本發明係部分地關於一種可用於抑制跨膜絲氨酸蛋白酶6（TMPRSS6）基因表達的組合物和方法。The present invention relates, in part, to compositions and methods useful for inhibiting the expression of the transmembrane serine protease 6 (TMPRSS6) gene.
TMPRSS6（跨膜蛋白酶，絲氨酸6），也稱為蛋白裂解酶-2，是一種II型絲氨酸蛋白酶。它主要在肝臟中表達，儘管在腎臟中也發現了高水平的TMPRSS6 mRNA，但在子宮中的水平較低，並且在許多其他組織中檢測到的量要少得多（Beliveau等人，2019年，Cell Chemical Biology 26, 1559-1572）。TMPRSS6通過調節鐵調素表達在鐵穩態中起關鍵作用。TMPRSS6通過結合鐵調素活化劑和BMP共受體HJV（血幼素）並以蛋白水解方式使兩者降解，導致鐵調素水平的下調，從而在鐵穩態中起作用。小鼠和人的遺傳數據表明，降低TMPRSS6表達可上調鐵調素，並改善與β-地中海貧血相關的許多疾病症狀。TMPRSS6 (transmembrane protease, serine 6), also known as matriptase-2, is a type II serine protease. It is primarily expressed in the liver. Although high levels of TMPRSS6 mRNA are also found in the kidney, levels are lower in the uterus and much lower in many other tissues (Beliveau et al., 2019, Cell Chemical Biology 26, 1559-1572). TMPRSS6 plays a key role in iron homeostasis by regulating hepcidin expression. TMPRSS6 plays a role in iron homeostasis by binding to the hepcidin activator and the BMP co-receptor HJV (hemojuvelin) and proteolytically degrading both, leading to downregulation of hepcidin levels. Genetic data from mice and humans indicate that reducing TMPRSS6 expression upregulates hepcidin and improves many disease symptoms associated with β-thalassemia.
目前對與鐵過載相關的障礙和障礙的治療並不總是有效的。因此，靶向TMPRSS6的新療法代表了降低TMPRSS6水平並治療TMPRSS6相關疾病諸如地中海貧血的新方法。Current treatments for disorders and conditions associated with iron overload are not always effective. Therefore, new therapies targeting TMPRSS6 represent a new approach to lower TMPRSS6 levels and treat TMPRSS6-related diseases such as thalassemia.
總體而言，本發明內容提供了新型TMPRSS6基因特異性RNAi藥劑、包含TMPRSS6 RNAi藥劑的組合物，以及使用該TMPRSS6 RNAi藥劑和包含本文所述的TMPRSS6 RNAi藥劑的組合物在體外和/或體內抑制TMPRSS6基因的表達的方法。本文所述的TMPRSS6 RNAi藥劑可選擇性地且有效地降低、抑制或沉默受試者（例如人或動物受試者）的TMPRSS6基因的表達。In general, the present invention provides novel RNAi agents specific for the TMPRSS6 gene, compositions comprising the TMPRSS6 RNAi agents, and methods of using the TMPRSS6 RNAi agents and compositions comprising the TMPRSS6 RNAi agents described herein to inhibit TMPRSS6 gene expression in vitro and/or in vivo. The TMPRSS6 RNAi agents described herein can selectively and effectively reduce, inhibit, or silence TMPRSS6 gene expression in a subject (e.g., a human or animal subject).
根據本發明的一個方面，提供了一種用於抑制跨膜絲氨酸蛋白酶6（TMPRSS6）的表達的雙鏈核糖核酸（dsRNA）藥劑，其中該dsRNA藥劑包含有義鏈和反義鏈，其中該有義鏈包含與SEQ ID NO: l、3或5的核苷酸序列相差不超過1、2或3個核苷酸的至少15個連續核苷酸，並且該反義鏈包含與SEQ ID NO: 2、4或6的核苷酸序列相差不超過1、2或3個核苷酸的至少15個連續核苷酸，其中該有義鏈和該反義鏈彼此能夠部分地、基本上或完全互補。According to one aspect of the present invention, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of transmembrane serine protease 6 (TMPRSS6) is provided, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 1, 3 or 5 by no more than 1, 2 or 3 nucleotides, and the antisense strand comprises at least 15 consecutive nucleotides that differ from the nucleotide sequence of SEQ ID NO: 2, 4 or 6 by no more than 1, 2 or 3 nucleotides, wherein the sense strand and the antisense strand are partially, substantially or completely complementary to each other.
在一些實施方案中，該dsRNA藥劑包含形成雙鏈區的有義鏈和反義鏈，其中所述反義鏈包含與編碼TMPRSS6的mRNA互補的區域，該互補區域包含與表1至表3中的任一個表中列出的反義序列中的任一者相差不超過1、2或3個核苷酸的至少15個連續核苷酸。在一些實施方案中，該dsRNA藥劑包含形成雙鏈區的有義鏈和反義鏈，其中所述反義鏈包含與編碼TMPRSS6的mRNA互補的區域，該互補區域包含來自表1至表3中的任一個表中列出的反義序列中的任一者的至少15個連續核苷酸。In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that form a duplex, wherein the antisense strand comprises a region that is complementary to an mRNA encoding TMPRSS6, the complementary region comprising at least 15 consecutive nucleotides that differ from any one of the antisense sequences listed in any one of Tables 1 to 3 by no more than 1, 2, or 3 nucleotides. In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand that form a duplex, wherein the antisense strand comprises a region that is complementary to an mRNA encoding TMPRSS6, the complementary region comprising at least 15 consecutive nucleotides from any one of the antisense sequences listed in any one of Tables 1 to 3.
在一些實施方案中，該dsRNA藥劑包含有義鏈和反義鏈，該反義鏈中的核苷酸位置2至18包含與TMPRSS6 RNA轉錄本互補的區域，其中該互補區域包含與表1至表3中的任一個表中列出的反義序列之一相差0、1、2或3個核苷酸的至少15、16、17、18、19、20或21個連續核苷酸，並且任選地包含靶向配體。In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2 to 18 of the antisense strand comprise a region complementary to the TMPRSS6 RNA transcript, wherein the complementary region comprises at least 15, 16, 17, 18, 19, 20, or 21 contiguous nucleotides that differ from one of the antisense sequences listed in any one of Tables 1 to 3 by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand.
在一些實施方案中，TMPRSS6 RNA轉錄本是SEQ ID NO: 1。In some embodiments, the TMPRSS6 RNA transcript is SEQ ID NO: 1.
在一些實施方案中，dsRNA藥劑的反義鏈與SEQ ID NO: 1的任一靶區域至少基本上互補，並且提供在表1至表3中的任一個表中。在一些實施方案中，dsRNA藥劑的反義鏈與SEQ ID NO: 1的任一靶區域完全互補，並且提供在表1至表3中的任一個表中。在一些實施方案中，dsRNA藥劑包含表1至表3中的任一個表中示出的有義鏈序列，其中該有義鏈序列與dsRNA藥劑中的反義鏈序列至少基本上互補。在某些實施方案中，dsRNA藥劑包含表1至表3中的任一個表中示出的有義鏈序列，其中該有義鏈序列與dsRNA藥劑中的反義鏈序列完全互補。在一些實施方案中，dsRNA藥劑包含表1至表3中的任一個表中示出的反義鏈序列。在一些實施方案中，dsRNA藥劑包含表1至表3中的任一個表中作為雙鏈體序列示出的序列。In some embodiments, the antisense strand of the dsRNA agent is at least substantially complementary to any of the target regions of SEQ ID NO: 1 and is provided in any of Tables 1 to 3. In some embodiments, the antisense strand of the dsRNA agent is fully complementary to any of the target regions of SEQ ID NO: 1 and is provided in any of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sense strand sequence shown in any of Tables 1 to 3, wherein the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In certain embodiments, the dsRNA agent comprises a sense strand sequence shown in any of Tables 1 to 3, wherein the sense strand sequence is fully complementary to the antisense strand sequence in the dsRNA agent. In some embodiments, the dsRNA agent comprises an antisense strand sequence shown in any one of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sequence shown in any one of Tables 1 to 3 as a duplex sequence.
在一些實施方案中，dsRNA藥劑包含至少一個經修飾的核苷酸。在某些實施方案中，反義鏈的所有或基本上所有核苷酸都是經修飾的核苷酸。在一些實施方案中，經修飾的核苷酸中的至少一者包括：2'-O-甲基核苷酸、2'-氟核苷酸、2'-脫氧核苷酸、2'3'-開環核苷酸模擬物、鎖核苷酸、非鎖核酸核苷酸（UNA）、乙二醇核酸核苷酸（GNA）、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、無鹼基核苷酸、核糖醇、反向核苷酸、反向無鹼基核苷酸、反向2'-Ome核苷酸、反向2'-脫氧核苷酸、異甘露醇核苷酸、經2'-氨基修飾的核苷酸、經2'-烷基修飾的核苷酸、嗎啉基核苷酸和3'-OMe核苷酸、包含5'-硫代磷酸酯基團的核苷酸、或與膽甾醇基衍生物或十二烷酸雙癸醯胺基團連接的末端核苷酸、經2'-氨基修飾的核苷酸、包含亞磷醯胺或非天然鹼基的核苷酸。在一些實施方案中，dsRNA藥劑還包含磷酸酯或磷酸酯模擬物。在一些實施方案中，該磷酸酯模擬物是5'-乙烯基膦酸酯（VP）。在一些實施方案中，dsRNA藥劑包含在引導鏈的5΄-末端處的E-乙烯基膦酸酯核苷酸。在一些實施方案中，有義鏈和反義鏈的所有或基本上所有核苷酸都是經修飾的核苷酸。在一些實施方案中，反義鏈包含15個或更多個獨立地選自2'-O-甲基核苷酸、2'-氟核苷酸和經UNA修飾的核苷酸的經修飾的核苷酸，其中少於6個經修飾的核苷酸是2'-氟核苷酸。在一些實施方案中，反義鏈包含3個或5個2'-氟核苷酸，優選地，反義鏈包含5個2'-氟核苷酸。在一些實施方案中，有義鏈包含15個或更多個獨立地選自2'-O-甲基核苷酸和2'-氟核苷酸的經修飾的核苷酸，其中少於4個經修飾的核苷酸是2'-氟核苷酸。在某些實施方案中，有義鏈包含3個2'-氟核苷酸。在一些實施方案中，反義鏈包含15個或更多個獨立地選自2'-O-甲基核苷酸和2'-氟核苷酸的經修飾的核苷酸，其中至少14個經修飾的核苷酸是2'-O-甲基核苷酸，並且在從反義鏈的5'端的第一匹配位置開始計數的位置2、5、7、12、14和/或16處的核苷酸獨立地是2'-氟核苷酸。在一些實施方案中，反義鏈包含至少一個經UNA修飾的核苷酸和5個2'-氟核苷酸。在一些實施方案中，反義鏈包含在從5'端的第一匹配位置開始計數的位置7處的一個經UNA修飾的核苷酸和位置2、5、12、14和16處的5個2'-氟核苷酸，以及其餘的2'-O-甲基核苷酸。在一些實施方案中，反義鏈包含在從5'端的第一匹配位置開始計數的位置2、7、12、14和16處的5個2'-氟核苷酸，以及其餘的2'-O-甲基核苷酸。在一些實施方案中，有義鏈包含15個或更多個獨立地選自2'-O-甲基核苷酸和2'-氟核苷酸的經修飾的核苷酸，優選地，其中至少18個經修飾的核苷酸是2'-O-甲基核苷酸，並且在從有義鏈的3'端的第一匹配位置開始計數的位置9、11和/或13處的核苷酸是2'-氟核苷酸。In some embodiments, the dsRNA agent comprises at least one modified nucleotide. In certain embodiments, all or substantially all nucleotides of the antisense strand are modified nucleotides. In some embodiments, at least one of the modified nucleotides comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxynucleotides, 2'3'-open ring nucleotide mimics, locking nucleotides, non-locking nucleic acid nucleotides (UNA), glycol nucleic acid nucleotides (GNA), 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, reversed nucleotides, reversed abasic nucleotides, Inverted 2'-Ome nucleotides, inverted 2'-deoxynucleotides, isomannitol nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinyl nucleotides and 3'-OMe nucleotides, nucleotides containing 5'-phosphorothioate groups, or terminal nucleotides linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group, 2'-amino modified nucleotides, nucleotides containing phosphoramidites or unnatural base groups. In some embodiments, the dsRNA agent further comprises a phosphate or a phosphate mimetic. In some embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP). In some embodiments, the dsRNA agent comprises an E-vinylphosphonate nucleotide at the 5'-terminus of the guide strand. In some embodiments, all or substantially all nucleotides of the sense and antisense strands are modified nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, and UNA-modified nucleotides, wherein fewer than 6 of the modified nucleotides are 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 3 or 5 2'-fluoro nucleotides, preferably, the antisense strand comprises 5 2'-fluoro nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein fewer than 4 of the modified nucleotides are 2'-fluoro nucleotides. In certain embodiments, the sense strand comprises 3 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least 14 of the modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 2, 5, 7, 12, 14, and/or 16, counting from the first matched position at the 5' end of the antisense strand, are independently 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises at least one UNA-modified nucleotide and five 2'-fluoro nucleotides. In some embodiments, the antisense strand comprises one UNA-modified nucleotide at position 7, counting from the first matched position at the 5' end, and five 2'-fluoro nucleotides at positions 2, 5, 12, 14, and 16, and the remainder are 2'-O-methyl nucleotides. In some embodiments, the antisense strand comprises five 2'-fluoro nucleotides at positions 2, 7, 12, 14, and 16, counted from the first matched position at the 5' end, and the remainder are 2'-O-methyl nucleotides. In some embodiments, the sense strand comprises 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, preferably, at least 18 of the modified nucleotides are 2'-O-methyl nucleotides, and the nucleotides at positions 9, 11, and/or 13, counted from the first matched position at the 3' end of the sense strand, are 2'-fluoro nucleotides.
在一些實施方案中，dsRNA藥劑包含至少一個經修飾的核苷酸，並且還包含一個或多個靶向基團或連接基團。在一些實施方案中，該一個或多個靶向基團或連接基團與有義鏈綴合。在一些實施方案中，該靶向基團或連接基團包括N-乙醯基-半乳糖胺（GalNAc）。In some embodiments, the dsRNA agent comprises at least one modified nucleotide and further comprises one or more targeting groups or linking groups. In some embodiments, the one or more targeting groups or linking groups are conjugated to a sense chain. In some embodiments, the targeting group or linking group comprises N-acetyl-galactosamine (GalNAc).
在一些實施方案中，靶向基團具有如式(X)的結構：In some embodiments, the targeting group has a structure of Formula (X):
式(X)Formula (X)
每個n''獨立地選自1或2。Each n'' is independently selected from 1 or 2.
在一些實施方案中，該靶向基團具有以下結構：
在一些實施方案中，dsRNA藥劑包含與有義鏈的5'-末端綴合的靶向基團。在一些實施方案中，dsRNA藥劑包含與有義鏈的3'-末端綴合的靶向基團。在一些實施方案中，反義鏈包含在3'-末端處的一個反向無鹼基殘基。在某些實施方案中，有義鏈包含在3'末端或/和5'末端處的一個或兩個反向無鹼基殘基和/或一個或兩個異甘露醇殘基。在某些實施方案中，有義鏈的每一端分別包含一個反向無鹼基殘基。在某些實施方案中，有義鏈的每一端分別包含一個異甘露醇殘基。在一些實施方案中，dsRNA藥劑含有兩個平端。在一些實施方案中，至少一條鏈包含至少1個核苷酸的3'突出端。在一些實施方案中，至少一條鏈包含至少2個核苷酸的3'突出端。In some embodiments, the dsRNA agent comprises a targeting group ligated to the 5'-end of the sense strand. In some embodiments, the dsRNA agent comprises a targeting group ligated to the 3'-end of the sense strand. In some embodiments, the antisense strand comprises an inverted abatic residue at the 3'-end. In certain embodiments, the sense strand comprises one or two inverted abatic residues and/or one or two isomannitol residues at the 3'-end or/and the 5'-end. In certain embodiments, each end of the sense strand comprises an inverted abatic residue. In certain embodiments, each end of the sense strand comprises an isomannitol residue. In some embodiments, the dsRNA agent contains two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.
在一些實施方案中，有義鏈和/或反義鏈的至少一個鍵是磷酸二酯（PO）鍵。在一些實施方案中，有義鏈和/或反義鏈的至少一個鍵是經修飾的鍵。在一些實施方案中，有義鏈和/或反義鏈的至少一個鍵是硫代磷酸酯（PS）鍵。在一些實施方案中，dsRNA藥劑包含至少一個硫代磷酸酯核苷間鍵。在某些實施方案中，有義鏈包含至少一個硫代磷酸酯核苷間鍵。在一些實施方案中，反義鏈包含至少一個硫代磷酸酯核苷間鍵。在一些實施方案中，有義鏈包含1、2、3、4、5或6個硫代磷酸酯核苷間鍵。在一些實施方案中，反義鏈包含1、2、3、4、5或6個硫代磷酸酯核苷間鍵。在一些實施方案中，在有義鏈和/或反義鏈的5'-端、3'-端或兩端引入至少一個硫代磷酸酯（PS）鍵。在一些實施方案中，在有義鏈和/或反義鏈的5'-端、3'-端或兩端獨立地引入1、2、3、4、5或6個硫代磷酸酯（PS）鍵。在一些實施方案中，反義鏈的一端或兩端的至少末端兩個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接。在一些實施方案中，反義鏈的一端或兩端的末端三個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接。在一些實施方案中，有義鏈的一端或兩端的至少末端兩個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接。在一些實施方案中，有義鏈的一端或兩端的末端三個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接。在一些實施方案中，有義鏈5'端的末端三個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接，並且有義鏈3'端的末端兩個經修飾或未修飾的核苷酸通過硫代磷酸酯鍵連接。在一些實施方案中，一個或多個反向無鹼基殘基或一個或多個異甘露醇殘基經由硫代磷酸酯鍵與有義鏈的任一端或兩端綴合。在一些實施方案中，靶向基團還經由硫代磷酸酯鍵與有義鏈的任一端綴合。在一些實施方案中，靶向基團還經由硫代磷酸酯鍵與有義鏈的5'-端綴合。In some embodiments, at least one bond in the sense and/or antisense strand is a phosphodiester (PO) bond. In some embodiments, at least one bond in the sense and/or antisense strand is a modified bond. In some embodiments, at least one bond in the sense and/or antisense strand is a phosphorothioate (PS) bond. In some embodiments, the dsRNA agent comprises at least one phosphorothioate internucleoside bond. In certain embodiments, the sense strand comprises at least one phosphorothioate internucleoside bond. In some embodiments, the antisense strand comprises at least one phosphorothioate internucleoside bond. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside bonds. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate (PS) linkage is introduced at the 5'-end, 3'-end, or both ends of the sense strand and/or the antisense strand. In some embodiments, 1, 2, 3, 4, 5, or 6 phosphorothioate (PS) linkages are independently introduced at the 5'-end, 3'-end, or both ends of the sense strand and/or the antisense strand. In some embodiments, at least the terminal two modified or unmodified nucleotides at one or both ends of the antisense strand are linked by phosphorothioate linkages. In some embodiments, the terminal three modified or unmodified nucleotides at one or both ends of the antisense strand are linked by phosphorothioate linkages. In some embodiments, at least the terminal two modified or unmodified nucleotides at one or both ends of the sense strand are linked by phosphorothioate bonds. In some embodiments, the terminal three modified or unmodified nucleotides at one or both ends of the sense strand are linked by phosphorothioate bonds. In some embodiments, the terminal three modified or unmodified nucleotides at the 5' end of the sense strand are linked by phosphorothioate bonds, and the terminal two modified or unmodified nucleotides at the 3' end of the sense strand are linked by phosphorothioate bonds. In some embodiments, one or more inverted abasic residues or one or more isomannitol residues are linked to either or both ends of the sense strand via phosphorothioate bonds. In some embodiments, the targeting group is further linked to either end of the sense chain via a phosphorothioate bond. In some embodiments, the targeting group is further linked to the 5'-end of the sense chain via a phosphorothioate bond.
在一些實施方案中，經修飾的有義鏈具有表2至表3中的任一個表中示出的修飾模式。在一些實施方案中，經修飾的反義鏈具有表2至表3中的任一個表中示出的修飾模式。在一些實施方案中，經修飾的有義鏈是表2至表3中的任一個表中示出的經修飾的有義鏈序列。在一些實施方案中，經修飾的反義鏈是表2至表3中的任一個表中示出的經修飾的反義鏈序列。In some embodiments, the modified sense chain has a modification pattern shown in any one of Tables 2 to 3. In some embodiments, the modified antisense chain has a modification pattern shown in any one of Tables 2 to 3. In some embodiments, the modified sense chain is a modified sense chain sequence shown in any one of Tables 2 to 3. In some embodiments, the modified antisense chain is a modified antisense chain sequence shown in any one of Tables 2 to 3.
在一些實施方案中，提供了一種用於抑制跨膜絲氨酸蛋白酶6（TMPRSS6）的表達的雙鏈核糖核酸（dsRNA）藥劑，其中該dsRNA藥劑包含有義鏈和反義鏈，其中該有義鏈與該反義鏈互補，其中該反義鏈包含與編碼TMPRSS6的mRNA的一部分互補的區域，其中每條鏈的長度為約14至約30個核苷酸，其中該有義鏈序列能夠由式(I)表示：In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of transmembrane serine protease 6 (TMPRSS6) is provided, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region that is complementary to a portion of an mRNA encoding TMPRSS6, wherein each strand is about 14 to about 30 nucleotides in length, wherein the sense strand sequence can be represented by formula (I):
5’-(N’L)n’N’LN’LN’N1N’N2N’N3N’N4N’LN’FN’LN’N5N’N6N’LN’LN’L(N’L)m’-3’ (I)5'-(N'L )n' N 'L N'L N'N1 N'N2 N'N3 N'N4 N'L N'F N'L N'N5 N'N6 N'L N'L N'L (N'L )m' -3' (I)
其中：in:
每個N’F表示經2'-氟修飾的核苷酸；每個N’N1、N’N2、N’N3、N’N4、N’N5和N’N6獨立地表示經修飾或未修飾的核苷酸；每個N’L獨立地表示經修飾或未修飾的核苷酸但不表示經2'-氟修飾的核苷酸，並且m’和n’各自獨立地為0至7的整數。EachN'F represents a 2'-fluorine-modified nucleotide; eachN'N1 ,N'N2 ,N'N3 ,N'N4 ,N'N5 andN'N6 independently represents a modified or unmodified nucleotide; eachN'L independently represents a modified or unmodified nucleotide but does not represent a 2'-fluorine-modified nucleotide, and m' and n' are each independently an integer from 0 to 7.
在一些實施方案中，經修飾的核苷酸是上文定義的經修飾的核苷酸。In some embodiments, the modified nucleotide is a modified nucleotide as defined above.
在一些實施方案中，經修飾的核苷酸是經2'-OMe修飾的核苷酸或經2'-F修飾的核苷酸。In some embodiments, the modified nucleotide is a 2'-OMe-modified nucleotide or a 2'-F-modified nucleotide.
在一些實施方案中，N’N2和N’N4各自獨立地表示經2'-氟修飾的核苷酸。In some embodiments,N'N2 andN'N4 each independently represent a 2'-fluoro modified nucleotide.
在一些實施方案中，N’N4和N’N5各自獨立地表示經2'-氟修飾的核苷酸。In some embodiments,N'N4 andN'N5 each independently represent a 2'-fluoro modified nucleotide.
在一些實施方案中，m'為2並且n'為3，或者m'為2並且n'為4，m'為2並且n'為5。In some embodiments, m' is 2 and n' is 3, or m' is 2 and n' is 4, or m' is 2 and n' is 5.
在一些實施方案中，m'為4並且n'為1，或者m'為4並且n'為2，m'為4並且n'為3。In some embodiments, m' is 4 and n' is 1, or m' is 4 and n' is 2, or m' is 4 and n' is 3.
在一些實施方案中，N’N4和N’N5各自獨立地表示經2'-氟修飾的核苷酸，m'為4，並且每個N’L、N’N1、N’N2、N’N3和N’N6獨立地表示2'-O-甲基核苷酸。In some embodiments,N'N4 andN'N5 each independently represent a 2'-fluoro modified nucleotide, m' is 4, and each ofN'L ,N'N1 ,N'N2 ,N'N3 andN'N6 independently represents a 2'-O-methyl nucleotide.
