死後,腎中天狼星紅(Sirius red)(上小圖)和KIM-1染色(下小圖)的代表性圖像。從上述圖可以清楚地看出,投與鹽水(0.9% NaCl)或ASO不會導致ASO相關的腎損傷的跡象。將圖像與陽性對照(缺血再灌注損傷;IRI)進行比較。B.第14天腎中天狼星紅染色的定量。接受ASO的三隻動物顯示腎膠原含量增加(HYB_03,n =2,SEQ ID 34,n=1),但此不能明確歸因於ASO的投與,並且可能是由於用於定量的圖像中潛在更高數量的主要血管。 Figure 16 : Repeated administration of SEQ ID NO: 30 , SEQ ID NO: 18 , SEQ ID NO: 34 or SEQ ID NO: 19 over 2 weeks did not result in signs of renal injury. 9-week-old male C57BL6 mice received 40 mg/kg HYB_03 (SEQ ID NO: 68), SEQ ID NO: 30, SEQ ID NO: 18, SEQ on days 0, 3, 7, and 10 ID NO: 34 or SEQ ID NO: 19 and compared to saline-treated animals (n = 5 mice per group). Representative images of Sirius red (upper panel) and KIM-1 staining (lower panel) in kidneys after sacrifice. It is clear from the above figure that administration of saline (0.9% NaCl) or ASO does not result in signs of ASO-related renal injury. Images were compared with positive control (ischemia-reperfusion injury; IRI). B. Quantification of Sirius red staining in kidneys on day 14. Three animals that received ASO showed increased renal collagen content (HYB_03, n = 2, SEQ ID 34, n = 1), but this cannot be clearly attributed to the administration of ASO and may be due to changes in the images used for quantification. Potentially higher number of major blood vessels.
圖17 :根據本發明的ASO 具有改進的抗病毒活性。在用BKV感染前24 h,將PTEC用ASO處理,並在感染後第5天定量BKV RNA表現。A) 用ASO8(SEQ ID NO: 8)、ASO23(WO 2019/168402的SEQ ID NO: 23)和ASO24(WO 2019/168402的SEQ ID NO: 24)處理的細胞的相對T-Ag和VP1 RNA表現。B) 用ASO8、ASO23和ASO24處理的細胞的相對VP1蛋白表現。 Figure 17 : ASOs according to the invention have improved antiviral activity. PTEC were treated with ASO 24 h before infection with BKV, and BKV RNA expression was quantified on day 5 postinfection. A) Relative T-Ag and VP1 RNA of cells treated with ASO8 (SEQ ID NO: 8), ASO23 (SEQ ID NO: 23 of WO 2019/168402) and ASO24 (SEQ ID NO: 24 of WO 2019/168402) Performance. B) Relative VP1 protein expression in cells treated with ASO8, ASO23 and ASO24.
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