在一些實施方案中，N’N2和N’N4各自獨立地表示經2'-氟修飾的核苷酸，m'為2，並且每個N’L、N’N1、N’N3、N’N5和N’N6獨立地表示2'-O-甲基核苷酸。In some embodiments,N'N2 andN'N4 each independently represent a 2'-fluoro modified nucleotide, m' is 2, and each ofN'L ,N'N1 ,N'N3 ,N'N5 andN'N6 independently represents a 2'-O-methyl nucleotide.
在一些實施方案中，dsRNA藥劑包含與有義鏈的5'-末端綴合的靶向基團，優選地，該靶向基團選自上述GLO-1至GLO-16以及GLS-1*至GLS-16*中的任一者，更優選地，該靶向基團是上述GLS-15*。在某些實施方案中，dsRNA藥劑包含與有義鏈的3'-末端綴合的靶向基團。在某些實施方案中，反義鏈包含在3'-末端處的一個反向無鹼基殘基。在某些實施方案中，有義鏈包含在3'末端或/和5'末端處的一個或兩個反向無鹼基殘基和/或一個或兩個異甘露醇殘基。在某些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含反向無鹼基殘基。在某些實施方案中，僅有義鏈的3'末端包含反向無鹼基殘基。在某些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含異甘露醇殘基。在某些實施方案中，有義鏈在3'末端和5'末端處分別包含一個反向無鹼基殘基，並且在3'末端或5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈在3'末端和5'末端處分別包含一個反向無鹼基殘基，並且在5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈包含在3'末端和5'末端處的一個反向無鹼基殘基，該反向無鹼基殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈在3'末端和5'末端處分別包含一個異甘露醇殘基，並且在3'末端或5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈在3'末端和5'末端處分別包含一個異甘露醇殘基，並且在5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5'-terminus of the sense strand. Preferably, the targeting group is selected from any one of GLO-1 to GLO-16 and GLS-1* to GLS-16*, and more preferably, the targeting group is GLS-15*. In certain embodiments, the dsRNA agent comprises a targeting group conjugated to the 3'-terminus of the sense strand. In certain embodiments, the antisense strand comprises an inverted abatic residue at the 3'-terminus. In certain embodiments, the sense strand comprises one or two inverted abatic residues and/or one or two isomannitol residues at the 3'-terminus and/or the 5'-terminus. In certain embodiments, each 3' and 5' terminus of the sense strand independently comprises an inverted abatic residue. In certain embodiments, only the 3' terminus of the sense strand comprises an inverted abatic residue. In certain embodiments, each 3' and 5' terminus of the sense strand independently comprises an isomannitol residue. In certain embodiments, the sense strand comprises one inverted abatic residue at each of its 3' and 5' termini, and the residue at either the 3' or 5' terminus is further conjugated to a targeting group, preferably GLS-15* as described above. In certain embodiments, the sense chain comprises an inverted abatic residue at each of the 3' and 5' termini, and the residue at the 5' terminus is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises an inverted abatic residue at each of the 3' and 5' termini, and the inverted abatic residue is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises an isomannitol residue at each of the 3' and 5' termini, and the residue at either the 3' or 5' terminus is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises an isomannitol residue at each of the 3' and 5' ends, and the residue at the 5' end is further conjugated to a targeting group, preferably GLS-15* as described above.
在一些實施方案中，提供了一種用於抑制跨膜絲氨酸蛋白酶6（TMPRSS6）的表達的雙鏈核糖核酸（dsRNA）藥劑，其中該dsRNA藥劑包含有義鏈和反義鏈，其中該有義鏈與該反義鏈互補，其中該反義鏈包含與編碼TMPRSS6的mRNA的一部分互補的區域，其中每條鏈的長度為約18至約30個核苷酸，其中該反義鏈序列能夠由式(II)表示：In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of transmembrane serine protease 6 (TMPRSS6) is provided, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region that is complementary to a portion of an mRNA encoding TMPRSS6, wherein each strand is about 18 to about 30 nucleotides in length, wherein the antisense strand sequence can be represented by formula (II):
3’-(NL)nNM1NLNM2NLNFNLNM3NLNM4NLNM5NM6NLNM7NM8NLNFNL-5’ (II)3'-(NL )n NM1 NL NM2 NL NF NL NM3 NL NM4 NL NM5 NM6 NL NM7 NM8 NL NF NL -5' (II)
其中：in:
每個NF表示經2'-氟修飾的核苷酸；每個NM1、NM2、NM3、NM4、NM5、NM6、NM7和NM8獨立地表示經修飾或未修飾的核苷酸；每個NL獨立地表示經修飾或未修飾的核苷酸但不表示經2'-氟修飾的核苷酸，並且n為0至7的整數。EachNF represents a 2'-fluorine-modified nucleotide; each NM1 , NM2 , NM3 , NM4 , NM5 , NM6 , NM7 and NM8 independently represents a modified or unmodified nucleotide; eachNL independently represents a modified or unmodified nucleotide but does not represent a 2'-fluorine-modified nucleotide, and n is an integer from 0 to 7.
在一些實施方案中，經修飾的核苷酸是上文定義的經修飾的核苷酸。In some embodiments, the modified nucleotide is a modified nucleotide as defined above.
在一些實施方案中，經修飾的核苷酸是經2'-OMe修飾的核苷酸或經2'-F修飾的核苷酸。In some embodiments, the modified nucleotide is a 2'-OMe-modified nucleotide or a 2'-F-modified nucleotide.
在一些實施方案中，NM2、NM3和NM6各自獨立地表示經2'-氟修飾的核苷酸；In some embodiments, NM2 , NM3 , and NM6 each independently represent a 2'-fluoro-modified nucleotide;
在一些實施方案中，NM2、NM3和NM7各自獨立地表示經2'-氟修飾的核苷酸，並且NM6表示經UNA修飾的核苷酸；In some embodiments, NM2 , NM3 , and NM7 each independently represent a 2'-fluorine-modified nucleotide, and NM6 represents a UNA-modified nucleotide;
在一些實施方案中，NM2、NM3和NM6各自獨立地表示經2'-氟修飾的核苷酸，並且每個NM1、NM4、NM5、NM7、NM8和NL獨立地表示2'-O-甲基核苷酸。In some embodiments, NM2 , NM3 , and NM6 each independently represent a 2'-fluoro modified nucleotide, and each NM1 , NM4 , NM5 , NM7 , NM8 , andNL independently represent a 2'-O-methyl nucleotide.
在一些實施方案中，NM2、NM3和NM7各自獨立地表示經2'-氟修飾的核苷酸，並且NM6表示經UNA修飾的核苷酸，並且每個NM1、NM4、NM5、NM8和NL各自獨立地表示2'-O-甲基核苷酸。In some embodiments, NM2 , NM3 , and NM7 each independently represent a 2'-fluoro modified nucleotide, and NM6 represents a UNA modified nucleotide, and each of NM1 , NM4 , NM5 , NM8 , andNL each independently represents a 2'-O-methyl nucleotide.
在一些實施方案中，n為1，或者n為2，或者n為3。In some embodiments, n is 1, or n is 2, or n is 3.
在一些實施方案中，NM6、NM3和NM2都是經2'-氟修飾的核苷酸。In some embodiments, NM6 , NM3 , and NM2 are all 2'-fluoro-modified nucleotides.
在一些實施方案中，提供了一種用於抑制跨膜絲氨酸蛋白酶6（TMPRSS6）的表達的雙鏈核糖核酸（dsRNA）藥劑，其中該dsRNA藥劑包含有義鏈和反義鏈，其中該有義鏈和該反義鏈形成dsRNA雙鏈體，其中所述有義鏈與該反義鏈互補，其中所述反義鏈包含與編碼TMPRSS6的mRNA互補的區域，其中該互補區域包含至少15個連續核苷酸，其中該dsRNA雙鏈體由式(III)表示：In some embodiments, a double-stranded ribonucleic acid (dsRNA) agent for inhibiting the expression of transmembrane serine protease 6 (TMPRSS6) is provided, wherein the dsRNA agent comprises a sense strand and an antisense strand, wherein the sense strand and the antisense strand form a dsRNA duplex, wherein the sense strand is complementary to the antisense strand, wherein the antisense strand comprises a region that is complementary to an mRNA encoding TMPRSS6, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNA duplex is represented by formula (III):
有義鏈：5’-(N’L)n’N’LN’LN’N1N’N2N’N3N’N4N’LN’FN’LN’N5N’N6N’LN’LN’L(N’L)m’-3’Sense chain: 5'-(N'L )n' N'L N'L N'N1 N'N2 N'N3 N'N4 N'L N'F N'L N'N5 N'N6 N'L N 'L N'L (N'L )m' -3'
反義鏈：3’-(NL)nNM1NLNM2NLNFNLNM3NLNM4NLNM5NM6NLNM7NM8NLNFNL-5’ (III)Antisense strand: 3'-(NL )n NM1 NL NM2 NL NF NL NM3 NL NM4 NL NM5 NM6 NL NM7 NM8 NL NF NL -5' (III)
其中：in:
每條鏈的長度為約17至約30個核苷酸；Each strand is about 17 to about 30 nucleotides in length;
每個NF和N’F獨立地表示經2'-氟修飾的核苷酸；NM1、NM2、NM3、NM4、NM5、NM6、NM7、NM8、N’N1、N’N2、N’N3、N’N4、N’N5和N’N6各自獨立地表示經修飾或未修飾的核苷酸；每個NL和N’L獨立地表示經修飾或未修飾的核苷酸但不表示經2'-氟修飾的核苷酸，並且m’、n’和n各自獨立地為0至7的整數。EachNF andN'F independently represents a 2'-fluorine-modified nucleotide; NM1 , NM2 , NM3 , NM4 , NM5 , NM6 , NM7 , NM8 , N'N1,N'N2 ,N'N3 ,N'N4 ,N'N5 andN'N6 independently represent a modified or unmodified nucleotide; eachNL andN'L independently represents a modified or unmodified nucleotide but does not represent a 2'-fluorine-modified nucleotide, and m', n' and n are each independentlyan integer from 0 to 7.
在一些實施方案中，經修飾的核苷酸是上文定義的經修飾的核苷酸。In some embodiments, the modified nucleotide is a modified nucleotide as defined above.
在一些實施方案中，經修飾的核苷酸是經2'-OMe修飾的核苷酸或經2'-F修飾的核苷酸。In some embodiments, the modified nucleotide is a 2'-OMe-modified nucleotide or a 2'-F-modified nucleotide.
在一些實施方案中，n'為1並且m'為2，或者n'為2並且m'為2，或者n'為1並且m'為4，或者n'為3並且m'為2，或者n'為3並且m'為4。In some embodiments, n' is 1 and m' is 2, or n' is 2 and m' is 2, or n' is 1 and m' is 4, or n' is 3 and m' is 2, or n' is 3 and m' is 4.
在一些實施方案中，N’N2和N’N4各自獨立地表示經2'-氟修飾的核苷酸。In some embodiments,N'N2 andN'N4 each independently represent a 2'-fluoro modified nucleotide.
在一些實施方案中，N’N4和N’N5各自獨立地表示經2'-氟修飾的核苷酸。In some embodiments,N'N4 andN'N5 each independently represent a 2'-fluoro modified nucleotide.
在一些實施方案中，N’N4和N’N5各自獨立地表示經2'-氟修飾的核苷酸，m'為4，並且每個N’L、N’N1、N’N2、N’N3和N’N6獨立地表示2'-O-甲基核苷酸。In some embodiments,N'N4 andN'N5 each independently represent a 2'-fluoro modified nucleotide, m' is 4, and each ofN'L ,N'N1 ,N'N2 ,N'N3 andN'N6 independently represents a 2'-O-methyl nucleotide.
在一些實施方案中，N’N2和N’N4各自獨立地表示經2'-氟修飾的核苷酸，m'為2，並且每個N’L、N’N1、N’N3、N’N5和N’N6獨立地表示2'-O-甲基核苷酸。In some embodiments,N'N2 andN'N4 each independently represent a 2'-fluoro modified nucleotide, m' is 2, and each ofN'L ,N'N1 ,N'N3 ,N'N5 andN'N6 independently represents a 2'-O-methyl nucleotide.
在一些實施方案中，n為1，或者n為2，或者n為3。In some embodiments, n is 1, or n is 2, or n is 3.
在一些實施方案中，N’N1N’N2N’N3和N’N4N’N5N’N6各自獨立地表示包含至少兩個不同經修飾的核苷酸的基序。In some embodiments,N'N1N'N2N'N3 andN'N4N'N5N'N6 each independently represent a motif comprising at least two different modified nucleotides.
在一些實施方案中，NM2、NM3和NM6各自獨立地表示經2'-氟修飾的核苷酸。In some embodiments, NM2 , NM3 , and NM6 each independently represent a 2'-fluoro modified nucleotide.
在一些實施方案中，NM2、NM3和NM7各自獨立地表示經2'-氟修飾的核苷酸，並且NM6表示經UNA修飾的核苷酸。In some embodiments, NM2 , NM3 , and NM7 each independently represent a 2'-fluoro modified nucleotide, and NM6 represents a UNA modified nucleotide.
在一些實施方案中，NM2、NM3和NM6各自獨立地表示經2'-氟修飾的核苷酸，並且每個NM1、NM4、NM5、NM7、NM8和NL獨立地表示2'-O-甲基核苷酸。In some embodiments, NM2 , NM3 , and NM6 each independently represent a 2'-fluoro modified nucleotide, and each NM1 , NM4 , NM5 , NM7 , NM8 , andNL independently represent a 2'-O-methyl nucleotide.
在一些實施方案中，NM2、NM3和NM7各自獨立地表示經2'-氟修飾的核苷酸，並且NM6表示經UNA修飾的核苷酸，並且每個NM1、NM4、NM5、NM8和NL各自獨立地表示2'-O-甲基核苷酸。In some embodiments, NM2 , NM3 , and NM7 each independently represent a 2'-fluoro modified nucleotide, and NM6 represents a UNA modified nucleotide, and each of NM1 , NM4 , NM5 , NM8 , andNL each independently represents a 2'-O-methyl nucleotide.
在式(II)或(III)的一些實施方案中，反義鏈包含在3'-末端處的一個反向無鹼基殘基。在式(I)或(III)的一些實施方案中，有義鏈包含在3'末端或/和5'末端處的一個或兩個反向無鹼基殘基和/或一個或兩個異甘露醇殘基。在某些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含反向無鹼基殘基。在式(I)或(III)的一些實施方案中，僅有義鏈的3'末端包含反向無鹼基殘基。在式(I)或(III)的一些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含異甘露醇殘基。在式(I)或(III)的一些實施方案中，有義鏈包含在3'末端和5'末端處的一個反向無鹼基殘基，該反向無鹼基殘基與介導遞送至肝臟組織的任選地為上述GLS-15*的靶向基團進一步綴合。在式(I)或(III)的一些實施方案中，有義鏈在3'末端和5'末端處分別包含一個反向無鹼基殘基，並且在3'末端或5'末端處的鹼基與介導遞送至肝臟組織的任選地為上述GLS-15*的靶向基團進一步綴合。在式(I)或(III)的一些實施方案中，有義鏈在3'末端和5'末端處分別包含一個異甘露醇殘基，並且在3'末端或5'末端處的鹼基與介導遞送至肝臟組織的任選地為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，上述dsRNA藥劑含有兩個平端。在式(I)、(II)或(III)的某些實施方案中，至少一條鏈包含至少1個核苷酸的3'突出端。在式(I)、(II)或(III)的某些實施方案中，至少一條鏈包含至少2個核苷酸的3'突出端。In some embodiments of formula (II) or (III), the antisense chain comprises an inverted abatic residue at the 3'-terminus. In some embodiments of formula (I) or (III), the sense chain comprises one or two inverted abatic residues and/or one or two isomannitol residues at the 3'-terminus and/or the 5'-terminus. In certain embodiments, each 3'-terminus and 5'-terminus of the sense chain independently comprises an inverted abatic residue. In some embodiments of formula (I) or (III), only the 3'-terminus of the sense chain comprises an inverted abatic residue. In some embodiments of formula (I) or (III), each 3'-terminus and 5'-terminus of the sense chain independently comprises an isomannitol residue. In some embodiments of Formula (I) or (III), the sense chain comprises an inverted abatic residue at the 3' terminus and an inverted abatic residue at the 5' terminus, which are further conjugated to a targeting group that mediates delivery to liver tissue, optionally GLS-15*. In some embodiments of Formula (I) or (III), the sense chain comprises an inverted abatic residue at the 3' terminus and an inverted abatic residue at the 5' terminus, respectively, and the abatic group at the 3' terminus or the 5' terminus is further conjugated to a targeting group that mediates delivery to liver tissue, optionally GLS-15*. In some embodiments of Formula (I) or (III), the sense strand comprises an isomannitol residue at each of the 3' and 5' ends, and the base at the 3' or 5' end is further conjugated to a targeting group that mediates delivery to liver tissue, optionally GLS-15*. In certain embodiments, the dsRNA agent comprises two blunt ends. In certain embodiments of Formula (I), (II), or (III), at least one strand comprises a 3' overhang of at least one nucleotide. In certain embodiments of Formula (I), (II), or (III), at least one strand comprises a 3' overhang of at least two nucleotides.
在一些實施方案中，dsRNA藥劑包含與有義鏈的5'-末端綴合的靶向基團，優選地，該靶向基團選自上述GLO-1至GLO-16以及GLS-1*至GLS-16*中的任一者，更優選地，該靶向基團是上述GLS-15*。在某些實施方案中，dsRNA藥劑包含與有義鏈的5'-末端綴合的靶向基團。在某些實施方案中，反義鏈包含在3'-末端處的一個反向無鹼基殘基。在某些實施方案中，有義鏈包含在3'末端或/和5'末端處的一個或兩個反向無鹼基殘基和/或一個或兩個異甘露醇殘基。在某些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含反向無鹼基殘基。在某些實施方案中，有義鏈的每個3'末端和5'末端獨立地包含異甘露醇殘基。在某些實施方案中，有義鏈包含在3'末端和5'末端處的兩個反向無鹼基殘基，並且在3'末端或5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈包含在3'末端和5'末端處的兩個反向無鹼基殘基，並且在5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈包含在3'末端和5'末端處的一個反向無鹼基殘基，該反向無鹼基殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈包含在3'末端和5'末端處的兩個異甘露醇殘基，並且在3'末端或5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。在某些實施方案中，有義鏈包含在3'末端和5'末端處的兩個異甘露醇殘基，並且在5'末端處的殘基與優選為上述GLS-15*的靶向基團進一步綴合。In some embodiments, the dsRNA agent comprises a targeting group conjugated to the 5'-terminus of the sense chain. Preferably, the targeting group is selected from any one of GLO-1 to GLO-16 and GLS-1* to GLS-16*, and more preferably, the targeting group is GLS-15*. In certain embodiments, the dsRNA agent comprises a targeting group conjugated to the 5'-terminus of the sense chain. In certain embodiments, the antisense chain comprises an inverted abatic residue at the 3'-terminus. In certain embodiments, the sense chain comprises one or two inverted abatic residues and/or one or two isomannitol residues at the 3'-terminus and/or the 5'-terminus. In certain embodiments, each 3' and 5' terminus of the sense chain independently comprises an inverted abatic residue. In certain embodiments, each 3' and 5' terminus of the sense chain independently comprises an isomannitol residue. In certain embodiments, the sense chain comprises two inverted abatic residues at the 3' and 5' termini, and the residue at the 3' or 5' terminus is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises two inverted abatic residues at the 3' and 5' termini, and the residue at the 5' terminus is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises an inverted abatic residue at the 3' and 5' ends, which is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises two isomannitol residues at the 3' and 5' ends, and the residue at the 3' or 5' end is further conjugated to a targeting group, preferably GLS-15*, as described above. In certain embodiments, the sense chain comprises two isomannitol residues at the 3' and 5' ends, and the residue at the 5' end is further conjugated to a targeting group, preferably GLS-15*, as described above.
在某些實施方案中，dsRNA藥劑含有兩個平端。在某些實施方案中，至少一條鏈包含至少1個核苷酸的3'突出端。在某些實施方案中，至少一條鏈包含至少2個核苷酸的3'突出端。In some embodiments, the dsRNA agent comprises two blunt ends. In some embodiments, at least one strand comprises a 3' overhang of at least 1 nucleotide. In some embodiments, at least one strand comprises a 3' overhang of at least 2 nucleotides.
在式(I)、(II)或(III)的一些實施方案中，NM1、NM2、NM3、NM4、NM5、NM6、NM7、NM8、N’N1、N’N2、N’N3、N’N4、N’N5、N’N6、N’L和NL各自獨立地經由磷酸二酯（PO）鍵與相鄰核苷酸連接。在式(I)、(II)或(III)的一些實施方案中，NM1、NM2、NM3、NM4、NM5、NM6、NM7、NM8、N’N1、N’N2、N’N3、N’N4、N’N5、N’N6、N’L和NL中的至少一者經由硫代磷酸酯（PS）鍵與相鄰核苷酸連接。在上述包括反向無鹼基殘基、異甘露醇殘基和/或靶向基團的式(I)、(II)或(III)的一些實施方案中，鏈的每一端的末端位置的位置1至10內的鍵獨立地包括1、2、3、4、5或6個硫代磷酸酯（PS）鍵。在上述包括反向無鹼基殘基、異甘露醇殘基和/或靶向基團的式(I)、(II)或(III)的一些實施方案中，鏈的每一端的末端位置的位置1至5內的鍵獨立地包括1、2或3個硫代磷酸酯（PS）鍵。在上述包括反向無鹼基殘基、異甘露醇殘基和/或靶向基團的式(I)、(II)或(III)的一些實施方案中，鏈的每一端的末端位置的位置1至3內的鍵獨立地包括1或2個硫代磷酸酯（PS）鍵。In some embodiments of Formula (I), (II) or (III),NM1 ,NM2 , NM3,NM4 ,NM5 ,NM6 ,NM7, NM8, N'N1, N'N2, N'N3, N'N4, N'N5, N'N6, N'L and NLareeachindependentlylinkedtoanadjacentnucleotideviaa phosphodiester (PO) bond. In some embodiments of Formula (I), (II), or (III), at least one of NM1 , NM2 , NM3 , NM4 , NM5 , NM6 , NM7 , NM8 , N'N1 , N'N2 , N'N3 , N'N4 , N'N5 , N'N6 , N'L , andNL is linked to an adjacent nucleotide via a phosphorothioate (PS) bond. In some embodiments of Formula (I), (II), or (III) described above that include an inverted abatic residue, an isomannitol residue, and/or a targeting group, the bonds within positions 1 to 10 at each end of the chain independently include 1, 2, 3, 4, 5, or 6 phosphorothioate (PS) bonds. In some embodiments of the above formula (I), (II) or (III) comprising a reversed abatic residue, an isomannol residue and/or a targeting group, the bonds within positions 1 to 5 of the terminal position at each end of the chain independently comprise 1, 2 or 3 phosphorothioate (PS) bonds. In some embodiments of the above formula (I), (II) or (III) comprising a reversed abatic residue, an isomannol residue and/or a targeting group, the bonds within positions 1 to 3 of the terminal position at each end of the chain independently comprise 1 or 2 phosphorothioate (PS) bonds.
在一些實施方案中，表1中的任一條有義鏈可以上述式(I)或(III)所示的模式進行進一步修飾。In some embodiments, any of the sense chains in Table 1 can be further modified as shown in formula (I) or (III) above.
在一些實施方案中，表1中的任一條反義鏈可以上述式(II)或(III)所示的模式進行進一步修飾。In some embodiments, any of the antisense chains in Table 1 can be further modified in the manner shown in formula (II) or (III) above.
在一些實施方案中，表1中的任一個雙鏈體可以上述式(III)所示的模式進行進一步修飾。In some embodiments, any of the diatoms in Table 1 can be further modified in the manner shown in Formula (III) above.
根據本發明的一個方面，提供了一種組合物，該組合物包含本發明的上述dsRNA藥劑方面的任何實施方案。在某些實施方案中，該組合物還包含藥學上可接受的載劑。在一些實施方案中，該組合物還包含一種或多種另外的治療劑。在某些實施方案中，該組合物被包裝在試劑盒、容器、包裝件、分配器、預填充注射器或小瓶中。在一些實施方案中，該組合物被配製用於皮下施用，或者被配製用於靜脈內（IV）施用。According to one aspect of the present invention, a composition is provided, comprising any of the embodiments of the dsRNA agent described above. In certain embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents. In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial. In some embodiments, the composition is formulated for subcutaneous administration or for intravenous (IV) administration.
根據本發明的另一個方面，提供了一種細胞，該細胞包含本發明的上述dsRNA藥劑方面的任何實施方案。在一些實施方案中，該細胞是哺乳動物細胞，任選地是人細胞。According to another aspect of the present invention, a cell is provided, comprising any of the embodiments of the dsRNA agent of the present invention. In some embodiments, the cell is a mammalian cell, optionally a human cell.
根據本發明的另一方面，提供了一種抑制細胞或受試者的TMPRSS6基因的表達的方法，該方法包括：(i)製備包含有效量的本發明的上述dsRNA藥劑方面的任何實施方案或本發明的上述組合物的任何實施方案的細胞。在某些實施方案中，該方法還包括：(ii)將所製備的細胞維持足夠長的時間以獲得TMPRSS6基因的mRNA轉錄本的降解，從而抑制該細胞中TMPRSS6基因的表達。在一些實施方案中，該細胞在受試者體內並且通過皮下將dsRNA藥劑施用於該受試者。在一些實施方案中，該細胞在受試者體內並且通過靜脈內施用將dsRNA藥劑施用於該受試者。在某些實施方案中，該方法還包括在將dsRNA藥劑施用於受試者後評估對TMPRSS6基因的抑制，其中用於該評估的方式包括：(i)測定受試者的TMPRSS6相關疾病或病症的一種或多種生理特徵，以及(ii)將所測定的生理特徵與該TMPRSS6相關疾病或病症的治療前基線生理特徵和/或該TMPRSS6相關疾病或病症的對照生理特徵進行比較，其中該比較表明對受試者的TMPRSS6基因表達的抑制存在或不存在中的一者或多者。在一些實施方案中，該生理特徵是以下項中的一者或多者：TMPRSS6 mRNA水平和TMPRSS6蛋白水平。TMPRSS6表達的降低還可通過測量TMPRSS6的生物活性的降低，例如以下項中的一者或多者的降低進行間接評估：鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平、Hb A2水平等。According to another aspect of the present invention, a method for inhibiting the expression of a TMPRSS6 gene in a cell or subject is provided, the method comprising: (i) preparing a cell comprising an effective amount of any embodiment of the dsRNA agent of the present invention or any embodiment of the composition of the present invention. In certain embodiments, the method further comprises: (ii) maintaining the prepared cell for a sufficient period of time to achieve degradation of the mRNA transcript of the TMPRSS6 gene, thereby inhibiting the expression of the TMPRSS6 gene in the cell. In some embodiments, the cell is in a subject and the dsRNA agent is administered to the subject subcutaneously. In some embodiments, the cell is in a subject and the dsRNA agent is administered to the subject intravenously. In certain embodiments, the method further comprises assessing suppression of the TMPRSS6 gene after administering the dsRNA agent to the subject, wherein the means for said assessment comprises: (i) measuring one or more physiological characteristics of a TMPRSS6-associated disease or condition in the subject, and (ii) comparing the measured physiological characteristics to a baseline physiological characteristic before treatment of the TMPRSS6-associated disease or condition and/or a control physiological characteristic of the TMPRSS6-associated disease or condition, wherein the comparison indicates one or more of the presence or absence of suppression of TMPRSS6 gene expression in the subject. In some embodiments, the physiological characteristics are one or more of: TMPRSS6 mRNA levels and TMPRSS6 protein levels. Reduction in TMPRSS6 expression can also be assessed indirectly by measuring a reduction in the biological activity of TMPRSS6, such as a reduction in one or more of the following: hepcidin levels, iron levels, transferrin saturation levels and red blood cell (RBC) count measurements, hemoglobin levels, ferritin levels, Hb F levels, Hb A2 levels, etc.
根據本發明的另一個方面，提供了一種抑制受試者的TMPRSS6基因的表達的方法，該方法包括向該受試者施用有效量的本發明的上述dsRNA藥劑方面的實施方案或本發明的上述組合物的實施方案。在一些實施方案中，通過皮下將dsRNA藥劑施用於受試者。在某些實施方案中，通過靜脈內施用將dsRNA藥劑施用於受試者。在一些實施方案中，該方法還包括：在施用dsRNA藥劑後評估對TMPRSS6基因的抑制，其中用於該評估的方式包括：(i)測定受試者的TMPRSS6相關疾病或病症的一種或多種生理特徵，以及(ii)將所測定的生理特徵與該TMPRSS6相關疾病或病症的治療前基線生理特徵和/或該TMPRSS6相關疾病或病症的對照生理特徵進行比較，其中該比較表明對受試者的TMPRSS6基因表達的抑制存在或不存在中的一者或多者。在一些實施方案中，TMPRSS6基因的表達可基於與TMPRSS6基因表達相關的任何變量的水平或水平變化（諸如TMPRSS6 mRNA水平、TMPRSS6蛋白水平）進行評估。TMPRSS6表達的降低還可通過測量TMPRSS6的生物活性的降低進行間接評估，例如鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平、Hb A2水平等中的一者或多者的降低。According to another aspect of the present invention, a method for inhibiting the expression of a TMPRSS6 gene in a subject is provided, the method comprising administering to the subject an effective amount of an embodiment of the dsRNA agent or the composition of the present invention. In some embodiments, the dsRNA agent is administered to the subject subcutaneously. In certain embodiments, the dsRNA agent is administered to the subject intravenously. In some embodiments, the method further comprises: assessing suppression of the TMPRSS6 gene after administration of the dsRNA agent, wherein the means for said assessment comprises: (i) measuring one or more physiological characteristics of a TMPRSS6-related disease or condition in the subject, and (ii) comparing the measured physiological characteristics to a baseline physiological characteristic before treatment of the TMPRSS6-related disease or condition and/or a control physiological characteristic of the TMPRSS6-related disease or condition, wherein the comparison indicates one or more of the presence or absence of suppression of TMPRSS6 gene expression in the subject. In some embodiments, expression of the TMPRSS6 gene can be assessed based on the level or change in the level of any variable associated with TMPRSS6 gene expression (e.g., TMPRSS6 mRNA level, TMPRSS6 protein level). Reduction in TMPRSS6 expression can also be assessed indirectly by measuring a reduction in the biological activity of TMPRSS6, such as a reduction in one or more of hepcidin levels, iron levels, transferrin saturation levels, and red blood cell (RBC) count measurements, hemoglobin levels, ferritin levels, Hb F levels, Hb A2 levels, etc.
根據本發明的另一個方面，提供了一種治療與TMPRSS6蛋白的存在相關的疾病或病症的方法，該方法包括：向受試者施用有效量的本發明的任何上述dsRNA藥劑方面的實施方案或本發明的任何上述組合物的實施方案，以抑制TMPRSS6基因表達。在一些實施方案中，與TMPRSS6相關的疾病、障礙或病症選自：血色素沉著症（例如遺傳性血色素沉著症、特發性血色素沉著症、原發性血色素沉著症、繼發性血色素沉著症、嚴重的青少年血色素沉著症、新生兒血色素沉著症）、鐵粒幼細胞性貧血、溶血性貧血、紅細胞生成異常性貧血、鐮狀細胞性貧血、血紅蛋白病、地中海貧血（例如β地中海貧血和α地中海貧血）、真性紅細胞增多症、骨髓增生異常綜合征、先天性紅細胞生成異常性貧血、丙酮酸激酶缺乏症、慢性肝病、遲發性皮膚卟啉症、紅細胞生成性卟啉症、轉鐵蛋白缺乏症、遺傳性酪氨酸血症、腦肝腎綜合征、特發性肺含鐵血黃素沉著症、腎含鐵血黃素沉著症、帕金森病、阿爾茨海默病、弗裡德賴希共濟失調，或其他與鐵過載相關的障礙和/或無效紅細胞生成障礙。According to another aspect of the present invention, a method for treating a disease or condition associated with the presence of TMPRSS6 protein is provided, the method comprising administering to a subject an effective amount of any of the above-mentioned dsRNA agent embodiments or any of the above-mentioned composition embodiments to inhibit TMPRSS6 gene expression. In some embodiments, the disease, disorder or condition associated with TMPRSS6 is selected from the group consisting of hemochromatosis (e.g., hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis), sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, hemoglobinopathy, thalassemia (e.g., beta thalassemia and alpha thalassemia), anemia), polycythemia vera, myelodysplastic syndrome, congenital erythropoietic anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea, erythropoietic porphyria, transferrin deficiency, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, or other disorders related to iron overload and/or ineffective erythropoiesis.
在一些實施方案中，該方法還包括：向受試者施用另外的治療方案。在一些實施方案中，該另外的治療方案包括治療TMPRSS6相關疾病或病症。在某些實施方案中，該另外的治療方案包括：向受試者施用一種或多種本發明的TMPRSS6反義多核苷酸，向受試者施用非TMPRSS6 dsRNA治療劑，以及對受試者的行為矯正。在一些實施方案中，非TMPRSS6 dsRNA治療劑是以下項中的一者或多者：鐵螯合劑（例如去鐵胺、地拉羅司（Exjade）、去鐵酮、維生素E、小麥胚芽油、生育酚和黃素）、葉酸、輸血、放血、止癢劑、收斂劑、局部麻醉劑或抗炎劑、治療潰瘍的藥劑、增加胎兒血紅蛋白水平的藥劑（例如羥基脲）、控制感染的藥劑（例如抗生素和抗病毒藥）、治療血栓形成狀態的藥劑。In some embodiments, the method further comprises administering an additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic regimen comprises treating a TMPRSS6-related disease or condition. In certain embodiments, the additional therapeutic regimen comprises administering to the subject one or more TMPRSS6 antisense polynucleotides of the present invention, administering to the subject a non-TMPRSS6 dsRNA therapeutic, and modifying the subject's behavior. In some embodiments, the non-TMPRSS6 dsRNA therapeutic agent is one or more of the following: iron chelators (e.g., deferoxamine, deferasirox (Exjade), deferoxone, vitamin E, wheat germ oil, tocopherols and flavins), folic acid, blood transfusions, phlebotomies, antipruritics, astringents, local anesthetics or anti-inflammatory agents, agents for treating ulcers, agents for increasing fetal hemoglobin levels (e.g., hydroxyureas), agents for controlling infection (e.g., antibiotics and antivirals), agents for treating thrombotic states.
根據本發明的另一個方面，提供了一種與受試者的TMPRSS6蛋白的治療前基線水平相比降低該受試者的TMPRSS6蛋白水平的方法，該方法包括向該受試者施用有效量的本發明的任何上述dsRNA藥劑的實施方案或本發明的任何上述組合物的實施方案，以降低TMPRSS6基因表達的水平。在一些實施方案中，通過皮下將dsRNA藥劑施用於受試者，或者通過靜脈內施用將其施用於受試者。According to another aspect of the present invention, a method is provided for reducing the level of TMPRSS6 protein in a subject compared to a pre-treatment baseline level of TMPRSS6 protein in the subject, the method comprising administering to the subject an effective amount of any of the aforementioned dsRNA agent embodiments or any of the aforementioned composition embodiments to reduce the level of TMPRSS6 gene expression. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or intravenously.
根據本發明的另一個方面，提供了一種與受試者的TMPRSS6相關疾病或病症的治療前基線生理特徵相比改變該受試者的該TMPRSS6相關疾病或病症的生理特徵的方法，該方法包括向該受試者施用有效量的本發明的任何上述dsRNA藥劑的實施方案或本發明的任何上述組合物的實施方案，以改變該受試者的該TMPRSS6相關疾病或病症的生理特徵。在一些實施方案中，通過皮下將dsRNA藥劑施用於受試者，或者通過靜脈內施用將其施用於受試者。在某些實施方案中，該生理特徵是以下項中的一者或多者：受試者的TMPRSS6 mRNA水平、TMPRSS6蛋白水平，或者鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平或Hb A2水平等。According to another aspect of the present invention, a method is provided for altering the physiological characteristics of a TMPRSS6-related disease or condition in a subject as compared to the subject's pre-treatment baseline physiological characteristics of the TMPRSS6-related disease or condition, the method comprising administering to the subject an effective amount of any of the aforementioned dsRNA agent embodiments of the present invention or any of the aforementioned composition embodiments of the present invention to alter the physiological characteristics of the TMPRSS6-related disease or condition in the subject. In some embodiments, the dsRNA agent is administered to the subject subcutaneously or intravenously. In certain embodiments, the physiological characteristic is one or more of the following: TMPRSS6 mRNA level, TMPRSS6 protein level, or hepcidin level, iron level, transferrin saturation level and red blood cell (RBC) count measurement, hemoglobin level, ferritin level, Hb F level or Hb A2 level in the subject.
根據本發明的另一個方面，提供了在治療與TMPRSS6蛋白的存在相關的疾病或病症的方法中使用的上述dsRNA藥劑。在一些實施方案中，疾病或病症是以下項中的一者或多者：血色素沉著症（例如遺傳性血色素沉著症、特發性血色素沉著症、原發性血色素沉著症、繼發性血色素沉著症、嚴重的青少年血色素沉著症、新生兒血色素沉著症）、鐵粒幼細胞性貧血、溶血性貧血、紅細胞生成異常性貧血、鐮狀細胞性貧血、血紅蛋白病、地中海貧血（例如β地中海貧血和α地中海貧血）、真性紅細胞增多症、骨髓增生異常綜合征、先天性紅細胞生成異常性貧血、丙酮酸激酶缺乏症、慢性肝病、遲發性皮膚卟啉症、紅細胞生成性卟啉症、轉鐵蛋白缺乏症、遺傳性酪氨酸血症、腦肝腎綜合征、特發性肺含鐵血黃素沉著症、腎含鐵血黃素沉著症、帕金森病、阿爾茨海默病、弗裡德賴希共濟失調，或其他與鐵過載相關的障礙和/或無效紅細胞生成障礙。According to another aspect of the present invention, the above-described dsRNA agent is provided for use in a method of treating a disease or condition associated with the presence of a TMPRSS6 protein. In some embodiments, the disease or condition is one or more of: hemochromatosis (e.g., hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis), sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, hemoglobinopathy, thalassemia (e.g., beta thalassemia and alpha thalassemia). , polycythemia vera, myelodysplastic syndrome, congenital erythropoietic anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea, erythropoietic porphyria, transferrin deficiency, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, or other disorders related to iron overload and/or ineffective erythropoiesis.
根據本發明的另一個方面，提供了一種用於抑制TMPRSS6蛋白的表達的反義多核苷酸藥劑，該藥劑包含10至30個連續核苷酸，其中這些連續核苷酸中的至少一個核苷酸是經修飾的核苷酸，並且其中該藥劑的核苷酸序列在其整個長度上與SEQ ID NO: 1的核苷酸序列的等同區域約80%互補。在一些實施方案中，該等同區域是SEQ ID NO: 1的任一靶區域，並且該互補序列是提供在表1至表3中的一個表中的互補序列。在某些實施方案中，該反義多核苷酸藥劑包含提供在表1至表3中的一個表中的反義序列中的一個反義序列。According to another aspect of the present invention, an antisense polynucleotide agent for inhibiting the expression of a TMPRSS6 protein is provided, the agent comprising 10 to 30 consecutive nucleotides, wherein at least one of the consecutive nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is approximately 80% complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 over its entire length. In some embodiments, the equivalent region is any target region of SEQ ID NO: 1, and the complementary sequence is a complementary sequence provided in one of Tables 1 to 3. In certain embodiments, the antisense polynucleotide agent comprises an antisense sequence among the antisense sequences provided in one of Tables 1 to 3.
根據本發明的另一個方面，提供了一種組合物，該組合物包含任何上述反義多核苷酸藥劑的實施方案。在一些實施方案中，該組合物還包含藥學上可接受的載劑。在一些實施方案中，該組合物還包含用於治療TMPRSS6相關疾病或病症的一種或多種另外的治療劑。在某些實施方案中，該組合物被包裝在試劑盒、容器、包裝件、分配器、預填充注射器或小瓶中。在某些實施方案中，該組合物被配製用於皮下或靜脈內施用。According to another aspect of the present invention, a composition is provided, comprising any of the above-described antisense polynucleotide agent embodiments. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises one or more additional therapeutic agents for treating a TMPRSS6-related disease or condition. In certain embodiments, the composition is packaged in a kit, container, package, dispenser, prefilled syringe, or vial. In certain embodiments, the composition is formulated for subcutaneous or intravenous administration.
根據本發明的另一個方面，提供了一種細胞，該細胞包含任何上述反義多核苷酸藥劑的實施方案。在一些實施方案中，該細胞是哺乳動物細胞，任選地是人細胞。According to another aspect of the present invention, a cell is provided, comprising any of the embodiments of the antisense polynucleotide agent described above. In some embodiments, the cell is a mammalian cell, optionally a human cell.
根據本發明的另一方面，提供了一種抑制細胞中TMPRSS6基因的表達的方法，該方法包括：(i)製備包含有效量的任何上述反義多核苷酸藥劑的實施方案的細胞。在一些實施方案中，該方法還包括(ii)將(i)中製備的細胞維持足夠長的時間以獲得TMPRSS6基因的mRNA轉錄本的降解，從而抑制細胞中TMPRSS6基因的表達。According to another aspect of the present invention, a method for inhibiting TMPRSS6 gene expression in a cell is provided, comprising: (i) preparing a cell comprising an effective amount of any of the above-described antisense polynucleotide embodiments. In some embodiments, the method further comprises (ii) maintaining the cell prepared in (i) for a period of time sufficient to degrade the mRNA transcript of the TMPRSS6 gene, thereby inhibiting TMPRSS6 gene expression in the cell.
根據本發明的另一個方面，提供了一種抑制受試者的TMPRSS6基因的表達的方法，該方法包括向受試者施用有效量的任何上述反義多核苷酸藥劑的實施方案。According to another aspect of the present invention, a method for inhibiting the expression of TMPRSS6 gene in a subject is provided, the method comprising administering to the subject an effective amount of any of the above-mentioned antisense polynucleotide agent embodiments.
根據本發明的另一個方面，一種治療與TMPRSS6蛋白的存在相關的疾病或病症的方法，該方法包括向受試者施用有效量的本發明的任何上述反義多核苷酸藥劑的實施方案或任何上述組合物的實施方案，以抑制TMPRSS6基因表達。在某些實施方案中，疾病或病症是以下項中的一者或多者：血色素沉著症（例如遺傳性血色素沉著症、特發性血色素沉著症、原發性血色素沉著症、繼發性血色素沉著症、嚴重的青少年血色素沉著症、新生兒血色素沉著症）、鐵粒幼細胞性貧血、溶血性貧血、紅細胞生成異常性貧血、鐮狀細胞性貧血、血紅蛋白病、地中海貧血（例如β地中海貧血和α地中海貧血）、真性紅細胞增多症、骨髓增生異常綜合征、先天性紅細胞生成異常性貧血、丙酮酸激酶缺乏症、慢性肝病、遲發性皮膚卟啉症、紅細胞生成性卟啉症、轉鐵蛋白缺乏症、遺傳性酪氨酸血症、腦肝腎綜合征、特發性肺含鐵血黃素沉著症、腎含鐵血黃素沉著症、帕金森病、阿爾茨海默病、弗裡德賴希共濟失調，或其他與鐵過載相關的障礙和/或無效紅細胞生成障礙。According to another aspect of the present invention, a method for treating a disease or condition associated with the presence of a TMPRSS6 protein comprises administering to a subject an effective amount of any of the above-described antisense polynucleotide agents or compositions to inhibit TMPRSS6 gene expression. In certain embodiments, the disease or condition is one or more of the following: hemochromatosis (e.g., hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis), sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, hemoglobinopathy, thalassemia (e.g., beta thalassemia and alpha thalassemia). , polycythemia vera, myelodysplastic syndrome, congenital erythropoietic anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea, erythropoietic porphyria, transferrin deficiency, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, or other disorders related to iron overload and/or ineffective erythropoiesis.
根據本發明的另一個方面，提供了一種與受試者的TMPRSS6蛋白的治療前基線水平相比降低該受試者的TMPRSS6蛋白水平的方法，該方法包括向該受試者施用有效量的本發明的任何上述反義多核苷酸藥劑的實施方案或任何上述組合物的實施方案，以降低TMPRSS6基因表達的水平。在某些實施方案中，通過皮下或靜脈內施用將反義多核苷酸藥劑施用於受試者。According to another aspect of the present invention, a method is provided for reducing the level of TMPRSS6 protein in a subject compared to a pre-treatment baseline level of TMPRSS6 protein in the subject, the method comprising administering to the subject an effective amount of any of the aforementioned antisense polynucleotide agent embodiments or any of the aforementioned composition embodiments to reduce the level of TMPRSS6 gene expression. In certain embodiments, the antisense polynucleotide agent is administered to the subject subcutaneously or intravenously.
根據本發明的另一個方面，提供了一種用於抑制TMPRSS6基因的表達的反義多核苷酸藥劑，該藥劑包含10至30個連續核苷酸，其中這些連續核苷酸中的至少一個核苷酸是經修飾的核苷酸，並且其中該藥劑的核苷酸序列在其整個長度上與SEQ ID NO: 1的核苷酸序列的等同區域約80%或約85%互補。According to another aspect of the present invention, an antisense polynucleotide agent for inhibiting the expression of the TMPRSS6 gene is provided, the agent comprising 10 to 30 consecutive nucleotides, wherein at least one nucleotide of the consecutive nucleotides is a modified nucleotide, and wherein the nucleotide sequence of the agent is complementary to the equivalent region of the nucleotide sequence of SEQ ID NO: 1 by about 80% or about 85% over its entire length.
根據本發明的另一個方面，提供了一種與受試者的TMPRSS6相關疾病或病症的治療前基線生理特徵相比改變該受試者的該TMPRSS6相關疾病或病症的生理特徵的方法，該方法包括向該受試者施用有效量的本發明的任何上述反義多核苷酸藥劑的實施方案或任何上述組合物的實施方案，以改變該受試者的該TMPRSS6相關疾病或病症的生理特徵。在一些實施方案中，通過皮下或靜脈內施用將反義多核苷酸藥劑施用於受試者。在一些實施方案中，該生理特徵是以下項中的一者或多者：受試者的TMPRSS6 mRNA水平、TMPRSS6蛋白水平，或者鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平或Hb A2水平等。 序列的簡要描述According to another aspect of the present invention, a method is provided for altering a physiological characteristic of a TMPRSS6-related disease or condition in a subject as compared to the subject's pre-treatment baseline physiological characteristic of the TMPRSS6-related disease or condition, the method comprising administering to the subject an effective amount of any of the aforementioned antisense polynucleotide agent embodiments or any of the aforementioned composition embodiments to alter the physiological characteristic of the TMPRSS6-related disease or condition in the subject. In some embodiments, the antisense polynucleotide agent is administered to the subject subcutaneously or intravenously. In some embodiments, the physiological characteristic is one or more of the following: TMPRSS6 mRNA level, TMPRSS6 protein level, or hepcidin level, iron level, transferrin saturation level, and red blood cell (RBC) count measurement, hemoglobin level, ferritin level, Hb F level, or Hb A2 level in the subject.Brief description of sequence
SEQ ID NO: 1和SEQ ID NO: 2（反向互補序列）是智人跨膜絲氨酸蛋白酶6（TMPRSS6）mRNA [NCBI參考序列：NM_001374504.1]。SEQ ID NO: 1 and SEQ ID NO: 2 (reverse complement sequences) are Homo sapiens transmembrane serine protease 6 (TMPRSS6) mRNA [NCBI Reference Sequence: NM_001374504.1].
SEQ ID NO: 3和SEQ ID NO: 4（反向互補序列）是預測的食蟹猴跨膜絲氨酸蛋白酶6（TMPRSS6）mRNA [NCBI參考序列：XM_005567384.2]。SEQ ID NO: 3 and SEQ ID NO: 4 (reverse complement sequence) are predicted cynomolgus monkey transmembrane serine protease 6 (TMPRSS6) mRNA [NCBI Reference Sequence: XM_005567384.2].
SEQ ID NO: 5和SEQ ID NO: 6（反向互補序列）是預測的獼猴跨膜絲氨酸蛋白酶6（TMPRSS6）mRNA [NCBI參考序列：XM_001085203.4]。SEQ ID NO: 5 and SEQ ID NO: 6 (reverse complement sequences) are predicted macaque transmembrane serine protease 6 (TMPRSS6) mRNAs [NCBI Reference Sequence: XM_001085203.4].
SEQ ID NO: 7-216、1553-1831示於表1中並且都是有義鏈序列。SEQ ID NOs: 7-216, 1553-1831 are shown in Table 1 and are all sense chain sequences.
SEQ ID NO: 217-426、1832-2110示於表1中並且都是反義鏈序列。SEQ ID NOs: 217-426, 1832-2110 are shown in Table 1 and are all antisense sequences.
SEQ ID NO: 427-636、893-1552示於表2中並且都含有化學修飾。SEQ ID NOs: 427-636, 893-1552 are shown in Table 2 and all contain chemical modifications.
SEQ ID NO: 637-892示於表3中。遞送分子在每條有義鏈的3'端或5'端處被表示為“GLX-_”。SEQ ID NOs: 637-892 are shown in Table 3. The delivery molecule is indicated as "GLX-_" at the 3' or 5' end of each sense strand.
本發明部分地包括RNAi藥劑，例如但不限於雙鏈（ds）RNAi藥劑，其能夠抑制跨膜絲氨酸蛋白酶6（TMPRSS6）基因表達。本發明還部分地包括包含TMPRSS6 RNAi藥劑的組合物以及使用這些組合物的方法。本文公開的TMPRSS6 RNAi藥劑可與遞送化合物連接以用於遞送至細胞，包括肝細胞。本發明的藥物組合物可包含至少一種dsRNA TMPRSS6藥劑和遞送化合物。在本發明的組合物和方法的一些實施方案中，遞送化合物是含GalNAc的遞送化合物。遞送至細胞的TMPRSS6 RNAi藥劑能夠抑制TMPRSS6基因表達，從而降低細胞中TMPRSS6基因蛋白產物的活性。本發明的dsRNAi藥劑可用於治療TMPRSS6相關疾病和病症。The present invention comprises, in part, RNAi agents, such as, but not limited to, double-stranded (ds) RNAi agents, that inhibit the expression of the transmembrane serine protease 6 (TMPRSS6) gene. The present invention also comprises, in part, compositions comprising the TMPRSS6 RNAi agents and methods of using these compositions. The TMPRSS6 RNAi agents disclosed herein can be linked to a delivery compound for delivery to cells, including hepatocytes. The pharmaceutical compositions of the present invention can comprise at least one dsRNA TMPRSS6 agent and a delivery compound. In some embodiments of the compositions and methods of the present invention, the delivery compound is a GalNAc-containing delivery compound. TMPRSS6 RNAi agents delivered to cells can inhibit TMPRSS6 gene expression, thereby reducing the activity of TMPRSS6 gene protein products in cells. The dsRNAi agents of the present invention can be used to treat TMPRSS6-related diseases and conditions.
在本發明的一些實施方案中，降低細胞或受試者的TMPRSS6表達分別治療與細胞或受試者的TMPRSS6表達相關的疾病或病症。可通過降低TMPRSS6活性治療的疾病和病症的非限制性示例是：血色素沉著症（例如遺傳性血色素沉著症、特發性血色素沉著症、原發性血色素沉著症、繼發性血色素沉著症、嚴重的青少年血色素沉著症、新生兒血色素沉著症）、鐵粒幼細胞性貧血、溶血性貧血、紅細胞生成異常性貧血、鐮狀細胞性貧血、血紅蛋白病、地中海貧血（例如β地中海貧血和α地中海貧血）、真性紅細胞增多症、骨髓增生異常綜合征、先天性紅細胞生成異常性貧血、丙酮酸激酶缺乏症、慢性肝病、遲發性皮膚卟啉症、紅細胞生成性卟啉症、轉鐵蛋白缺乏症、遺傳性酪氨酸血症、腦肝腎綜合征、特發性肺含鐵血黃素沉著症、腎含鐵血黃素沉著症、帕金森病、阿爾茨海默病、弗裡德賴希共濟失調，或降低TMPRSS6蛋白的水平和活性從而在醫學上獲益的其他疾病。在一些實施方案中，地中海貧血主要是α地中海貧血。在一些實施方案中，地中海貧血是β中間型地中海貧血。In some embodiments of the present invention, reducing TMPRSS6 expression in a cell or subject treats a disease or condition associated with TMPRSS6 expression in the cell or subject, respectively. Non-limiting examples of diseases and conditions that can be treated by reducing TMPRSS6 activity are: hemochromatosis (e.g., hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis), sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, hemoglobinopathy, thalassemia (e.g., beta thalassemia and alpha thalassemia), Polycythemia vera, myelodysplastic syndrome, congenital erythropoietic anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea, erythropoietic porphyria, transferrin deficiency, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, or other diseases where reducing the level or activity of TMPRSS6 protein would be medically beneficial. In some embodiments, the thalassemia is primarily alpha thalassemia. In some embodiments, the thalassemia is beta intermedia thalassemia.
如本文所用，“G”、“C”、“A”和“U”各自通常分別代表含有鳥嘌呤、胞嘧啶、腺嘌呤和尿嘧啶作為鹼基的核苷酸。然而，應當理解，術語“核糖核苷酸”或“核苷酸”也可以是指經修飾的核苷酸，如下文進一步詳述，或替代替換部分。技術人員應當理解，鳥嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可被其他部分替換，而基本上不改變包含帶有此類替換部分的核苷酸的寡核苷酸的鹼基配對特性。例如，非限制性地，包含肌苷作為其鹼基的核苷酸可與含有腺嘌呤、胞嘧啶或尿嘧啶的核苷酸進行鹼基配對。因此，本發明的核苷酸序列中含有尿嘧啶、鳥嘌呤或腺嘌呤的核苷酸可被含有例如肌苷的核苷酸替換。包含此類替換部分的序列是本發明的實施方案。As used herein, "G," "C," "A," and "U" each generally represent a nucleotide containing guanine, cytosine, adenine, and uracil as a base, respectively. However, it will be understood that the term "ribonucleotide" or "nucleotide" may also refer to a modified nucleotide, as described in further detail below, or an alternative replacement moiety. A skilled artisan will appreciate that guanine, cytosine, adenine, and uracil may be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide containing a nucleotide bearing such a replacement moiety. For example, and without limitation, a nucleotide containing inosine as its base may base pair with a nucleotide containing adenine, cytosine, or uracil. Thus, a nucleotide containing uracil, guanine, or adenine in the nucleotide sequence of the present invention may be replaced by a nucleotide containing, for example, inosine. Sequences containing such replacement moieties are embodiments of the present invention.
如本文所用，可與術語“TMPRSS6”互換使用的“跨膜絲氨酸蛋白酶6”是指編碼來自任何脊椎動物或哺乳動物來源的跨膜絲氨酸蛋白酶6蛋白的天然存在的基因，該脊椎動物或哺乳動物來源包括但不限於人、牛、雞、齧齒類動物、小鼠、大鼠、豬、綿羊、靈長類動物、猴和豚鼠，除非另有說明。該術語也指天然TMPRSS6的保持天然TMPRSS6的至少一種體內或體外活性的片段和變體。人TMPRSS6基因的參考序列的氨基酸和完整編碼序列可參見例如GenBank Ref Seq登錄號NM_001374504.1（SEQ ID NO: 1和SEQ ID NO: 2）。人TMPRSS6基因的哺乳動物直系同源物可參見例如GenBank Ref Seq登錄號XM_005567384.2，即食蟹猴（SEQ ID NO: 3和SEQ ID NO: 4）；GenBank Ref Seq登錄號XM_001085203.4，即恒河猴（SEQ ID NO: 5和SEQ ID NO: 6）。TMPRSS6 mRNA序列的其他示例可容易地使用公眾可獲得的數據庫獲得，例如GenBank、UniProt、Ensembl和OMIM。As used herein, the term "transmembrane serine protease 6," which is used interchangeably with the term "TMPRSS6," refers to a naturally occurring gene encoding a transmembrane serine protease 6 protein from any vertebrate or mammalian source, including, but not limited to, humans, cows, chickens, rodents, mice, rats, pigs, sheep, primates, monkeys, and guinea pigs, unless otherwise indicated. The term also refers to fragments and variants of native TMPRSS6 that retain at least one in vivo or in vitro activity of native TMPRSS6. The amino acid and complete coding sequence of the reference sequence of the human TMPRSS6 gene can be found, for example, in GenBank Ref Seq Accession No. NM_001374504.1 (SEQ ID NO: 1 and SEQ ID NO: 2). Mammalian orthologs of the human TMPRSS6 gene can be found, for example, in GenBank Ref Seq Accession No. XM_005567384.2, from cynomolgus monkeys (SEQ ID NOs: 3 and 4); and GenBank Ref Seq Accession No. XM_001085203.4, from rhesus monkeys (SEQ ID NOs: 5 and 6). Additional examples of TMPRSS6 mRNA sequences can be readily obtained using publicly available databases such as GenBank, UniProt, Ensembl, and OMIM.
下文描述了如何製備和使用包含TMPRSS6單鏈（ssRNA）和dsRNA藥劑的組合物以抑制TMPRSS6基因表達，以及用於治療由TMPRSS6基因表達引起或調節的疾病和病症的組合物和方法。術語“RNAi”也是本領域已知的，並且可稱為“siRNA”。The following describes how to prepare and use compositions containing TMPRSS6 single stranded (ssRNA) and dsRNA agents to inhibit TMPRSS6 gene expression, as well as compositions and methods for treating diseases and conditions caused or regulated by TMPRSS6 gene expression. The term "RNAi" is also known in the art and may also be referred to as "siRNA."
如本文所用，術語“RNAi”是指包含RNA並經由RNA誘導的沉默複合物（RISC）途徑來介導RNA轉錄本的靶向切割的藥劑。如本領域所知，RNAi靶區域是指在基因轉錄期間形成的mRNA分子的核苷酸序列的連續部分，包括作為初級轉錄產物的RNA加工產物的信使RNA（mRNA）。該序列的靶部分至少足夠長，以便在該部分或其附近用作RNAi指導的切割的底物。靶序列可為8至30個核苷酸長（包括端值）、10至30個核苷酸長（包括端值）、12至25個核苷酸長（包括端值）、15至23個核苷酸長（包括端值）、16至23個核苷酸長（包括端值）、或18至23個核苷酸長（包括端值），包括在每個所述範圍內的所有更短的長度。在本發明的一些實施方案中，靶序列為9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25或26個核苷酸長。在某些實施方案中，靶序列的長度為9至26個核苷酸之間（包括端值），包括其間的所有子範圍和整數。例如，儘管並非旨在限制，但在本發明的某些實施方案中，靶序列為8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個核苷酸長，其中該序列與TMPRSS6基因的RNA轉錄本的至少一部分完全或至少基本上互補。本發明的一些方面包括藥物組合物，這些藥物組合物包含一種或多種TMPRSS6 dsRNA藥劑和藥學上可接受的載劑。在本發明的某些實施方案中，本文所述的TMPRSS6 RNAi抑制TMPRSS6蛋白的表達。As used herein, the term "RNAi" refers to an agent that contains RNA and mediates the targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. As known in the art, an RNAi target region refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during gene transcription, including messenger RNA (mRNA), which is a product of RNA processing of the primary transcription product. The target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion. The target sequence can be 8 to 30 nucleotides long (inclusive), 10 to 30 nucleotides long (inclusive), 12 to 25 nucleotides long (inclusive), 15 to 23 nucleotides long (inclusive), 16 to 23 nucleotides long (inclusive), or 18 to 23 nucleotides long (inclusive), including all shorter lengths within each of these ranges. In some embodiments of the present invention, the target sequence is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides in length. In certain embodiments, the target sequence is between 9 and 26 nucleotides in length (inclusive), including all subranges and integers therebetween. For example, although not intended to be limiting, in certain embodiments of the present invention, the target sequence is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, wherein the sequence is completely or at least substantially complementary to at least a portion of the RNA transcript of the TMPRSS6 gene. Some aspects of the present invention include pharmaceutical compositions comprising one or more TMPRSS6 dsRNA agents and a pharmaceutically acceptable carrier. In certain embodiments of the present invention, the TMPRSS6 RNAi described herein inhibits the expression of TMPRSS6 protein.
如本文所用，“dsRNA藥劑”意指含有RNA或RNA樣（例如，經化學修飾的RNA）寡核苷酸分子的組合物，該組合物能夠以序列特異性方式降解或抑制靶mRNA的信使RNA（mRNA）轉錄本的翻譯。儘管不希望受到特定理論的限制，但本發明的dsRNA藥劑可通過RNA干擾機制（即，通過與哺乳動物細胞的RNA干擾途徑機制（RNA誘導的沉默複合物或RISC）進行相互作用來誘導RNA干擾）或者通過任何替代機制或途徑起作用。使植物、無脊椎動物和脊椎動物細胞中的基因沉默的方法是本領域熟知的[參見，例如，（Sharp等人，Genes Dev.2001, 15:485；Bernstein等人，(2001) Nature 409:363；Nykanen等人，(2001) Cell 107:309；以及Elbashir等人，(2001) Genes Dev.15:188），這些文獻中的每一篇的公開內容以引用方式全文併入本文]。本領域已知的基因沉默方法可與本文提供的公開內容聯合使用，以抑制TMPRSS6的表達。As used herein, a "dsRNA agent" refers to a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting translation of the messenger RNA (mRNA) transcript of a target mRNA in a sequence-specific manner. While not wishing to be bound by a particular theory, the dsRNA agents of the present invention may act through an RNA interference mechanism (i.e., by inducing RNA interference by interacting with the RNA interference pathway machinery of mammalian cells (RNA-induced silencing complex or RISC)) or through any alternative mechanism or pathway. Methods for silencing genes in plant, invertebrate, and vertebrate cells are well known in the art [see, for example, (Sharp et al., Genes Dev. 2001, 15:485; Bernstein et al., (2001) Nature 409:363; Nykanen et al., (2001) Cell 107:309; and Elbashir et al., (2001) Genes Dev. 15:188), the disclosures of each of which are incorporated herein by reference in their entireties]. Gene silencing methods known in the art can be used in combination with the disclosure provided herein to inhibit the expression of TMPRSS6.
本文公開的dsRNA藥劑由有義鏈和反義鏈組成，並且包含但不限於：短干擾RNA（siRNA）、RNAi藥劑、微小RNA（miRNA）、短髮夾RNA（shRNA）和dicer底物。本文所述的dsRNA藥劑的反義鏈與所靶向的mRNA至少部分地互補。本領域應當理解，不同長度的dsRNA雙鏈體結構可用於抑制靶基因表達。例如，已知含有19、20、21、22和23個鹼基對的雙鏈體結構的dsRNA可有效誘導RNA干擾（Elbashir等人，EMBO 2001, 20:6877-6888）。本領域還已知的是，更短或更長的RNA雙鏈體結構也會有效誘導RNA干擾。在一些實施方案中，有義鏈和反義鏈可具有相同長度或不同長度。在一些實施方案中，每條鏈的長度不超過40個核苷酸。在一些實施方案中，每條鏈的長度不超過30個核苷酸。在一些實施方案中，每條鏈的長度不超過25個核苷酸。在一些實施方案中，每條鏈的長度不超過23個核苷酸。在一些實施方案中，每條鏈的長度不超過21個核苷酸。在一些實施方案中，RNAi藥劑的有義鏈和反義鏈的長度可各自為15至49個核苷酸。在一些實施方案中，反義鏈的長度獨立地為15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個核苷酸。在一些實施方案中，有義鏈的長度獨立地為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或49個核苷酸。在一些實施方案中，有義鏈和反義鏈的長度均為21個核苷酸。在一些實施方案中，有義鏈與反義鏈互補或基本上互補，並且互補區域的長度為15至23個核苷酸。在一些實施方案中，互補區域的長度為19至21個核苷酸。在一些實施方案中，互補區域的長度是14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個核苷酸。在本發明的某些實施方案中，TMPRSS6 dsRNA可包含至少一條長度為最少21個核苷酸的鏈，或者可含有基於表1至表3中的任一個表中示出的序列之一的較短雙鏈體，但與表1至表3中分別示出的dsRNA相比，在一端或兩端減去1、2、3或4個核苷酸也可能是有效的。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑可含有來自表1至表3的一個或多個序列的至少15、16、17、18、19、20個或更多個連續核苷酸的部分序列，並且這些藥劑抑制TMPRSS6基因表達的能力與由包含完整序列的dsRNA產生的抑制水平相差不超過5%、10%、15%、20%、25%或30%。表1至表3中公開的有義序列、反義序列和雙鏈體在本文中可稱為“親本”序列，這意味著表1至表3中公開的序列可如本文所述進行修飾、縮短、延長、包括取代等，其中所得序列在本發明的方法和組合物中保留了其親本序列的全部或至少一部分功效。包含在本發明dsRNA中的有義鏈和反義鏈是獨立選擇的。如本文所用，術語“獨立選擇”意指兩個或更多個類似元件中的每一者均可獨立於其他元件的選擇而被選擇。例如，儘管並非旨在進行限制，但在製備本發明的dsRNA時，可選擇兩條鏈的“元件”以包含在雙鏈體中。例如，一個所選的元件，有義序列可以是SEQ ID NO: 427（示於表2中），以及其他所選的元件，反義序列可以是SEQ ID NO: 532或者可以是經修飾、縮短、延長的SEQ ID NO: 532，並且/或與其親本序列SEQ ID NO: 427相比包含1、2或3個取代。應當理解，本發明的雙鏈體不一定包含表1至表3中的雙鏈體配對所示的有義序列和反義序列兩者。這些表中的每個有義鏈序列和反義鏈序列後面緊接著其SEQ ID NO。The dsRNA agents disclosed herein are composed of a sense strand and an antisense strand and include, but are not limited to, short interfering RNAs (siRNAs), RNAi agents, microRNAs (miRNAs), short hairpin RNAs (shRNAs), and dicer substrates. The antisense strand of the dsRNA agents described herein is at least partially complementary to the targeted mRNA. It is understood in the art that dsRNA duplexes of varying lengths can be used to inhibit target gene expression. For example, dsRNAs containing duplexes of 19, 20, 21, 22, and 23 base pairs are known to effectively induce RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). It is also known in the art that shorter or longer RNA duplexes can also effectively induce RNA interference. In some embodiments, the sense and antisense strands may be of the same length or of different lengths. In some embodiments, each strand is no longer than 40 nucleotides. In some embodiments, each strand is no longer than 30 nucleotides. In some embodiments, each strand is no longer than 25 nucleotides. In some embodiments, each strand is no longer than 23 nucleotides. In some embodiments, each strand is no longer than 21 nucleotides. In some embodiments, the sense and antisense strands of an RNAi agent may each be 15 to 49 nucleotides in length. In some embodiments, the antisense strand is independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the sense strand is independently 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, or 49 nucleotides in length. In some embodiments, the sense and antisense strands are both 21 nucleotides in length. In some embodiments, the sense and antisense strands are complementary or substantially complementary, and the complementary region is 15 to 23 nucleotides in length. In some embodiments, the complementary region is 19 to 21 nucleotides in length. In some embodiments, the complementary region is 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In certain embodiments of the invention, the TMPRSS6 dsRNA may comprise at least one chain of at least 21 nucleotides in length, or may comprise a shorter duplex based on one of the sequences shown in any one of Tables 1 to 3, although it may also be effective if 1, 2, 3, or 4 nucleotides are missing from one or both ends compared to the dsRNAs shown in Tables 1 to 3, respectively. In some embodiments of the invention, the TMPRSS6 dsRNA agents may comprise a partial sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one or more sequences in Tables 1 to 3, and the ability of these agents to inhibit TMPRSS6 gene expression does not differ by more than 5%, 10%, 15%, 20%, 25%, or 30% from the level of inhibition produced by dsRNA comprising the complete sequence. The sense sequences, antisense sequences, and duplexes disclosed in Tables 1 to 3 may be referred to herein as "parent" sequences, meaning that the sequences disclosed in Tables 1 to 3 may be modified, shortened, extended, including substitutions, etc., as described herein, wherein the resulting sequences retain all or at least a portion of the efficacy of their parent sequences in the methods and compositions of the present invention. The sense and antisense strands included in the dsRNA of the present invention are independently selected. As used herein, the term "independently selected" means that each of two or more similar elements can be selected independently of the selection of the other elements. For example, although not intended to be limiting, when preparing the dsRNA of the present invention, "elements" of both strands can be selected for inclusion in the duplex. For example, for one selected element, the sense sequence can be SEQ ID NO: 427 (shown in Table 2), and for another selected element, the antisense sequence can be SEQ ID NO: 532, or can be a modified, shortened, or extended version of SEQ ID NO: 532 and/or contain one, two, or three substitutions compared to its parent sequence, SEQ ID NO: 427. It should be understood that the duplexes of the present invention do not necessarily contain both the sense and antisense sequences shown in the duplex pairs in Tables 1 to 3. Each sense and antisense sequence in these tables is followed by its SEQ ID NO.
本發明的組合物和方法的某些實施方案包括組合物中的和/或施用於受試者的單鏈RNA。例如，反義鏈諸如表1至表3中的任一個表中列出的反義鏈可以是組合物或施用於受試者以降低該受試者的TMPRSS6多肽活性和/或TMPRSS6基因表達的組合物。表1示出了某些TMPRSS6 dsRNA藥劑反義鏈和有義鏈核心延伸鹼基序列。可包含在某些組合物中和/或在本發明的某些方法中施用的單鏈反義分子在本文中稱為“單鏈反義藥劑”或“反義多核苷酸藥劑”。可包含在某些組合物中和/或在本發明的某些方法中施用的單鏈有義分子在本文中稱為“單鏈有義藥劑”或“有義多核苷酸藥劑”。術語“鹼基序列”在本文中用於指沒有化學修飾或遞送化合物的多核苷酸序列。例如，示於表1中的有義鏈GGCUACUCUGGUAUUUCCUAA（SEQ ID NO: 12）是表2中的SEQ ID NO: 432和表3中的SEQ ID NO: 639的鹼基序列，其中SEQ ID NO: 432和SEQ ID NO: 639示出了它們的化學修飾和遞送化合物。本文公開的序列可被分配標識符。例如，單鏈有義序列可用“有義鏈SS#”標識；單鏈反義序列可用“反義鏈AS#”標識，並且包含有義鏈和反義鏈的雙鏈體可用“雙鏈體AD#/AV#”標識。Certain embodiments of the compositions and methods of the present invention include single-stranded RNA in the compositions and/or administered to a subject. For example, an antisense strand such as those listed in any of Tables 1 to 3 can be a composition or a composition administered to a subject to reduce TMPRSS6 polypeptide activity and/or TMPRSS6 gene expression in the subject. Table 1 shows the core extension base sequences of certain TMPRSS6 dsRNA agent antisense strands and sense strands. Single-stranded antisense molecules that can be included in certain compositions and/or administered in certain methods of the present invention are referred to herein as "single-stranded antisense agents" or "antisense polynucleotide agents." Single-stranded sense molecules that can be included in certain compositions and/or administered in certain methods of the present invention are referred to herein as "single-stranded sense agents" or "sense polynucleotide agents." The term "base sequence" is used herein to refer to a polynucleotide sequence without chemical modifications or delivery compounds. For example, the sense strand GGCUACUCUGGUAUUUCCUAA (SEQ ID NO: 12) shown in Table 1 is the base sequence of SEQ ID NO: 432 in Table 2 and SEQ ID NO: 639 in Table 3, where SEQ ID NO: 432 and SEQ ID NO: 639 are shown with their chemical modifications and delivery compounds. Sequences disclosed herein may be assigned identifiers. For example, a single-stranded sense sequence may be identified by "sense strand SS#"; a single-stranded antisense sequence may be identified by "antisense strand AS#"; and a duplex comprising the sense and antisense strands may be identified by "duplex AD#/AV#."
表1包括有義鏈和反義鏈，並提供了由表1中同一條線上的有義鏈和反義鏈形成的雙鏈體的標識編號。在本發明的某些實施方案中，反義序列包含在反義序列的位置1中的核鹼基U或核鹼基A。在本發明的某些實施方案中，反義序列包含在反義序列的位置1中的核鹼基U。如本文所用，有義鏈和反義鏈中的術語“匹配位置”是當兩條鏈為雙鏈時每條鏈中“配對”的位置。例如，在含有21個核鹼基的有義鏈和含有21個核鹼基的反義鏈中，有義鏈的位置1中的核鹼基和反義鏈的位置21中的核鹼基處於“匹配位置”。在又一個非限制性示例中，在含有23個核鹼基的有義鏈和含有23個核鹼基的反義鏈中，有義鏈的核鹼基2和反義鏈的位置22處於匹配位置。在另一個非限制性示例中，在含有18個核鹼基的有義鏈和含有18個核鹼基的反義鏈中，有義鏈的位置1中的核鹼基和反義鏈中的核鹼基18處於匹配位置，並且有義鏈中的核鹼基4和反義鏈中的核鹼基15位於匹配位置。本領域技術人員應當理解如何標識是或者將是雙鏈和配對鏈的有義鏈和反義鏈中的匹配位置。Table 1 includes the sense and antisense chains and provides identification numbers for duplexes formed by the sense and antisense chains on the same line in Table 1. In certain embodiments of the present invention, the antisense sequence comprises a nucleobase U or a nucleobase A at position 1 of the antisense sequence. In certain embodiments of the present invention, the antisense sequence comprises a nucleobase U at position 1 of the antisense sequence. As used herein, the term "matching position" in the sense and antisense chains refers to the position in each chain that "pairs" when the two chains are a duplex. For example, in a sense chain containing 21 nucleobases and an antisense chain containing 21 nucleobases, the nucleobase at position 1 of the sense chain and the nucleobase at position 21 of the antisense chain are in "matching positions." In yet another non-limiting example, in a sense chain containing 23 nucleobases and an antisense chain containing 23 nucleobases, nucleobase 2 of the sense chain and position 22 of the antisense chain are in matching positions. In another non-limiting example, in a sense chain containing 18 nucleobases and an antisense chain containing 18 nucleobases, the nucleobase in position 1 of the sense chain and nucleobase 18 in the antisense chain are in matching positions, and nucleobase 4 in the sense chain and nucleobase 15 in the antisense chain are in matching positions. One skilled in the art will understand how to identify matching positions in a sense chain and an antisense chain that are or will be duplexes and paired chains.
表1中的第一列表示雙鏈體AV#，即包含同一表行中的有義序列和反義序列的雙鏈體。例如，表1公開了分配雙鏈體的雙鏈體AV# AV01369.um，其包含有義鏈SEQ ID NO: 7和反義鏈SEQ ID NO: 217。因此，表1中的每一行標識本發明的雙鏈體，每個雙鏈體包含同一行所示的有義序列和反義序列，其中每個雙鏈體的所分配標識符示於該行的第一列中。The first column in Table 1 represents duplex AV#, i.e., a duplex comprising the sense and antisense sequences in the same row. For example, Table 1 discloses the assigned duplex AV# AV01369.um, which comprises the sense strand SEQ ID NO: 7 and the antisense strand SEQ ID NO: 217. Thus, each row in Table 1 identifies a duplex of the present invention, each duplex comprising the sense and antisense sequences shown in the same row, wherein the assigned identifier for each duplex is shown in the first column of that row.
在本發明的方法的一些實施方案中，將包含表1至表3中的任一個表所示的多核苷酸序列的RNAi藥劑施用於受試者。在本發明的一些實施方案中，施用於受試者的RNAi藥劑包括雙鏈體，該雙鏈體包含表1中示出的鹼基序列中的至少一者，包括0、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24個序列修飾。在本發明的方法的一些實施方案中，將包含表1至表3中的任一個表所示的多核苷酸序列的RNAi藥劑與遞送分子連接，該遞送分子的非限制性示例為包括GalNAc化合物或GLS-15*化合物的遞送化合物。In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in any one of Tables 1 to 3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to the subject comprises a duplex comprising at least one of the base sequences shown in Table 1, including 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 sequence modifications. In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in any one of Tables 1 to 3 is linked to a delivery molecule, non-limiting examples of which include a GalNAc compound or a GLS-15* compound.
表1：未修飾的TMPRSS6 RNAi藥劑反義鏈和有義鏈序列。所有序列均以5'至3'方向示出。雙鏈體AV#是分配給該表的同一行中的兩條鏈的雙鏈體的編號。
表2示出了本發明的某些經化學修飾的TMPRSS6 RNAi藥劑反義鏈序列和有義鏈序列。在本發明的方法的一些實施方案中，將含有表2所示的多核苷酸序列的RNAi藥劑施用於細胞和/或受試者。在本發明的方法的一些實施方案中，將含有表2所示的多核苷酸序列的RNAi藥劑施用於受試者。在本發明的一些實施方案中，施用於受試者的RNAi藥劑包含表2第一列的某一行中標識的雙鏈體，並且包含表2第三列和第六列的同一行中的有義鏈序列和反義鏈序列分別所示的序列修飾。在本發明的方法的一些實施方案中，表2所示的序列可與能夠將RNAi藥劑遞送至受試者體內的細胞和/或組織的化合物連接（本文也稱為“綴合”）。可用於本發明的某些實施方案的遞送化合物的非限制性示例是含GalNAc的化合物或含GLS-15*的化合物。在表2中，第一列表示含有如表1所示的鹼基序列的雙鏈體AV#。表2公開了雙鏈體AV#，並且還示出了包含在雙鏈體的有義序列和反義序列中的化學修飾。例如，表1示出了鹼基單鏈序列SEQ ID NO: 12（有義鏈）和SEQ ID NO: 222（反義鏈），它們一起構成被標識為雙鏈體AV# AV01374.um的雙鏈體，並且表2列出了Duplex AV# AV01374，其表明SEQ ID NO: 432和SEQ ID NO: 537的雙鏈體分別包含SEQ ID NO: 12和SEQ ID NO: 222的鹼基序列，但含有如第三列所示的有義序列和第六列所示的反義序列分別所示的化學修飾。表2第二列中的“有義鏈SS#”是同一行中第3列所示的有義序列（包括修飾）的所分配的標識符。表2第五列中的“反義鏈AS#”是第六列所示的反義序列（包括修飾）的所分配的標識符。Table 2 shows the antisense and sense strand sequences of certain chemically modified TMPRSS6 RNAi agents of the present invention. In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in Table 2 is administered to a cell and/or a subject. In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in Table 2 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to the subject comprises the duplex identified in a row of the first column of Table 2 and comprises the sequence modifications shown in the sense and antisense strand sequences in the same row of the third and sixth columns of Table 2, respectively. In some embodiments of the methods of the present invention, the sequences shown in Table 2 can be linked (also referred to herein as "conjugated") to a compound capable of delivering an RNAi agent to cells and/or tissues within a subject. Non-limiting examples of delivery compounds useful in certain embodiments of the present invention are GalNAc-containing compounds or GLS-15*-containing compounds. In Table 2, the first column represents duplex AV# containing the base sequence shown in Table 1. Table 2 discloses duplex AV# and also shows the chemical modifications contained in the sense and antisense sequences of the duplex. For example, Table 1 shows the base single strand sequences of SEQ ID NO: 12 (sense strand) and SEQ ID NO: 222 (antisense strand), which together form a duplex identified as duplex AV# AV01374.um, and Table 2 lists Duplex AV# AV01374, which indicates that the duplexes of SEQ ID NO: 432 and SEQ ID NO: 537 comprise the base sequences of SEQ ID NO: 12 and SEQ ID NO: 222, respectively, but contain the chemical modifications indicated in the sense sequence shown in the third column and the antisense sequence shown in the sixth column, respectively. "Sense Strand SS#" in the second column of Table 2 is the assigned identifier for the sense sequence (including modifications) shown in the third column of the same row. "Antisense chain AS#" in the fifth column of Table 2 is the assigned identifier of the antisense sequence (including modifications) shown in the sixth column.
表2：提供了經化學修飾的TMPRSS6 RNAi藥劑反義鏈序列和有義鏈序列。所有序列均以5'至3'示出。這些序列用於本文所述的某些體外測試研究。
表3示出了本發明的某些經化學修飾的TMPRSS6 RNAi藥劑反義鏈序列和有義鏈序列。在本發明的方法的一些實施方案中，將表3所示的RNAi藥劑施用於細胞和/或受試者。在本發明的方法的一些實施方案中，將含有表3所示的多核苷酸序列的RNAi藥劑施用於受試者。在本發明的一些實施方案中，施用於受試者的RNAi藥劑包含表3第一列的某一行中標識的雙鏈體，並且包含表3第三列和第六列的同一行中的有義鏈序列和反義鏈序列分別所示的序列修飾和/或遞送化合物。這些序列用於本文別處所述的某些體內測試研究。在本發明的方法的一些實施方案中，表3所示的序列可與遞送化合物連接（本文也稱為“綴合”），該遞送化合物的非限制性示例是含GalNAc的化合物，其中遞送化合物在表3第三列中的有義鏈上被標識為“GLX-n”。如本文所用，“GLX-n”用於表示“GLS-n*”遞送化合物或“GLO-n”遞送化合物（“X”可以是“S”或“O”），並且GLX-0可以是可在合成期間與寡核苷酸的3'-端連接的“GLS-n*”遞送化合物和“GLO-n”遞送化合物中的任一者。如本文所用並且如表3所示，用於表示所連接的含GalNAc的化合物的“GLX-n”是化合物GLS-1*、GLS-2*、GLS-3*、GLS-4*、GLS-5*、GLS-6*、GLS-7*、GLS-8*、GLS-9*、GLS-10*、GLS-11*、GLS-12*、GLS-13*、GLS-14*、GLS-15*、GLS-16*、GLO-1、GLO-2、GLO-3、GLO-4、GLO-5、GLO-6、GLO-7、GLO-8、GLO-9、GLO-10、GLO-11、GLO-12、GLO-13、GLO-14、GLO-15和GLO-16的任一者，這些化合物中的每一者的結構均提供於本文別處。本領域技術人員能夠製備和使用本發明的dsRNA化合物，其中所連接的遞送化合物是GLS-1*、GLS-2*、GLS-3*、GLS-4*、GLS-5*、GLS-6*、GLS-7*、GLS-8*、GLS-9*、GLS-10*、GLS-11*、GLS-12*、GLS-13*、GLS-14*、GLS-15*、GLS-16*、GLO-1、GLO-2、GLO-3、GLO-4、GLO-5、GLO-6、GLO-7、GLO-8、GLO-9、GLO-10、GLO-11、GLO-12、GLO-13、GLO-14、GLO-15和GLO-16中的一者。表3的第一列提供了分配給該表的某一行中的有義序列和反義序列的雙鏈體的雙鏈體AD#。例如，雙鏈體AD# AD00874是有義鏈SEQ ID NO: 637和反義鏈SEQ ID NO: 677的雙鏈體。表3中的每一行提供了有義鏈和反義鏈，並公開了含有所示有義鏈和反義鏈的雙鏈體。表3第二列中的“有義鏈SS#”是同一行中第3列所示的有義序列（包括修飾）的所分配的標識符。表3第五列中的“反義鏈AS#”是第六列所示的反義序列（包括修飾）的所分配的標識符。某些連接的含GalNAc的“GLO-n”或“GLS-n*”化合物的標識符以GLS-5*或GLS-15*的形式顯示。Table 3 shows the antisense and sense strand sequences of certain chemically modified TMPRSS6 RNAi agents of the present invention. In some embodiments of the methods of the present invention, the RNAi agents shown in Table 3 are administered to cells and/or subjects. In some embodiments of the methods of the present invention, an RNAi agent comprising a polynucleotide sequence shown in Table 3 is administered to a subject. In some embodiments of the present invention, the RNAi agent administered to a subject comprises a duplex identified in a row of the first column of Table 3 and comprises sequence modifications and/or delivery compounds as shown in the sense and antisense strand sequences in the third and sixth columns of Table 3, respectively, on the same row. These sequences were used in certain in vivo testing studies described elsewhere herein. In some embodiments of the methods of the present invention, the sequences shown in Table 3 can be linked (also referred to herein as "ligated") to a delivery compound, a non-limiting example of which is a GalNAc-containing compound, wherein the delivery compound is identified as "GLX-n" on the sense strand in the third column of Table 3. As used herein, "GLX-n" is used to refer to either a "GLS-n*" delivery compound or a "GLO-n" delivery compound ("X" can be "S" or "O"), and GLX-0 can be either a "GLS-n*" delivery compound or a "GLO-n" delivery compound that can be linked to the 3'-end of an oligonucleotide during synthesis. As used herein and as shown in Table 3, "GLX-n" used to represent the attached GalNAc-containing compound is any of compounds GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16, the structures of each of which are provided elsewhere herein. Those skilled in the art can prepare and use the dsRNA compounds of the present invention wherein the linked delivery compound is one of GLS-1*, GLS-2*, GLS-3*, GLS-4*, GLS-5*, GLS-6*, GLS-7*, GLS-8*, GLS-9*, GLS-10*, GLS-11*, GLS-12*, GLS-13*, GLS-14*, GLS-15*, GLS-16*, GLO-1, GLO-2, GLO-3, GLO-4, GLO-5, GLO-6, GLO-7, GLO-8, GLO-9, GLO-10, GLO-11, GLO-12, GLO-13, GLO-14, GLO-15, and GLO-16. The first column of Table 3 provides the duplex AD# assigned to the sense and antisense duplexes in a row of the table. For example, duplex AD# AD00874 is a duplex comprising the sense and antisense chains of SEQ ID NO: 637 and SEQ ID NO: 677. Each row in Table 3 provides a sense and antisense chain and discloses a duplex containing the indicated sense and antisense chains. "Sense Chain SS#" in the second column of Table 3 is the assigned identifier for the sense sequence (including modifications) shown in the third column of the same row. "Antisense Chain AS#" in the fifth column of Table 3 is the assigned identifier for the antisense sequence (including modifications) shown in the sixth column. The identifiers for certain linked GalNAc-containing "GLO-n" or "GLS-n*" compounds are shown as GLS-5* or GLS-15*.
表3提供了經化學修飾的TMPRSS6 RNAi藥劑反義鏈序列和有義鏈序列。所有序列均以5'至3'示出。這些序列用於本文別處所述的某些體內測試研究。用於體內研究的遞送分子在每條有義鏈的3'-端或5'-端處被表示為“GLO-n”或“GLS-n*”。
在本發明的某些實施方案中，dsRNA（本文也稱為“雙鏈體”）是表1至表3中的任一個表中公開的一種。表1至表3中的每一行公開了包含該表行中的有義鏈序列和反義鏈序列的雙鏈體。除了表1至表3中公開的雙鏈體之外，應當理解，在一些實施方案中，本發明的雙鏈體可包含表1至表3所示的有義序列和反義序列，這些有義序列和反義序列與表1至表3所示的序列相差零、一、二或三個核苷酸。因此，作為非限制性示例，在一些實施方案中，本發明的雙鏈體中的反義鏈可以是分別與SEQ ID NO: 687、688、689、690、691、692、693、694、695或696中的核苷酸含有零、一、兩或三個不同核苷酸的SEQ ID NO: 687、688、689、690、691、692、693、694、695或696。In certain embodiments of the present invention, the dsRNA (also referred to herein as a "duplex") is one disclosed in any one of Tables 1 to 3. Each row in Tables 1 to 3 discloses a duplex comprising the sense and antisense sequences in that row. In addition to the duplexes disclosed in Tables 1 to 3, it should be understood that in some embodiments, the duplexes of the present invention may comprise the sense and antisense sequences shown in Tables 1 to 3, where these sense and antisense sequences differ from the sequences shown in Tables 1 to 3 by zero, one, two, or three nucleotides. Thus, as a non-limiting example, in some embodiments, the antisense strand in the duplex of the present invention can be SEQ ID NO: 687, 688, 689, 690, 691, 692, 693, 694, 695, or 696, which contains zero, one, two, or three different nucleotides from the nucleotides in SEQ ID NO: 687, 688, 689, 690, 691, 692, 693, 694, 695, or 696, respectively.
應當理解，本發明的雙鏈體中的有義鏈序列和反義鏈序列可以是獨立選擇的。因此，本發明的dsRNA可包含表1至表3中的某一行中公開的雙鏈體的有義鏈和反義鏈。另選地，在本發明的dsRNA中，dsRNA中所選的有義鏈和反義鏈中的一者或兩者可包括表1至表3所示的序列，但有義序列和反義序列中的一者或兩者包含來自親本序列的1、2、3個或更多個核鹼基取代。在一些實施方案中，所選的序列可比它們的親本序列更長或更短。因此，包括在本發明中的dsRNA藥劑可以但不一定包含表1至表3中公開為雙鏈體的有義和反義對的精確序列。It should be understood that the sense and antisense sequences in the duplexes of the present invention can be independently selected. Therefore, the dsRNA of the present invention may comprise the sense and antisense sequences of the duplexes disclosed in a row of Tables 1 to 3. Alternatively, in the dsRNA of the present invention, one or both of the sense and antisense sequences selected in the dsRNA may include the sequences shown in Tables 1 to 3, but one or both of the sense and antisense sequences may include 1, 2, 3 or more nucleobase substitutions from the parent sequence. In some embodiments, the selected sequences may be longer or shorter than their parent sequences. Therefore, the dsRNA agents included in the present invention may, but do not necessarily, comprise the exact sequences of the sense and antisense pairs disclosed as duplexes in Tables 1 to 3.
在一些實施方案中，dsRNA藥劑包含有義鏈和反義鏈，該反義鏈中的核苷酸位置2至18包含與TMPRSS6 RNA轉錄本互補的區域，其中該互補區域包含與表1至表3中的任一個表中列出的反義序列之一相差0、1、2或3個核苷酸的至少15個連續核苷酸，並且任選地包含靶向配體。在一些情況下，與該TMPRSS6 RNA轉錄本互補的區域包含與表1至表3中的任一個表中列出的反義序列之一相差不超過3個核苷酸的至少15、16、17、18、或19個連續核苷酸。在本發明的dsRNA藥劑的一些實施方案中，dsRNA藥劑的反義鏈與SEQ ID NO: 1的任一靶區域至少基本上互補，並且提供在表1至表3中的任一個表中。在一些實施方案中，本發明的dsRNA藥劑的反義鏈與SEQ ID NO: 1的任一靶區域完全互補，並且提供在表1至表3中的任一個表中。在一些實施方案中，dsRNA藥劑包含表1至表3中的任一個表中示出的有義鏈序列，並且該有義鏈序列與該dsRNA藥劑中的反義鏈序列至少基本上互補。在其他實施方案中，本發明的dsRNA藥劑包含表1至表3中的任一個表中示出的有義鏈序列，並且該有義鏈序列與該dsRNA藥劑中的反義鏈序列完全互補。在一些情況下，本發明的dsRNA藥劑包含表1至表3中的任一個表中示出的反義鏈序列。本發明的dsRNA藥劑的一些實施方案包含表1至表3中的任一個表中公開為雙鏈體的有義序列和反義序列。如本文所述，應當理解本發明的雙鏈體中的有義鏈和反義鏈可以是獨立選擇的。錯配In some embodiments, the dsRNA agent comprises a sense strand and an antisense strand, wherein nucleotide positions 2 to 18 in the antisense strand comprise a region complementary to a TMPRSS6 RNA transcript, wherein the complementary region comprises at least 15 contiguous nucleotides that differ from one of the antisense sequences listed in any one of Tables 1 to 3 by 0, 1, 2, or 3 nucleotides, and optionally comprises a targeting ligand. In some cases, the region complementary to the TMPRSS6 RNA transcript comprises at least 15, 16, 17, 18, or 19 contiguous nucleotides that differ from one of the antisense sequences listed in any one of Tables 1 to 3 by no more than 3 nucleotides. In some embodiments of the dsRNA agents of the present invention, the antisense strand of the dsRNA agent is at least substantially complementary to any of the target regions of SEQ ID NO: 1 and is provided in any of Tables 1 to 3. In some embodiments, the antisense strand of the dsRNA agent of the present invention is fully complementary to any of the target regions of SEQ ID NO: 1 and is provided in any of Tables 1 to 3. In some embodiments, the dsRNA agent comprises a sense strand sequence shown in any of Tables 1 to 3, and the sense strand sequence is at least substantially complementary to the antisense strand sequence in the dsRNA agent. In other embodiments, the dsRNA agent of the present invention comprises a sense strand sequence shown in any one of Tables 1 to 3, and the sense strand sequence is completely complementary to the antisense strand sequence in the dsRNA agent. In some cases, the dsRNA agent of the present invention comprises an antisense strand sequence shown in any one of Tables 1 to 3. Some embodiments of the dsRNA agent of the present invention comprise a sense sequence and an antisense sequence disclosed as a duplex in any one of Tables 1 to 3. As described herein, it should be understood that the sense strand and the antisense strand in the duplex of the present invention can be independently selected.Mismatch
本領域技術人員已知，就dsRNA的功效而言，錯配是可被容忍的，尤其是錯配位於dsRNA的末端區域內時。某些錯配可被較好容忍，例如就功效而言，含有擺動鹼基對G:U和A:C的錯配可被較好容忍（Du等人，“一種針對所有單核苷酸錯配靶位點處的活性siRNA的沉默效應的系統性分析（A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.）”，Nucleic Acids Res.2005 Mar 21;33(5):1671-7. Doi: 10.1093/nar/gki312. Nucleic Acids Res. 2005;33(11):3698）。在本發明的方法和化合物的一些實施方案中，TMPRSS6 dsRNA藥劑可含有一個或多個與TMPRSS6靶序列的錯配。在一些實施方案中，本發明的TMPRSS6 dsRNA藥劑不包含錯配。在某些實施方案中，本發明的TMPRSS6 dsRNA藥劑包含不超過1個錯配。在一些實施方案中，本發明的TMPRSS6 dsRNA藥劑包含不超過2個錯配。在某些實施方案中，本發明的TMPRSS6 dsRNA藥劑包含不超過3個錯配。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑的反義鏈含有與TMPRSS6靶序列的不位於互補區域中心的錯配。在一些實施方案中，TMPRSS6 dsRNA藥劑的反義鏈包含位於互補區域的5'端或3'端中的一者或兩者的最後5、4、3、2或1個核苷酸內的1、2、3、4個或更多個錯配。本文所述的方法和/或本領域已知的方法可用於測定含有與TMPRSS6靶序列的錯配的TMPRSS6 dsRNA藥劑是否有效抑制TMPRSS6基因的表達。It is known to those skilled in the art that mismatches are tolerated for dsRNA efficacy, particularly when located in the terminal regions of the dsRNA. Certain mismatches are better tolerated, for example, mismatches containing dangling base pairs G:U and A:C are better tolerated for efficacy (Du et al., "A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites." Nucleic Acids Res. 2005 Mar 21;33(5):1671-7. Doi: 10.1093/nar/gki312. Nucleic Acids Res. 2005;33(11):3698). In some embodiments of the methods and compounds of the invention, a TMPRSS6 dsRNA agent may contain one or more mismatches with a TMPRSS6 target sequence. In some embodiments, a TMPRSS6 dsRNA agent of the invention contains no mismatches. In certain embodiments, a TMPRSS6 dsRNA agent of the invention contains no more than one mismatch. In some embodiments, a TMPRSS6 dsRNA agent of the invention contains no more than two mismatches. In certain embodiments, a TMPRSS6 dsRNA agent of the invention contains no more than three mismatches. In some embodiments of the invention, the antisense strand of the TMPRSS6 dsRNA agent contains a mismatch with a TMPRSS6 target sequence that is not located in the center of the complementary region. In some embodiments, the antisense strand of the TMPRSS6 dsRNA agent comprises 1, 2, 3, 4, or more mismatches within the last 5, 4, 3, 2, or 1 nucleotides of one or both of the 5' or 3' ends of the complementary region. The methods described herein and/or methods known in the art can be used to determine whether a TMPRSS6 dsRNA agent containing mismatches to a TMPRSS6 target sequence is effective in inhibiting expression of the TMPRSS6 gene.
互補Mutual Complementation
如本文所用，除非另有說明，否則術語“互補”在用於描述與第二核苷酸序列（例如，TMPRSS6 dsRNA藥劑反義鏈或單鏈反義多核苷酸）相關的第一核苷酸序列（例如，TMPRSS6 dsRNA藥劑有義鏈或靶向TMPRSS6 mRNA）時，意指包含該第一核苷酸序列的寡核苷酸或多核苷酸在某些條件下與包含該第二核苷酸序列的寡核苷酸或多核苷酸雜交[在哺乳動物生理條件（或體外類似條件）下形成鹼基對氫鍵]並形成雙鏈體或雙螺旋結構的能力。可應用其他條件，諸如在生物體內部可能遇到的生理相關條件。技術人員將能夠根據所雜交的核苷酸的最終應用來確定最適合用於測試兩個序列的互補性的一組條件。互補序列包含沃森-克裡克鹼基對或非沃森-克裡克鹼基對，並且包含至少達到滿足上述雜交要求的程度的天然的或經修飾的核苷酸或核苷酸模擬物。序列同一性或互補性不依賴於修飾。As used herein, unless otherwise indicated, the term "complementary," when used to describe a first nucleotide sequence (e.g., a TMPRSS6 dsRNA agent sense strand or a single-stranded antisense polynucleotide) relative to a second nucleotide sequence (e.g., a TMPRSS6 dsRNA agent antisense strand or a single-stranded antisense polynucleotide), refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-to-hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a duplex or double helical structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Other conditions may apply, such as physiologically relevant conditions that may be encountered within an organism. A skilled artisan will be able to determine the most appropriate set of conditions for testing the complementarity of two sequences, depending on the ultimate application of the nucleotides being hybridized. Complementary sequences contain Watson-Crick base pairs or non-Watson-Crick base pairs and contain natural or modified nucleotides or nucleotide mimetics at least to the extent that the hybridization requirements described above are met. Sequence identity or complementarity is independent of modification.
例如，在本文所述的TMPRSS6 dsRNA內的互補序列包括包含第一核苷酸序列的寡核苷酸或多核苷酸與包含第二核苷酸序列的寡核苷酸或多核苷酸在一個或兩個核苷酸序列的整個長度上的鹼基配對。此類序列在本文中可被稱為相對於彼此“完全互補”。應當理解，在實施方案中，當兩個寡核苷酸被設計成在雜交時形成一個或多個單鏈突出端時，此類突出端在本文中不被視為關於確定互補性的錯配。例如，TMPRSS6 dsRNA藥劑包含一個長度為19個核苷酸的寡核苷酸和另一個長度為20個核苷酸的寡核苷酸，其中較長的寡核苷酸包含與較短的寡核苷酸完全互補的含有19個核苷酸的序列，為了本文所述的目的，仍可被稱為“完全互補”。因此，如本文所用，“完全互補”意指第一多核苷酸的連續序列中全部（100%）的鹼基將與第二多核苷酸的連續序列中相同數目的鹼基雜交。這些連續序列可包含第一核苷酸序列或第二核苷酸序列的全部或一部分。For example, complementary sequences within the TMPRSS6 dsRNA described herein include base pairing of an oligonucleotide or polynucleotide comprising a first nucleotide sequence with an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences may be referred to herein as "fully complementary" with respect to each other. It should be understood that, in embodiments, when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs are not considered mismatches for purposes of determining complementarity. For example, a TMPRSS6 dsRNA agent comprising one oligonucleotide of 19 nucleotides in length and another oligonucleotide of 20 nucleotides in length, where the longer oligonucleotide comprises a 19-nucleotide sequence that is fully complementary to the shorter oligonucleotide, may still be referred to as "fully complementary" for the purposes described herein. Thus, as used herein, "completely complementary" means that all (100%) of the bases in a contiguous sequence of a first polynucleotide will hybridize with the same number of bases in a contiguous sequence of a second polynucleotide. These contiguous sequences may comprise all or part of the first nucleotide sequence or the second nucleotide sequence.
如本文所用，術語“基本上互補”意指在核鹼基序列的雜交對中，第一多核苷酸的連續序列中至少約85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%但不是全部的鹼基將與第二多核苷酸的連續序列中相同數目的鹼基雜交。如果兩個序列在雜交時包含一個或多個（例如至少1、2、3、4或5個）錯配鹼基對，形成多達15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個鹼基對（bp）的雙鏈體，同時保留在與其最終應用最相關的條件下雜交的能力（例如，經由RISC途徑抑制TMPRSS6基因表達），則術語“基本上互補”可用於指第一序列相對於第二序列。As used herein, the term "substantially complementary" means that in a hybrid pair of nucleic acid base sequences, at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, but not all, of the bases in the contiguous sequence of a first polynucleotide will hybridize with the same number of bases in the contiguous sequence of a second polynucleotide. The term "substantially complementary" may be used to refer to a first sequence relative to a second sequence if the two sequences, upon hybridization, contain one or more (e.g., at least 1, 2, 3, 4, or 5) mismatched base pairs to form a duplex of up to 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs (bp) while retaining the ability to hybridize under conditions most relevant to their ultimate application (e.g., inhibition of TMPRSS6 gene expression via the RISC pathway).
術語“部分互補”在本文中可用於指核鹼基序列的雜交對，其中第一多核苷酸的連續序列中至少75%但不是全部的鹼基將與第二多核苷酸的連續序列中相同數目的鹼基雜交。在一些實施方案中，“部分互補”意指第一多核苷酸的連續序列中至少76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的鹼基將與第二多核苷酸的連續序列中相同數目的鹼基雜交。The term "partially complementary" may be used herein to refer to a hybridization of nucleic acid base sequences wherein at least 75%, but not all, of the bases in the contiguous sequence of a first polynucleotide will hybridize with the same number of bases in the contiguous sequence of a second polynucleotide. In some embodiments, "partially complementary" means that at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the bases in the contiguous sequence of the first polynucleotide will hybridize with the same number of bases in the contiguous sequence of the second polynucleotide.
本文所用的術語“互補”、“完全互補”、“基本上互補”和“部分互補”是指TMPRSS6 dsRNA藥劑的有義鏈和反義鏈之間、TMPRSS6 dsRNA藥劑的反義鏈和靶TMPRSS6 mRNA的序列之間、或單鏈反義寡核苷酸和靶TMPRSS6 mRNA的序列之間的鹼基匹配。應當理解，術語“TMPRSS6 dsRNA藥劑的反義鏈”可以指“TMPRSS6反義多核苷酸藥劑”的相同序列。As used herein, the terms "complementary," "completely complementary," "substantially complementary," and "partially complementary" refer to base matches between the sense and antisense strands of a TMPRSS6 dsRNA agent, between the antisense strand of a TMPRSS6 dsRNA agent and the sequence of a target TMPRSS6 mRNA, or between a single-stranded antisense oligonucleotide and the sequence of a target TMPRSS6 mRNA. It will be understood that the term "antisense strand of a TMPRSS6 dsRNA agent" can refer to the same sequence of a "TMPRSS6 antisense polynucleotide agent."
如本文所用，關於核酸序列所用的術語“基本上相同”或“基本同一性”意指包括與參考序列相比具有至少約85%序列同一性或更高序列同一性（優選至少90%、至少91%、至少92%、至少93%、至少94%、至少95%、至少96%、至少97%、至少98%或至少99%）的序列的核酸序列。通過在比較窗口上比較兩個最佳比對的序列來測定序列同一性的百分比。該百分比的計算方法是：測定兩個序列中出現相同核酸鹼基的位置數，得到匹配位置數，用匹配位置數除以比較窗口中的位置總數，然後將結果乘以100，得到序列同一性的百分比。本文公開的發明涵蓋與本文公開的那些核苷酸序列（例如，表1至表3中的核苷酸序列）基本上相同的核苷酸序列。在一些實施方案中，本文公開的序列與本文公開的那些序列（例如，表1至表3中的序列）完全相同，或者至少約85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%相同。As used herein, the term "substantially identical" or "substantial identity" with respect to nucleic acid sequences is intended to include nucleic acid sequences having at least about 85% sequence identity or greater (preferably at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) sequence identity compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions at which the identical nucleic acid base occurs in the two sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and then multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences that are substantially identical to those disclosed herein (e.g., the nucleotide sequences in Tables 1 to 3). In some embodiments, the sequences disclosed herein are identical, or at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to those disclosed herein (e.g., the sequences in Tables 1 to 3).
如本文所用，術語“包含序列的鏈”意指包含由使用標準核苷酸命名法提及的序列描述的核苷酸鏈的寡核苷酸。如本文所用，術語“雙鏈RNA”或“dsRNA”是指包括含有雜交雙鏈體區域的RNA分子或分子複合物的RNAi，該雜交雙鏈體區域包含兩條反向平行且基本上或完全互補的核酸鏈，這兩條核酸鏈相對於靶TMPRSS6 RNA被稱為具有“有義”和“反義”取向。雙鏈體區域可具有允許期望的靶TMPRSS6 RNA通過RISC途徑進行特異性降解的任何長度，但通常長度範圍為9至30個鹼基對，例如15至30個鹼基對。考慮到雙鏈體含有9至30個鹼基對，該雙鏈體可具有該範圍內的任何長度，例如9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30個鹼基對以及其中的任何子範圍，包括但不限於15至30個鹼基對、15至26個鹼基對、15至23個鹼基對、15至22個鹼基對、15至21個鹼基對、15至20個鹼基對、15至19個鹼基對、15至18個鹼基對、15至17個鹼基對、18至30個鹼基對、18至26個鹼基對、18至23個鹼基對、18至22個鹼基對、18至21個鹼基對、18至20個鹼基對、19至30個鹼基對、19至26個鹼基對、19至23個鹼基對、19至22個鹼基對、19至21個鹼基對、19至20個鹼基對、20至30個鹼基對、20至26個鹼基對、20至25個鹼基對、20至24個鹼基對、20至23個鹼基對、20至22個鹼基對、20至21個鹼基對、21至30個鹼基對、21至26個鹼基對、21至25個鹼基對、21至24個鹼基對、21至23個鹼基對、或21至22個鹼基對。在細胞中通過用Dicer和類似酶加工而產生的TMPRSS6 dsRNA藥劑的長度範圍通常為19至22個鹼基對。TMPRSS6 dsDNA藥劑的雙鏈體區域的一條鏈包含與靶TMPRSS6 RNA的區域基本上互補的序列。形成雙鏈體結構的兩條鏈可來自含有至少一個自身互補區域的單個RNA分子，或者可由兩個或更多個單獨的RNA分子形成。當雙鏈體區域由單個分子的兩條鏈形成時，該分子可在形成雙鏈體結構的一條鏈的3'-端和相應另一條鏈的5'-端之間含有由單鏈核苷酸鏈分隔的雙鏈體區域（本文中稱為“髮夾環”）。在本發明的一些實施方案中，髮夾環包含至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個或更多個未配對的核苷酸。當TMPRSS6 dsRNA藥劑的兩條基本上互補的鏈由單獨的RNA分子組成時，這些分子不一定但可以共價連接。當兩條鏈以非髮夾環的方式共價連接時，連接結構被稱為“接頭”。術語“siRNA”在本文中也用於指如本文所述的dsRNA藥劑。As used herein, the term "sequence-containing strand" refers to an oligonucleotide comprising a nucleotide strand described by a sequence referenced using standard nucleotide nomenclature. As used herein, the term "double-stranded RNA" or "dsRNA" refers to RNAi comprising an RNA molecule or complex of molecules containing a hybrid duplex region comprising two antiparallel and substantially or completely complementary nucleic acid strands, which are referred to as having "sense" and "antisense" orientations relative to the target TMPRSS6 RNA. The duplex region can be of any length that allows for specific degradation of the desired target TMPRSS6 RNA via the RISC pathway, but typically ranges from 9 to 30 base pairs in length, such as 15 to 30 base pairs. Considering that the duplex contains 9 to 30 base pairs, the duplex can have any length within this range, for example, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs and any subranges therein, including but not limited to 15 to 30 base pairs, 15 to 26 base pairs, 15 to 23 base pairs, 15 to 22 base pairs, 15 to 21 base pairs, 15 to 20 base pairs, 15 to 19 base pairs, 15 to 18 base pairs, 15 to 17 base pairs, 18 to 30 base pairs, 18 to 26 base pairs, 18 to 23 base pairs, 18 to 22 base pairs, 18 to 21 base pairs, 18 to 20 base pairs, 19 to 30 base pairs, 19 to 26 base pairs, 19 to 23 base pairs, 19 to 22 base pairs, 19 to 21 base pairs, 19 to 20 base pairs, 20 to 30 base pairs, 20 to 26 20 to 25 base pairs, 20 to 24 base pairs, 20 to 23 base pairs, 20 to 22 base pairs, 20 to 21 base pairs, 21 to 30 base pairs, 21 to 26 base pairs, 21 to 25 base pairs, 21 to 24 base pairs, 21 to 23 base pairs, or 21 to 22 base pairs. TMPRSS6 dsRNA agents produced in cells by processing with Dicer and similar enzymes typically have a length ranging from 19 to 22 base pairs. One strand of the duplex region of the TMPRSS6 dsDNA agent comprises a sequence that is substantially complementary to a region of the target TMPRSS6 RNA. The two chains forming the duplex structure can be derived from a single RNA molecule containing at least one self-complementary region, or can be formed from two or more separate RNA molecules. When the duplex region is formed from two chains of a single molecule, the molecule may contain a duplex region separated by a single nucleotide strand between the 3'-end of one chain forming the duplex structure and the 5'-end of the corresponding other chain (referred to herein as a "hairpin"). In some embodiments of the present invention, the hairpin comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more unpaired nucleotides. When the two substantially complementary strands of a TMPRSS6 dsRNA agent are composed of separate RNA molecules, these molecules are not necessarily but may be covalently linked. When the two strands are covalently linked in a non-hairpin manner, the linked structure is referred to as a "linker." The term "siRNA" is also used herein to refer to dsRNA agents as described herein.
在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑可包含在dsRNA藥劑的一個或兩個末端處沒有未配對核苷酸或核苷酸類似物的有義序列和反義序列。沒有未配對核苷酸的一端被稱為“平端”並且沒有核苷酸突出端。如果dsRNA藥劑的兩端都是平的，則dsRNA被稱為“平端的”。在本發明的一些實施方案中，dsRNA藥劑的第一端是平的，在一些實施方案中，dsRNA藥劑的第二端是平的，並且在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑的兩端都是平的。In some embodiments of the invention, a TMPRSS6 dsRNA agent may comprise a sense sequence and an antisense sequence that have no unpaired nucleotides or nucleotide analogs at one or both ends of the dsRNA agent. An end without unpaired nucleotides is referred to as "blunt-ended" and has no nucleotide overhangs. If both ends of a dsRNA agent are blunt, the dsRNA is referred to as "blunt-ended." In some embodiments of the invention, the first end of the dsRNA agent is blunt, in some embodiments, the second end of the dsRNA agent is blunt, and in certain embodiments of the invention, both ends of the TMPRSS6 dsRNA agent are blunt.
在本發明的dsRNA藥劑的一些實施方案中，dsRNA不含一個或兩個平端。在此類情況下，在dsRNA藥劑的鏈的末端處存在至少一個未配對的核苷酸。例如，當dsRNA的一條鏈的3'-端延伸超過另一條鏈的5'-端時（或者反之亦然），存在核苷酸突出端。dsRNA可包含含有至少1、2、3、4、5、6個或更多個核苷酸的突出端。核苷酸突出端可包含核苷酸/核苷類似物（包括脫氧核苷酸/核苷）或由其組成。應當理解，在一些實施方案中，核苷酸突出端在dsRNA藥劑的有義鏈上，在dsRNA藥劑的反義鏈上，或者在dsRNA藥劑的兩端上，並且突出端的核苷酸可存在於dsRNA的反義鏈或有義鏈的5'端、3'端或兩端上。在本發明的某些實施方案中，突出端中的一個或多個核苷酸被核苷硫代磷酸酯替換。In some embodiments of the dsRNA agents of the present invention, the dsRNA does not contain one or two blunt ends. In such cases, at least one unpaired nucleotide is present at the terminus of the strand of the dsRNA agent. For example, a nucleotide overhang is present when the 3'-end of one strand of the dsRNA extends beyond the 5'-end of the other strand (or vice versa). The dsRNA may comprise an overhang comprising at least 1, 2, 3, 4, 5, 6, or more nucleotides. The nucleotide overhang may comprise or consist of nucleotide/nucleoside analogs (including deoxynucleotides/nucleosides). It should be understood that in some embodiments, the nucleotide overhang is on the sense strand of the dsRNA agent, on the antisense strand of the dsRNA agent, or on both ends of the dsRNA agent, and the nucleotides of the overhang can be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the dsRNA. In certain embodiments of the invention, one or more nucleotides in the overhang are replaced by nucleoside phosphorothioates.
如本文所用，術語“反義鏈”或“引導鏈”是指TMPRSS6 dsRNA藥劑的包含與TMPRSS6靶序列基本上互補的區域的鏈。如本文所用，術語“有義鏈”或“過客鏈”指TMPRSS6 dsRNA藥劑的包含與TMPRSS6 dsRNA藥劑的反義鏈的區域基本上互補的區域的鏈。As used herein, the term "antisense strand" or "guide strand" refers to the strand of a TMPRSS6 dsRNA agent that comprises a region that is substantially complementary to the TMPRSS6 target sequence. As used herein, the term "sense strand" or "passenger strand" refers to the strand of a TMPRSS6 dsRNA agent that comprises a region that is substantially complementary to a region of the antisense strand of the TMPRSS6 dsRNA agent.
修飾Renovation
在本發明的一些實施方案中，TMPRSS6 RNAi藥劑的RNA經化學修飾以增強穩定性和/或一種或多種其他有益特徵。本發明的某些實施方案中的核酸可通過本領域熟知的方法進行合成和/或修飾，例如，描述於“核酸化學實驗室指南（Current protocols in Nucleic Acid Chemistry）”，Beaucage, S. L.等人（編輯），John Wiley & Sons, Inc., New York, N.Y., USA，該文獻以引用方式併入本文。可存在於本發明的TMPRSS6 dsRNA藥劑的某些實施方案中的修飾包括，例如，(a)末端修飾，例如5'端修飾（磷酸化、綴合、反向連接等）、3'端修飾（綴合、DNA核苷酸、反向連接等），(b)鹼基修飾，例如用穩定的鹼基、去穩定的鹼基或與擴展的配體庫鹼基配對的鹼基進行置換，鹼基（無鹼基核苷酸）或綴合鹼基的去除，(c)糖修飾（例如，在2'位置或4'位置處）或糖的置換，以及(d)主鏈修飾，包括磷酸二酯鍵的修飾或替換。可用於本發明的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和TMPRSS6有義多核苷酸的某些實施方案中的RNA化合物的具體示例，包括但不限於包含經修飾的主鏈或沒有天然核苷間鍵的RNA。作為非限制性示例，含有經修飾的主鏈的RNA可在該主鏈中不含磷原子。在其核苷間主鏈中不含磷原子的RNA可被稱為寡核苷。在本發明的某些實施方案中，經修飾的RNA在其核苷間主鏈中含有磷原子。In some embodiments of the present invention, the RNA of the TMPRSS6 RNAi agent is chemically modified to enhance stability and/or one or more other beneficial properties. The nucleic acids used in certain embodiments of the present invention can be synthesized and/or modified by methods well known in the art, such as those described in "Current Protocols in Nucleic Acid Chemistry," Beaucage, S.L. et al. (eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is incorporated herein by reference. Modifications that may be present in certain embodiments of the TMPRSS6 dsRNA agents of the invention include, for example, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation, conjugation, inverted ligation, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, inverted ligation, etc.), (b) base modifications, such as replacement with a stable base, a destabilized base, or a base that pairs with a base of an expanded ligand library, removal of a base (abasic nucleotides) or a conjugated base, (c) sugar modifications (e.g., at the 2' position or the 4' position) or replacement of sugars, and (d) backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNA compounds useful in certain embodiments of the TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and TMPRSS6 sense polynucleotides of the present invention include, but are not limited to, RNAs comprising modified backbones or lacking natural internucleoside linkages. As a non-limiting example, RNAs comprising modified backbones may not contain a phosphorus atom in the backbone. RNAs that do not contain a phosphorus atom in their internucleoside backbones may be referred to as oligonucleosides. In certain embodiments of the present invention, modified RNAs contain a phosphorus atom in their internucleoside backbones.
應當理解，術語“RNA分子”或“RNA”或“核糖核酸分子”不僅涵蓋如在自然界中表達或發現的RNA分子，而且還涵蓋如本文所述或如本領域已知的包含一種或多種核糖核苷酸/核糖核苷類似物或衍生物的RNA類似物和衍生物。術語“核糖核苷”和“核糖核苷酸”在本文中可互換使用。RNA分子可在核鹼基結構或核糖-磷酸酯主鏈結構中進行修飾，例如，如本文下文所述，並且包含核糖核苷類似物或衍生物的分子必須保留形成雙鏈體的能力。作為非限制性示例，RNA分子還可包含至少一個經修飾的核糖核苷，包括但不限於經2'-O-甲基修飾的核苷、包含5'硫代磷酸酯基團的核苷、與膽甾醇基衍生物或十二烷酸雙癸醯胺基團連接的末端核苷、鎖核苷、無鹼基核苷、經2'-脫氧-2'-氟修飾的核苷、經2'-氨基修飾的核苷、經2'-烷基修飾的核苷、嗎啉基核苷、包含氨基磷酸酯或非天然鹼基的核苷或它們的任何組合。在本發明的一些實施方案中，RNA分子包含至少1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個或至多全長的TMPRSS6 dsRNA藥劑分子的核糖核苷，其為經修飾的核糖核苷。對於RNA分子中的此類多個經修飾的核糖核苷中的每一者，修飾不一定是相同的。It should be understood that the terms "RNA molecule," "RNA," or "ribonucleic acid molecule" encompass not only RNA molecules as expressed or found in nature, but also encompass RNA analogs and derivatives comprising one or more ribonucleotide/ribonucleoside analogs or derivatives, as described herein or as known in the art. The terms "ribonucleoside" and "ribonucleotide" are used interchangeably herein. RNA molecules can be modified in the nucleobase structure or the ribose-phosphate backbone structure, for example, as described herein below, and molecules comprising ribonucleoside analogs or derivatives must retain the ability to form duplexes. By way of non-limiting example, the RNA molecule may further comprise at least one modified ribonucleoside, including but not limited to 2'-O-methyl modified nucleosides, nucleosides comprising a 5' phosphorothioate group, terminal nucleosides linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group, lock nucleosides, abasic nucleosides, 2'-deoxy-2'-fluoro modified nucleosides, 2'-amino modified nucleosides, 2'-alkyl modified nucleosides, morpholinyl nucleosides, nucleosides comprising phosphoramidates or unnatural bases, or any combination thereof. In some embodiments of the invention, the RNA molecule comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or up to the full length of the TMPRSS6 dsRNA agent molecule, which are modified ribonucleosides. The modification need not be the same for each of these multiple modified ribonucleosides in the RNA molecule.
在一些實施方案中，本發明的DsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸可包含一個或多個獨立選擇的經修飾的核苷酸和/或一個或多個獨立選擇的非磷酸二酯鍵。如本文所用，術語“核苷酸間鍵”、“核苷間鍵”、“主鏈鍵”和“接頭”可互換使用，並且是指本發明的dsRNA主鏈中的連接基團，其可特異性地表示未修飾或經修飾的核苷之間的鍵，和/或未修飾或經修飾的核苷與一個或多個殘基之間的鍵，和/或未修飾或經修飾的核苷與寡核苷酸鏈中的一個或多個靶向基團之間的鍵。在一些實施方案中，該鍵可獨立地選自單鏈或雙鏈寡核苷酸的任何位置處的二核苷酸的磷酸二酯（PO）鍵、硫代磷酸酯（PS）鍵和/或二硫代磷酸酯（PS2）鍵。如本文所用，術語“獨立選擇”用於指所選的元件，諸如經修飾的核苷酸、非磷酸二酯鍵等，意指兩個或更多個所選的元件可以但不一定彼此相同。In some embodiments, the dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention may comprise one or more independently selected modified nucleotides and/or one or more independently selected non-phosphodiester bonds. As used herein, the terms "internucleotide bond," "internucleoside bond," "backbone bond," and "linker" are used interchangeably and refer to linking groups in the dsRNA backbone of the present invention, which may specifically refer to bonds between unmodified or modified nucleosides, and/or bonds between an unmodified or modified nucleoside and one or more residues, and/or bonds between an unmodified or modified nucleoside and one or more targeting groups in the oligonucleotide chain. In some embodiments, the bond can be independently selected from phosphodiester (PO) bonds, phosphorothioate (PS) bonds, and/or phosphorodithioate (PS2) bonds of dinucleotides at any position in a single-stranded or double-stranded oligonucleotide. As used herein, the term "independently selected" is used to refer to selected elements, such as modified nucleotides, non-phosphodiester bonds, etc., meaning that two or more selected elements can, but need not, be identical to each other.
如本文所用，“核苷酸鹼基”、“核苷酸”或“核鹼基”是雜環嘧啶或嘌呤化合物，是所有核酸的標準組成部分，並且包括形成核苷酸腺嘌呤、鳥嘌呤、胞嘧啶、胸腺嘧啶和尿嘧啶的鹼基。核鹼基可進一步被修飾以包括（儘管並非旨在進行限制）：通用鹼基、疏水性鹼基、混雜鹼基、尺寸擴大的鹼基和氟化鹼基。術語“核糖核苷酸”或“核苷酸”可在本文中用於指未修飾的核苷酸、經修飾的核苷酸、核苷酸類似物或替代替換部分。本領域技術人員將認識到鳥嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可被其他部分替換，而基本上不改變包含帶有此類置換部分的核苷酸的寡核苷酸的鹼基配對特性。As used herein, "nucleobase," "nucleotide," or "nucleobase" is a heterocyclic pyrimidine or purine compound that is a standard component of all nucleic acids and includes the bases that form the nucleotides adenine, guanine, cytosine, thymine, and uracil. Nucleobases can be further modified to include (although not intended to be limiting): universal bases, hydrophobic bases, mixed bases, size-expanded bases, and fluorinated bases. The term "ribonucleotide" or "nucleotide" may be used herein to refer to unmodified nucleotides, modified nucleotides, nucleotide analogs, or alternative substitution moieties. Those skilled in the art will recognize that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such a replacement moiety.
如本文所用，“任選地”或“任選的”意指後面描述的事件或環境可以但不一定發生，包括該事件或環境發生或不發生的情況。例如，“C1-6烷基任選地被鹵素或氰基取代”意指鹵素或氰基可以但不一定存在，包括烷基被鹵素或氰基取代的情況以及烷基未被鹵素和氰基取代的情況。As used herein, "optionally" or "optional" means that the event or circumstances described later may but need not occur, including cases where the event or circumstances occur or do not occur. For example, "C1-6 alkyl is optionally substituted with a halogen or a cyano group" means that a halogen or a cyano group may but need not be present, including cases where the alkyl group is substituted with a halogen or a cyano group and cases where the alkyl group is not substituted with a halogen or a cyano group.
如本文所用，在本發明內容的化合物的化學結構中，鍵表示未指定的構型，即，如果該化學結構中存在手性異構體，則鍵可以是“”或“”或者“”和“”兩種構型。儘管為了簡單起見，上述一些結構式被描述為一些異構形式，但本發明內容可包括所有異構體，諸如互變異構體、旋轉異構體以及它們的混合物。合適的手性化合物包括：幾何異構體、非對映異構體、外消旋體和對映異構體。As used herein, in the chemical structures of the compounds of the present invention, the bond Indicates an unspecified configuration, i.e., if chiral isomers exist in the chemical structure, the bond It can be " "or" "or" "and" "Two configurations. Although some of the above structural formulas are described as some isomeric forms for simplicity, the present invention may include all isomers, such as tautomers, rotational isomers, and mixtures thereof. Suitable chiral compounds include: geometric isomers, diastereomers, racemates, and enantiomers.
如本文所用，本發明內容的化學式中使用的“”或“”可根據本文所述的發明範圍與任一個或多個基團連接。As used herein, the chemical formulas used in the present invention are “ "or" " can be linked to any one or more groups according to the scope of the invention described herein.
在一個實施方案中，考慮了用於本文所述的方法和組合物的經修飾的RNA是肽核酸（PNA），其具有形成期望的雙鏈體結構的能力並且允許或介導經由RISC途徑特異性降解靶RNA。在本發明的某些實施方案中，TMPRSS6 RNA干擾劑包含與靶TMPRSS6 RNA序列相互作用以指導靶TMPRSS6 RNA的切割的單鏈RNA。In one embodiment, the modified RNA contemplated for use in the methods and compositions described herein is a peptide nucleic acid (PNA), which has the ability to form a desired duplex structure and allows or mediates the specific degradation of the target RNA via the RISC pathway. In certain embodiments of the invention, the TMPRSS6 RNA interferor comprises a single-stranded RNA that interacts with a target TMPRSS6 RNA sequence to direct the cleavage of the target TMPRSS6 RNA.
經修飾的RNA主鏈可包括，例如，硫代磷酸酯、手性硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基磷酸三酯、甲基和其他烷基膦酸酯（包括3'-亞烷基膦酸酯和手性膦酸酯）、亞膦酸酯、氨基磷酸酯（包括3'-氨基磷酸酯和氨基烷基氨基磷酸酯）、硫代氨基磷酸酯、硫代烷基膦酸酯、硫代烷基磷酸三酯和含有正常3'-5'鍵的硼代磷酸酯、這些硼代磷酸酯的2'-5'連接的類似物，以及那些具有相反極性的化合物，其中相鄰的核苷單元對是3'-5'連接至5'-3'或者2'-5'連接至5'-2'。還包括各種鹽、混合鹽和游離酸形式。製備含磷鍵的方式是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑、某些經修飾的TMPRSS6反義多核苷酸和/或某些經修飾的TMPRSS6有義多核苷酸。Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-phosphoramidates and aminoalkylphosphoramidates), thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, and boronates containing normal 3'-5' linkages, 2'-5' linked analogs of these boronates, and those with opposite polarity, where adjacent nucleoside unit pairs are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts, and free acid forms are also included. Methods for preparing phospho-bond-containing proteins are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents, certain modified TMPRSS6 antisense polynucleotides, and/or certain modified TMPRSS6 sense polynucleotides of the present invention.
其中不包含磷原子的經修飾的RNA主鏈含有由短鏈烷基或環烷基核苷間鍵、混合雜原子和烷基或環烷基核苷間鍵、或一個或多個短鏈雜原子或雜環核苷間鍵形成的主鏈。這些主鏈包括含有嗎啉鍵（部分地由核苷的糖部分形成）的那些主鏈；矽氧烷主鏈；硫化物、亞碸和碸主鏈；甲醯基和硫代甲醯基主鏈；亞甲基甲醯基和硫代甲醯基主鏈；含烯烴的主鏈；氨基磺酸鹽主鏈；亞甲基亞氨基和亞甲基肼基主鏈；磺酸鹽和磺醯胺主鏈；醯胺主鏈；以及其他含有混合的N、O、S和CH2組成部分的主鏈。製備不包含磷原子的經修飾的RNA主鏈的方式是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑、某些經修飾的TMPRSS6反義多核苷酸和/或某些經修飾的TMPRSS6有義多核苷酸。The modified RNA backbone that does not contain a phosphorus atom contains a backbone formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatom or heterocyclic internucleoside linkages. These backbones include those containing morpholino bonds (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfone, and sulfonium backbones; formyl and thioformyl backbones; methyleneformyl and thioformyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and other backbones containing mixed N, O, S, andCH₂ moieties. Methods for preparing modified RNA backbones that do not contain phosphorus atoms are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents, certain modified TMPRSS6 antisense polynucleotides, and/or certain modified TMPRSS6 sense polynucleotides of the present invention.
在本發明的某些實施方案中，RNA模擬物被包含在TMPRSS6 dsRNA、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸中，諸如但不限於：用新的基團替換核苷酸單元的糖和核苷間鍵，即主鏈。在此類實施方案中，保持鹼基單位用於與合適的TMPRSS6核酸靶標化合物雜交。一種這樣的寡聚化合物，已被證明具有優異雜交特性的RNA模擬物，被稱為肽核酸（PNA）。在PNA化合物中，RNA的糖主鏈被含有醯胺的主鏈，特別是氨基乙基甘氨酸主鏈替換。核鹼基被保留並直接或間接地結合到主鏈的醯胺部分的氮雜氮原子。製備RNA模擬物的方式是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑。In certain embodiments of the present invention, RNA mimetics are included in TMPRSS6 dsRNA, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides, such as, but not limited to, by replacing the sugar and internucleoside linkages of the nucleotide units, i.e., the backbone, with novel moieties. In such embodiments, the basic units are retained for hybridization with appropriate TMPRSS6 nucleic acid target compounds. One such oligomeric compound, an RNA mimetic that has been demonstrated to possess excellent hybridization properties, is known as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of RNA is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly bound to nitrogen atoms of the amide portion of the backbone. Methods for preparing RNA mimetics are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents of the present invention.
本發明的一些實施方案包括含有硫代磷酸酯主鏈的RNA和含有雜原子主鏈的寡核苷，並且特別是-CH2-NH-CH2-、-CH2-N(CH3)-O-CH2-[已知為亞甲基(甲基亞氨基)或MMI主鏈]、-CH2-O-N(CH3)-CH2-、-CH2-N(CH3)-N(CH3)-CH2-和-N(CH3)-CH2-[其中天然磷酸二酯主鏈被表示為-O-P-O-CH2-]。製備含有硫代磷酸酯主鏈的RNA和含有雜原子主鏈的寡核苷的方式是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑、某些TMPRSS6反義多核苷酸和/或某些TMPRSS6有義多核苷酸。Some embodiments of the invention include RNAs containing phosphorothioate backbones and oligonucleosides containing heteroatom backbones, and particularly-CH2- NH-CH2- , -CH2-N (CH3 )-O-CH2- [known as the methylene(methylimino) or MMI backbone], -CH2-ON (CH3)-CH2- ,-CH2 -N(CH3 )-N(CH3 )-CH2- , and -N(CH3 )-CH2-[ where the native phosphodiester backbone is represented as -OPO-CH2- ]. Methods for preparing RNA containing phosphorothioate backbones and oligonucleotides containing heteroatom backbones are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents, certain TMPRSS6 antisense polynucleotides, and/or certain TMPRSS6 sense polynucleotides of the present invention.
經修飾的RNA還可包含一個或多個經取代的糖部分。本發明的TMPRSS6 dsRNA、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸可在2'位置處包含以下項中的一者：OH；F；O-、S-或N-烷基；O-、S-或N-烯基；O-、S-或N-炔基；或O-烷基-O-烷基，其中該烷基、烯基和炔基可以是經取代或未取代的C1至C10烷基或C2至C10烯基和炔基。示例性的合適修飾包括O[(CH2)nO]mCH3、O(CH2)nOCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2和O(CH2)nON[(CH2)nCH3)]2，其中n和m為1至約10。在其他實施方案中，dsRNA在2'位置處包括以下項中的一者：C1至C10低級烷基、經取代的低級烷基、烷芳基、芳烷基、O-烷芳基或O-芳烷基、SH、SCH3、OCN、Cl、Br、CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2、雜環烷基、雜環烷芳基、氨基烷基氨基、聚烷基氨基、經取代的甲矽烷基、RNA切割基團、報告基團、嵌入劑、用於改善TMPRSS6 dsRNA藥劑的藥代動力學特性的基團，或用於改善TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的藥效學特性的基團，以及具有類似特性的其他取代基。在一些實施方案中，修飾包括2'-甲氧基乙氧基（2'-O-CH2CH2OCH3，也稱為2'-O-(2-甲氧基乙基)或2'-MOE）（Martin等人，Helv.Chim.Acta, 1995, 78:486-504），即烷氧基-烷氧基基團。另一個示例性的修飾是2'-二甲基氨基氧基乙氧基，即O(CH2)2ON(CH3)2基團（也稱為2'-DMAOE，如本文下文的示例所述），和2'-二甲基氨基乙氧基乙氧基（本領域也稱為2'-O-二甲基氨基乙氧基乙基或2'-DMAEOE），即2'-O-CH2-O-CH2-N(CH2)2。製備經修飾的RNA的方式（諸如那些描述的方式）是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑。The modified RNA may also comprise one or more substituted sugar moieties. The TMPRSS6 dsRNA, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention may comprise one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl groups may be substituted or unsubstitutedC1 toC10 alkyl orC2 toC10 alkenyl and alkynyl groups. Exemplary suitable modifications include O[(CH2 )nO ]mCH3 , O(CH2 )nOCH3, O(CH2)nNH2 , O(CH2 )nCH3 , O(CH2 )nONH2, and O(CH2)nON[(CH2)nCH3)]2,wheren and m are 1 to about 10. In other embodiments, the dsRNA includes one of the following at the 2' position:C1 toC10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH,SCH3 , OCN, Cl, Br, CN, CF3,OCF3 ,SOCH3 ,SO2CH3 ,ONO2 ,NO2 ,N3 ,NH2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkylamino , substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of TMPRSS6 dsRNA agent, or group for improvingthe pharmacokinetic properties of TMPRSS6 The invention relates to groups that enhance the pharmacodynamic properties of dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides, as well as other substituents with similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy group (2' -O-CH2CH2OCH3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2 )2 ON(CH3 )2 group (also known as 2'-DMAOE, as described in the examples below), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-CH2 -O-CH2 -N(CH2 )2. Methods for preparing modified RNAs, such as those described, are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents of the invention.
其他修飾包括2'-甲氧基（2'-OCH3）、2'-氨基丙氧基（2'-OCH2CH2CH2NH2）和2'-氟（2'-F）。類似的修飾還可在本發明的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的RNA上的其他位置處進行，特別是在3'末端核苷酸上或在2'-5'連接的TMPRSS6 dsRNA、TMPRSS6反義多核苷酸或TMPRSS6有義多核苷酸中的糖的3'位置，以及5'末端核苷酸的5'位置。TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸還可含有糖模擬物，諸如用環丁基部分代替呋喃戊糖基糖。製備經修飾的RNA的方式（諸如那些描述的方式）是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸。Other modifications include 2'-methoxy (2'-OCH3 ), 2'-aminopropoxy (2'-OCH2 CH2 CH2 NH2 ), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA of the TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention, particularly at the 3' position of the sugar at the 3' terminal nucleotide or in 2'-5' linked TMPRSS6 dsRNAs, TMPRSS6 antisense polynucleotides, or TMPRSS6 sense polynucleotides, and at the 5' position of the 5' terminal nucleotide. TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides can also contain sugar mimetics, such as replacing the pentofuranosyl sugar with a cyclobutyl moiety. Methods for preparing modified RNA, such as those described, are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention.
在一些實施方案中，TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸可包含核鹼基（在本領域中經常簡稱為“鹼基”）修飾或取代。如本文所用，“未修飾的”或“天然的”核鹼基包括嘌呤鹼基腺嘌呤和鳥嘌呤，以及嘧啶鹼基胸腺嘧啶、胞嘧啶和尿嘧啶。經修飾的核鹼基包括其他合成的核鹼基和天然的核鹼基，諸如5-甲基胞嘧啶（5-Me-C），5-羥甲基胞嘧啶，黃嘌呤，次黃嘌呤，2-氨基腺嘌呤，腺嘌呤和鳥嘌呤的6-甲基和其他烷基衍生物，腺嘌呤和鳥嘌呤的2-丙基和其他烷基衍生物，2-硫代尿嘧啶，2-硫代胸腺嘧啶和2-硫代胞嘧啶，5-鹵代尿嘧啶和胞嘧啶，5-丙炔基尿嘧啶和胞嘧啶，6-偶氮尿嘧啶、胞嘧啶和胸腺嘧啶，5-尿嘧啶（假尿嘧啶），4-硫代尿嘧啶，8-鹵代、8-氨基、8-硫醇、8-硫代烷基、8-羥基和其他8-取代的腺嘌呤和鳥嘌呤，5-鹵代（特別是5-溴代）、5-三氟甲基和其他5-取代的尿嘧啶和胞嘧啶，7-甲基鳥嘌呤和7-甲基腺嘌呤，8-氮雜鳥嘌呤和8-氮雜腺嘌呤，7-脫氮鳥嘌呤和7-脫氮腺嘌呤，以及3-脫氮鳥嘌呤和3-脫氮腺嘌呤。可包含在本發明的TMPRSS6 dsRNA藥劑的某些實施方案中的其他核鹼基是本領域已知的，參見例如：“生物化學、生物技術和醫學中的經修飾的核苷（Modified Nucleosides in Biochemistry, Biotechnology and Medicine）”，Herdewijn, P.編輯Wiley-VCH, 2008；“聚合物科學與工程的簡明百科全書（The Concise Encyclopedia Of Polymer Science And Engineering）”，第858-859頁，Kroschwitz, J. L編輯，John Wiley & Sons, 1990；English等人，“應用化學，國際版（Angewandte Chemie, International Edition）”，1991, 30, 613；Sanghvi, Y S.，“dsRNA研究與應用（dsRNA Research and Applications）”，第15章第289-302頁，Crooke, S. T.和Lebleu, B.編輯，CRC Press, 1993。製備包含核鹼基修飾和/或取代（諸如本文所述的核鹼基修飾和/或取代）的dsRNA、TMPRSS6反義鏈多核苷酸和/或TMPRSS6有義鏈多核苷酸的方式是本領域常規實踐的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑、TMPRSS6有義多核苷酸和/或TMPRSS6反義多核苷酸。In some embodiments, TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides may comprise modifications or substitutions of nucleobases (often referred to in the art as simply "bases"). As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine and guanine, and the pyrimidine bases thymine, cytosine, and uracil. Modified nucleobases include other synthetic nucleobases and natural nucleobases such as 5-methylcytosine (5-Me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halogenated uracil and cytosine, 5-propynyl uracil and cytosine, 6-azouracil, cytosine, thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halogenated (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Other nucleoside groups that may be included in certain embodiments of the TMPRSS6 dsRNA agents of the present invention are known in the art, see, for example, "Modified Nucleosides in Biochemistry, Biotechnology and Medicine," Herdewijn, P. ed. Wiley-VCH, 2008; "The Concise Encyclopedia of Polymer Science And Engineering," pp. 858-859, Kroschwitz, J. L. ed., John Wiley & Sons, 1990; English et al., "Angewandte Chemie, International Edition," 1991, 30, 613; Sanghvi, Y S., "dsRNA Research and Applications," pp. 106-107. Crooke, S. T. and Lebleu, B., eds., CRC Press, 1993, Chapter 15, pp. 289-302. Methods for preparing dsRNA, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides comprising nucleobase modifications and/or substitutions, such as those described herein, are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents, TMPRSS6 sense polynucleotides, and/or TMPRSS6 antisense polynucleotides of the present invention.
本發明的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的某些實施方案包括經修飾以包含一個或多個鎖核酸（LNA）的RNA。鎖核酸是含有經修飾的核糖部分的核苷酸，該經修飾的核糖部分包含連接2'碳和4'碳的額外的橋。該結構有效地將核糖“鎖定”在3'-內結構構象中。在本發明的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸中添加鎖核酸可增加血清的穩定性，並降低脫靶效應（Elmen, J.等人，(2005) Nucleic Acids Research 33(1):439-447；Mook, O R.等人，(2007)Mol Canc Ther 6(3):833-843；Grunweller, A.等人，(2003) Nucleic Acids Research 31(12):3185-3193）。製備包含鎖核酸的dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的方式是本領域常規實施的，並且此類方法可用於製備本發明的某些經修飾的TMPRSS6 dsRNA藥劑。Certain embodiments of the TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention include RNA modified to include one or more locked nucleic acids (LNAs). LNAs are nucleotides containing a modified ribose moiety that includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-endo conformation. The addition of a locking nucleic acid to the TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the present invention can increase serum stability and reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Methods for preparing dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides containing a locking nucleic acid are routinely practiced in the art, and such methods can be used to prepare certain modified TMPRSS6 dsRNA agents of the present invention.
本發明的TMPRSS6 dsRNA化合物、有義多核苷酸和/或反義多核苷酸的某些實施方案包含至少一個經修飾的核苷酸，其中該至少一個經修飾的核苷酸包括：2'-O-甲基核苷酸、2'-氟核苷酸、2'-脫氧核苷酸、2'3'-開環核苷酸模擬物、鎖核苷酸、2'-F-阿拉伯糖核苷酸、2'-甲氧基乙基核苷酸、經2'-氨基修飾的核苷酸、經2'-烷基修飾的核苷酸、嗎啉基核苷酸和3'-OMe核苷酸，包含5'-硫代磷酸酯基團的核苷酸、包含乙烯基膦酸酯的核苷酸、包含腺苷-乙二醇核酸（GNA）的核苷酸、包含胸苷-乙二醇核酸（GNA）S-異構體的核苷酸、包含2-羥甲基-四氫呋喃-5-磷酸酯的核苷酸、包含2'-脫氧胸苷-3'磷酸酯的核苷酸、包含2'-脫氧鳥苷-3'-磷酸酯的核苷酸、或與膽甾醇基衍生物或十二烷酸雙癸醯胺基團連接的末端核苷酸、經2'-氨基修飾的核苷酸、包含氨基磷酸酯或非天然鹼基的核苷酸。在一些實施方案中，TMPRSS6 dsRNA化合物包含在反義鏈（在本文也稱為引導鏈）的5΄-端處的E-乙烯基膦酸酯核苷酸。Certain embodiments of the TMPRSS6 dsRNA compounds, sense polynucleotides, and/or antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxynucleotides, 2'3'-open nucleotide mimetics, locking nucleotides, 2'-F-arabinose nucleotides, 2'-methoxyethyl nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholinyl nucleotides, and 3'-OMe nucleotides, including 5'-phosphorothioate groups. In some embodiments, the TMPRSS6 dsRNA compound comprises an E-vinylphosphonate nucleotide at the 5Y-terminus of the antisense strand (also referred to herein as the guide strand).
本發明的TMPRSS6 dsRNA化合物、有義多核苷酸的3'端和5'端和/或反義多核苷酸的3'端的某些實施方案包含至少一個經修飾的核苷酸，其中該至少一個經修飾的核苷酸包括：無鹼基核苷酸、核糖醇、反向核苷酸、反向無鹼基核苷酸、反向2'-OMe核苷酸、反向2'-脫氧核苷酸。本領域技術人員已知的是，在寡核苷酸的末端處包含無鹼基核苷酸或反向無鹼基核苷酸增強了穩定性（Czauderna等人，“哺乳動物細胞中合成siRNA的結構變異和穩定修飾（Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells.）”，Nucleic Acids Res.2003;31(11):2705-2716. doi:10.1093/nar/gkg393）。在一些實施方案中，TMPRSS6 dsRNA化合物在3'-端或5'-端處或者3'-端和5'-端兩者處包含一個或多個反向無鹼基殘基（invab）。示例性的反向無鹼基殘基（invab）包括但不限於以下項：Certain embodiments of the TMPRSS6 dsRNA compounds, the 3' and 5' ends of the sense polynucleotides, and/or the 3' end of the antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises: an abasic nucleotide, a ribitol, an inverted nucleotide, an inverted abasic nucleotide, an inverted 2'-OMe nucleotide, or an inverted 2'-deoxynucleotide. It is known to those skilled in the art that inclusion of abatic or inverted abatic nucleotides at the termini of oligonucleotides enhances stability (Czauderna et al., "Structural variations and stabilizing modifications of synthetic siRNAs in mammalian cells." Nucleic Acids Res. 2003;31(11):2705-2716. doi:10.1093/nar/gkg393). In some embodiments, the TMPRSS6 dsRNA compound comprises one or more inverted abatic residues (invabs) at the 3'-end or the 5'-end, or at both the 3'-end and the 5'-end. Exemplary inverted abatic residues (invabs) include, but are not limited to, the following:
本發明的TMPRSS6 dsRNA化合物、有義多核苷酸的3’端和5'端和/或反義多核苷酸的3'端的某些實施方案包含至少一個經修飾的核苷酸，其中該至少一個經修飾的核苷酸包括：異甘露醇核苷酸或所述異甘露醇核苷酸的立體異構體。異甘露醇核苷酸或所述異甘露醇核苷酸的立體異構體的具體示例包括但不限於：、、、、、、、、、、和，其中短語“寡核苷酸”各自獨立地表示多核苷酸部分。示例性的異甘露醇殘基（imann）包括但不限於以下項：Certain embodiments of the TMPRSS6 dsRNA compounds, the 3' and 5' ends of the sense polynucleotides, and/or the 3' end of the antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises an isomannitol nucleotide or a stereoisomer of the isomannitol nucleotide. Specific examples of isomannitol nucleotides or stereoisomers of the isomannitol nucleotides include, but are not limited to: 、 、 、 、 、 、 、 、 、 、 and , wherein the phrase "oligonucleotide" independently refers to a polynucleotide portion. Exemplary isomannide residues (imann) include, but are not limited to, the following:
或。or .
在某些實施方案中，這些異甘露醇核苷酸可與一個或多個靶向基團或遞送分子（諸如GalNAc部分）進一步綴合。In certain embodiments, these isomannitol nucleotides can be further conjugated to one or more targeting groups or delivery molecules, such as a GalNAc moiety.
本發明的TMPRSS6 dsRNA化合物、反義多核苷酸的某些實施方案包含至少一個經修飾的核苷酸，其中該至少一個經修飾的核苷酸包括非鎖核酸核苷酸（UNA）或/和乙二醇核酸核苷酸（GNA）。本領域技術人員已知的是，UNA和GNA是能夠顯著改善siRNA化合物的脫靶分佈的熱不穩定的化學修飾（Janas等人，“對具有有限脫靶驅動的大鼠肝毒性的GalNAc-綴合的siRNA選擇（Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity.）”，Nat Commun.2018;9(1):723. doi:10.1038/s41467-018-02989-4；Laursen等人，“利用非鎖核酸（UNA）來增強siRNA在體外和體內的性能（Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo.）”，Mol BioSyst.2010;6:862–70）。Certain embodiments of the TMPRSS6 dsRNA compounds and antisense polynucleotides of the present invention comprise at least one modified nucleotide, wherein the at least one modified nucleotide comprises a non-locking nucleic acid nucleotide (UNA) and/or a glycol nucleic acid nucleotide (GNA). It is known to those skilled in the art that UNA and GNA are thermolabile chemical modifications that can significantly improve the off-target distribution of siRNA compounds (Janas et al., "Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity." Nat Commun. 2018;9(1):723. doi:10.1038/s41467-018-02989-4; Laursen et al., "Utilization of unlocked nucleic acid (UNA) to enhance siRNA performance in vitro and in vivo." Mol BioSyst. 2010;6:862–70).
本發明的TMPRSS6 dsRNA化合物、反義多核苷酸的某些實施方案還包含磷酸酯模擬物。如本文所用，磷酸酯部分是指磷酸酯基團，包括與核苷酸的糖部分（例如核糖或脫氧核糖或它們的類似物）連接的磷酸酯或磷酸酯模擬物。包含磷酸酯模擬物的核苷酸也可被定義為經膦酸酯修飾的核苷酸。Certain embodiments of the TMPRSS6 dsRNA compounds and antisense polynucleotides of the present invention further comprise a phosphate mimetic. As used herein, a phosphate moiety refers to a phosphate group, including a phosphate or phosphate mimetic attached to a sugar moiety of a nucleotide (e.g., ribose or deoxyribose, or analogs thereof). Nucleotides comprising a phosphate mimetic may also be defined as phosphonate-modified nucleotides.
在一些實施方案中，該磷酸酯模擬物是5'-乙烯基膦酸酯（VP）。在示例性的實施方案中，本發明內容的乙烯基膦酸酯具有以下結構：In some embodiments, the phosphate mimetic is 5'-vinylphosphonate (VP). In an exemplary embodiment, the vinylphosphonate of the present invention has the following structure:
本發明內容的乙烯基膦酸酯可與本發明內容的dsRNA的反義鏈或有義鏈連接。在某些優選的實施方案中，本發明內容的乙烯基膦酸酯與dsRNA的反義鏈連接，任選地位於該dsRNA的反義鏈的5'端處。The vinylphosphonate of the present invention can be linked to the antisense or sense strand of the dsRNA of the present invention. In certain preferred embodiments, the vinylphosphonate of the present invention is linked to the antisense strand of the dsRNA, optionally at the 5' end of the antisense strand of the dsRNA.
在某些實施方案中，本發明內容的經乙烯基膦酸酯修飾的核苷酸具有式(IV)的結構：In certain embodiments, the vinylphosphonate-modified nucleotides of the present invention have the structure of Formula (IV):
(IV)(IV)
其中X為O或S；Where X is O or S;
R為氫、羥基、氟或C1-20烷氧基（例如，甲氧基或正十六烷氧基）；R is hydrogen, hydroxy, fluorine or C1-20 alkoxy (e.g., methoxy or n-hexadecyloxy);
R5'為=C(H)-P(O)(OH)2，並且C5'碳和R5'之間的雙鍵處於E或Z取向（例如，E取向）；並且R5' is =C(H)-P(O)(OH)2 , and the double bond between the C5' carbon and R5' is in the E or Z orientation (e.g., the E orientation); and
B為核鹼基或經修飾的核鹼基，任選地其中B為腺嘌呤、鳥嘌呤、胞嘧啶、胸腺嘧啶或尿嘧啶。B is a nucleobase or a modified nucleobase, optionally wherein B is adenine, guanine, cytosine, thymine or uracil.
在某些實施方案中，R5'為=C(H)-P(O)(OH)2，並且C5'碳和R5'之間的雙鍵處於E取向。在某些實施方案中，R為甲氧基，R5'為=C(H)-P(O)(OH)2，並且C5'碳和R5'之間的雙鍵處於E取向。在某些實施方案中，X為S，R為甲氧基，R5'為=C(H)-P(O)(OH)2，並且C5'碳和R5'之間的雙鍵處於E取向。In certain embodiments, R5' is =C(H)-P(O)(OH)2 , and the double bond between the C5' carbon and R5' is in the E orientation. In certain embodiments, R is methoxy, R5' is =C(H)-P(O)(OH)2 , and the double bond between the C5' carbon and R5' is in the E orientation. In certain embodiments, X is S, R is methoxy, R5' is =C(H)-P(O)(OH)2 , and the double bond between the C5' carbon and R5' is in the E orientation.
對於本發明內容的dsRNA、組合物和方法，還考慮了乙烯基磷酸酯修飾。示例性的乙烯基磷酸酯結構是：Vinyl phosphate modifications are also contemplated for use in the dsRNA, compositions, and methods of the present invention. An exemplary vinyl phosphate structure is:
在某些實施方案中，經乙烯基膦酸酯修飾的核苷酸是具有如下結構的VPu*：In certain embodiments, the vinylphosphonate-modified nucleotide is V Pu * having the structure:
在許多情況下，保護基團在本發明化合物的製備期間使用。如本文所用，術語“受保護的”意指所指定的部分含有添加在其上的保護基團。在本發明的一些實施方案中，化合物含有一個或多個保護基團。在本發明的方法中可採用各種保護基團。一般來講，保護基團使得化學官能團對特定反應條件呈惰性，並且可在分子中添加或去除此類官能團上而基本上不破壞該分子的其餘部分。一般來講，保護基團（特別是羥基保護基團）是本領域熟知的（Greene和Wuts，“有機合成中的保護基團（Protective Groups in Organic Synthesis）”，第2章，第2版，John Wiley & Sons, New York, 1991）。In many cases, protecting groups are used during the preparation of the compounds of the present invention. As used herein, the term "protected" means that the designated moiety has a protecting group added thereto. In some embodiments of the present invention, the compound contains one or more protecting groups. A variety of protecting groups can be employed in the methods of the present invention. Generally speaking, protecting groups render a chemical functional group inert to specific reaction conditions and allow such functional groups to be added or removed from a molecule without substantially disrupting the remainder of the molecule. In general, protecting groups, particularly hydroxy protecting groups, are well known in the art (Greene and Wuts, "Protective Groups in Organic Synthesis," Chapter 2, 2nd ed., John Wiley & Sons, New York, 1991).
如本文所用，保護基團（例如，羥基保護基團）的示例包括但不限於甲基、乙基、苄基（Bn）、苯基、異丙基、叔丁基、乙醯基、氯乙醯基、三氯乙醯基、新戊醯基、叔丁氧基甲基、甲氧基甲基、1-乙氧基乙基、1-(2-氯乙氧基)乙基、烯丙基、環己基、9-芴基甲氧基羰基（Fmoc）、甲磺酸酯、甲苯磺酸酯、三氟甲磺酸酯、苯甲醯基、苯甲醯基甲酸酯、對苯基苯甲醯基、4-甲氧基苄基、單甲氧基三苯甲基、二甲氧基三苯甲基、三甲氧基三苯甲基、4-氯苄基、4-硝基苄基、2,4-二硝基苯基、4-醯氧基苄基、2-甲基苯基、2,6-二甲基苯基、2-氯苯基、2,6-二氯苄基、二苯基甲基、三苯基甲基、4-甲硫基-1-丁基、S-乙醯基硫代乙酸酯（SATA）、2-氰基乙基、2-氰基、1-二甲基乙基（CDM）、4-氰基-2-丁烯基、2-(三甲基甲矽烷基)乙基（TSE）、2-(苯硫基)乙基、2-(三苯基甲矽烷基)乙基、2-(苄基磺醯基)乙基、2,2,2-三氯乙基、2,2,2-三溴乙基、2,3-二溴丙基、2,2,2-三氟乙基、苯硫基、2-氯-4-三苯甲基苯基、2-溴苯基、2-[N-異丙基-N-(4-甲氧基苯甲醯基)氨基]乙基、4-(N-三氟乙醯基氨基)丁基、4-氧代戊基、4-三苯甲基氨基苯基、4-苄基氨基苯基、四氫吡喃基、嗎啉基、三甲基甲矽烷基、三乙基甲矽烷基、叔丁基二甲基甲矽烷基、叔丁基二苯基甲矽烷基、三苯基甲矽烷基、三異丙基甲矽烷基、新戊醯基氧基甲基（POM）和9-苯基黃嘌呤-9-基。As used herein, examples of protecting groups (e.g., hydroxy protecting groups) include, but are not limited to, methyl, ethyl, benzyl (Bn), phenyl, isopropyl, tert-butyl, acetyl, chloroacetyl, trichloroacetyl, neopentanoyl, tert-butoxymethyl, methoxymethyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, allyl, cyclohexyl, 9-fluorenylmethoxycarbonyl (Fmoc), mesylate, tosylate, triflate, benzyl, phenyl Formyl formate, p-phenylbenzyl, 4-methoxybenzyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 4-chlorobenzyl, 4-nitrobenzyl, 2,4-dinitrophenyl, 4-acyloxybenzyl, 2-methylphenyl, 2,6-dimethylphenyl, 2-chlorophenyl, 2,6-dichlorobenzyl, diphenylmethyl, triphenylmethyl, 4-methylthio-1-butyl, S-acetylthioacetate (SATA), 2-cyano Ethyl, 2-cyano, 1-dimethylethyl (CDM), 4-cyano-2-butenyl, 2-(trimethylsilyl)ethyl (TSE), 2-(phenylthio)ethyl, 2-(triphenylsilyl)ethyl, 2-(benzylsulfonyl)ethyl, 2,2,2-trichloroethyl, 2,2,2-tribromoethyl, 2,3-dibromopropyl, 2,2,2-trifluoroethyl, phenylthio, 2-chloro-4-tritylphenyl, 2-bromophenyl, 2-[N- isopropyl-N-(4-methoxybenzyl)amino]ethyl, 4-(N-trifluoroacetylamino)butyl, 4-oxopentyl, 4-tritylaminophenyl, 4-benzylaminophenyl, tetrahydropyranyl, morpholinyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, triphenylsilyl, triisopropylsilyl, neopentanoyloxymethyl (POM), and 9-phenylxanthin-9-yl.
如本文所用，氨基保護基團的示例包括但不限於氨基甲酸保護基團，諸如2-三甲基甲矽烷基乙氧基羰基（Teoc）、1-甲基-1-(4-聯苯)乙氧基羰基（Bpoc）、叔丁氧基羰基（BOC）、烯丙基氧基羰基（Alloc）、9-芴基-甲氧基羰基（Fmoc）、苄氧基羰基（Cbz）；醯胺保護基團，諸如甲醯基、乙醯基、新戊醯基、三鹵代乙醯基、苯甲醯基、2-硝基苯磺醯基；以及亞胺和環狀醯亞胺保護基團，諸如鄰苯二甲醯亞胺基和二硫代琥珀醯基。這些氨基保護基團的等同物也被涵蓋在本發明的化合物和方法中。As used herein, examples of amino protecting groups include, but are not limited to, carbamate protecting groups such as 2-trimethylsilylethoxycarbonyl (Teoc), 1-methyl-1-(4-biphenyl)ethoxycarbonyl (Bpoc), tert-butyloxycarbonyl (BOC), allyloxycarbonyl (Alloc), 9-fluorenyl-methoxycarbonyl (Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups such as formyl, acetyl, neopentanoyl, trihaloacetyl, benzyl, and 2-nitrobenzenesulfonyl; and imine and cyclic imide protecting groups such as phthalimido and disulfosuccinyl. Equivalents of these amino protecting groups are also encompassed in the compounds and methods of the present invention.
可包含在本發明的TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的某些實施方案的RNA中的另一種修飾包括將一個或多個配體、部分或綴合物以化學方式連接至RNA，這些配體、部分或綴合物分別增強TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的一種或多種特徵。可被增強的特徵的非限制性示例是：TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸活性，細胞分佈，TMPRSS6 dsRNA藥劑的遞送，TMPRSS6 dsRNA藥劑的藥代動力學特性，以及TMPRSS6 dsRNA藥劑的細胞攝取。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑包含一個或多個在本發明的TMPRSS6 dsRNA藥劑的某些實施方案中與有義鏈綴合的靶向基團或連接基團。靶向基團的非限制性示例是包含N-乙醯基-半乳糖胺（GalNAc）的化合物。術語“靶向基團”、“靶向劑”、“連接劑”、“靶向化合物”、“遞送分子”、“遞送化合物”和“靶向配體”在本文中可互換使用。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑包含與有義鏈的5'-末端綴合的靶向化合物。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑包含與有義鏈的3'-末端綴合的靶向化合物。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑包含含有GalNAc的靶向基團。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑不包含與有義鏈的3'-末端和5'-末端中的一者或兩者綴合的靶向化合物。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑不包含與有義鏈的5'-末端和3'-末端中的一者或兩者綴合的含GalNAc的靶向化合物。Another modification that may be included in the RNA of certain embodiments of the TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides of the invention includes chemically linking to the RNA one or more ligands, moieties, or conjugates that enhance one or more characteristics of the TMPRSS6 dsRNA agent, TMPRSS6 antisense polynucleotide, and/or TMPRSS6 sense polynucleotide, respectively. Non-limiting examples of characteristics that can be enhanced include: TMPRSS6 dsRNA agent, TMPRSS6 antisense polynucleotide, and/or TMPRSS6 sense polynucleotide activity, cellular distribution, delivery of TMPRSS6 dsRNA agent, pharmacokinetic properties of TMPRSS6 dsRNA agent, and cellular uptake of TMPRSS6 dsRNA agent. In some embodiments of the present invention, the TMPRSS6 dsRNA agent comprises one or more targeting groups or linker groups that are conjugated to the sense strand in certain embodiments of the TMPRSS6 dsRNA agent of the present invention. A non-limiting example of a targeting group is a compound comprising N-acetyl-galactosamine (GalNAc). The terms "targeting group," "targeting agent," "linker," "targeting compound," "delivery molecule," "delivery compound," and "targeting ligand" are used interchangeably herein. In certain embodiments of the present invention, a TMPRSS6 dsRNA agent comprises a targeting compound conjugated to the 5'-end of the sense strand. In certain embodiments of the present invention, a TMPRSS6 dsRNA agent comprises a targeting compound conjugated to the 3'-end of the sense strand. In certain embodiments of the present invention, a TMPRSS6 dsRNA agent comprises a targeting group comprising GalNAc. In certain embodiments of the present invention, a TMPRSS6 dsRNA agent does not comprise a targeting compound conjugated to one or both of the 3'-end and the 5'-end of the sense strand. In certain embodiments of the invention, the TMPRSS6 dsRNA agent does not comprise a GalNAc-containing targeting compound conjugated to one or both of the 5'-end and the 3'-end of the sense strand.
另外的靶向劑和連接劑是本領域熟知的，例如，可用於本發明的某些實施方案的靶向劑和連接劑包括但不限於脂質部分，諸如膽固醇部分（Letsinger等人，Proc.Natl.Acid.Sci. USA, 1989, 86: 6553-6556）；膽酸（Manoharan等人，Biorg.Med.Chem.Let., 1994, 4:1053-1060）；硫醚，例如綠柱石-S-三苯甲基硫醇（Manoharan等人，Ann.N.Y.Acad.Sci., 1992, 660:306-309；Manoharan等人，Biorg.Med.Chem.Let., 1993, 3:2765-2770）；硫代膽固醇（Oberhauser等人，Nucl.Acids Res., 1992, 20:533-538）；脂族鏈，例如十二烷二醇或十一烷基殘基（Saison-Behmoaras等人，EMBO J, 1991, 10:1111-1118；Kabanov等人，FEBS Lett., 1990, 259:327-330；Svinarchuk等人，Biochimie, 1993, 75:49-54）；磷脂，例如二-十六烷基-外消旋-甘油或三乙基-銨1,2-二-O-十六烷基-外消旋-甘油-3-膦酸酯（Manoharan等人，Tetrahedron Lett., 1995, 36:3651-3654；Shea等人，Nucl.Acids Res., 1990, 18:3777-3783）；聚胺或聚乙二醇鏈（Manoharan等人，Nucleosides & Nucleotides, 1995, 14:969-973）；或金剛烷乙酸（Manoharan等人，Tetrahedron Lett., 1995, 36:3651-3654）；棕櫚醯基部分（Mishra等人，Biochim.Biophys.Acta, 1995, 1264:229-237）；或十八胺或己基氨基-羰基氧基膽固醇部分（Crooke等人，J. Pharmacol.Exp.Ther., 1996, 277:923-937）。Additional targeting agents and linkers are well known in the art. For example, targeting agents and linkers that can be used in certain embodiments of the present invention include, but are not limited to, lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4: 1053-1060); thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660: 306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); aliphatic chains, such as dodecandiol or undecyl residue (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); a polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
包含TMPRSS6 dsRNA藥劑、TMPRSS6反義多核苷酸和/或TMPRSS6有義多核苷酸的組合物的某些實施方案可包含改變TMPRSS6 dsRNA藥劑的分佈、靶向等的配體。在包含本發明的TMPRSS6 dsRNA藥劑的組合物的一些實施方案中，例如與缺乏此類配體的物種相比，該配體增加了對所選的靶標的親和力，所選的靶標例如分子、細胞或細胞類型、區室，例如細胞或器官區室、組織、器官或身體區域。可用於本發明的組合物和/或方法的配體可以是天然存在的物質，諸如蛋白質（例如人血清白蛋白（HSA）、低密度脂蛋白（LDL）或球蛋白）；碳水化合物（例如葡聚糖、普魯蘭多糖、幾丁質、脫乙醯殼多糖、菊粉、環糊精或透明質酸）；或脂質。配體也可以是重組分子或合成分子，諸如合成聚合物，例如合成聚氨基酸或聚胺。聚氨基酸的示例是聚賴氨酸（PLL）、聚L-天冬氨酸、聚L-谷氨酸、苯乙烯-馬來酸酐共聚物、聚(L-丙交酯-共-乙醇酸)共聚物、二乙烯基醚-馬來酸酐共聚物、N-(2-羥丙基)甲基丙烯醯胺共聚物（HMPA）、聚乙二醇（PEG）、聚乙烯醇（PVA）、聚氨酯、聚(2-乙基丙烯酸)、N-異丙基丙烯醯胺聚合物或聚膦嗪。聚胺的示例包括：聚乙烯亞胺、聚賴氨酸（PLL）、精胺、亞精胺、聚胺、偽肽-聚胺、肽模擬物聚胺、樹枝狀聚胺、精氨酸、脒、魚精蛋白、陽離子脂質、陽離子卟啉、聚胺的季鹽、或α螺旋肽。Certain embodiments of compositions comprising TMPRSS6 dsRNA agents, TMPRSS6 antisense polynucleotides, and/or TMPRSS6 sense polynucleotides may comprise a ligand that alters the distribution, targeting, etc., of the TMPRSS6 dsRNA agent. In some embodiments of the compositions comprising the TMPRSS6 dsRNA agents of the invention, the ligand increases affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a body region, e.g., compared to a species lacking such ligand. Ligands useful in the compositions and/or methods of the present invention can be naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, deacetylated chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, e.g., synthetic polyamino acids or polyamines. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolic acid) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.
包含在本發明的組合物和/或方法中的配體可包括靶向基團，其非限制性示例是細胞或組織靶向劑，例如凝集素、糖蛋白、脂質或蛋白質，例如與特定細胞類型諸如腎臟細胞或肝臟細胞結合的抗體。靶向基團可以是促甲狀腺素、促黑素、凝集素、糖蛋白、表面活性劑蛋白A、粘蛋白碳水化合物、多價乳糖、多價半乳糖、N-乙醯基-半乳糖胺、N-乙醯基-古洛糖胺多價甘露糖、多價岩藻糖、糖基化聚氨基酸、多價半乳糖、轉鐵蛋白、雙膦酸鹽、聚谷氨酸鹽、聚天冬氨酸鹽、脂質、膽固醇、類固醇、膽汁酸、葉酸鹽、維生素B12、維生素A、生物素或RGD肽或RGD肽模擬物。Ligands included in the compositions and/or methods of the present invention may include targeting groups, non-limiting examples of which are cell or tissue targeting agents, such as lectins, glycoproteins, lipids, or proteins, such as antibodies that bind to specific cell types, such as kidney cells or liver cells. The targeting group can be thyrotropin, melanocyte-stimulating hormone, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-gulosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or RGD peptide or RGD peptide mimetic.
配體的其他示例包括染料、嵌入劑（例如吖啶）、交聯劑（例如補骨脂素、絲裂黴素C）、卟啉（TPPC4、德克薩斯卟啉（texaphyrin）、噻啉（sapphyrin））、多環芳烴（例如吩嗪、二氫吩嗪）、人工核酸內切酶（例如EDTA）、親脂性分子（例如膽固醇、膽酸、金剛烷乙酸、1-芘丁酸、二氫睾酮、1,3-雙-O(十六烷基)甘油、香葉氧基己基基團、十六烷基甘油、冰片、薄荷醇、1,3-丙二醇、十七烷基基團、棕櫚酸、肉豆蔻酸、O3-(油醯基)石膽酸、O3-(油醯基)膽烯酸、二甲氧基三苯甲基或吩噁嗪）和肽綴合物（例如觸足肽、Tat肽）、烷化劑、磷酸酯、氨基、巰基、PEG（例如PEG-40K）、MPEG、[MPEG]2、聚氨基、烷基、經取代的烷基、放射性標記物、酶、半抗原（例如，生物素）、轉運/吸收促進劑（例如，阿司匹林、維生素E、葉酸）、合成核糖核酸酶（例如，咪唑、雙咪唑、組胺、咪唑簇、吖啶-咪唑綴合物、四氮雜大環的Eu3+複合物）、二硝基苯基、HRP或AP。Other examples of ligands include dyes, intercalators (e.g., acridine), crosslinkers (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-dihydrotestosterone), -O(hexadecyl)glycerin, geranyloxyhexyl group, hexadecylglycerin, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid, O3-(oleyl)cholestyric acid, O3-(oleyl)cholestyric acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., cataplasm peptide, Tat peptide), alkylating agents, phosphate esters, amino groups, hydroxyl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2. Polyamino, alkyl, substituted alkyl, radiolabel, enzyme, hapten (e.g., biotin), transport/absorption enhancer (e.g., aspirin, vitamin E, folic acid), synthetic ribonuclease (e.g., imidazole, bisimidazole, histamine, imidazole cluster, acridine-imidazole conjugate, tetrazamacrocyclic Eu3+ complex), dinitrophenyl, HRP, or AP.
包含在本發明的組合物和/或方法中的配體可以是蛋白質（例如糖蛋白或肽，例如對共配體具有特定親和力的分子），或抗體，例如與特定細胞類型諸如癌細胞、內皮細胞、心臟細胞或骨細胞結合的抗體。可用於本發明的組合物和/或方法的實施方案中的配體可以是激素或激素受體。可用於本發明的組合物和/或方法的實施方案中的配體可以是脂質、凝集素、碳水化合物、維生素、輔因子、多價乳糖、多價半乳糖、N-乙醯基-半乳糖胺、N-乙醯基-古洛糖胺多價甘露糖、或多價岩藻糖。可用於本發明的組合物和/或方法的實施方案中的配體可以是能夠促進細胞攝取TMPRSS6 dsRNA藥劑的物質，例如通過破壞細胞的細胞骨架，例如通過破壞細胞的微管、微絲和/或中間絲。這種類型的藥劑的非限制性示例是：taxon、長春新鹼、長春花鹼、細胞鬆弛素、諾考達唑、促進微絲聚合劑（japlakinolide）、微絲解聚素A、鬼筆環肽、微絲細胞骨架解聚劑（swinholide）A、印丹諾辛（indanocine）和myoservin。The ligands included in the compositions and/or methods of the present invention can be proteins (e.g., glycoproteins or peptides, e.g., molecules with a specific affinity for a co-ligand), or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, heart cells, or bone cells. The ligands useful in embodiments of the compositions and/or methods of the present invention can be hormones or hormone receptors. The ligands useful in embodiments of the compositions and/or methods of the present invention can be lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulosamine, multivalent mannose, or multivalent fucose. Ligands useful in embodiments of the compositions and/or methods of the present invention can be substances that promote cellular uptake of TMPRSS6 dsRNA agents, for example, by disrupting the cell's cytoskeleton, for example, by disrupting microtubules, microfilaments, and/or intermediate filaments. Non-limiting examples of such agents are: taxon, vincristine, catharanthus alkaloids, cytorelaxant, nocodazole, japlakinolide, swinholide A, phalloidin, swinholide A, indanocine, and myoservin.
在一些實施方案中，與本發明的TMPRSS6 dsRNA藥劑連接的配體作為藥代動力學（PK）調節劑起作用。可用於本發明的組合物和方法中的PK調節劑的一個示例包括但不限於：親油基、膽汁酸、類固醇、磷脂類似物、肽、蛋白質結合劑、PEG、維生素、膽固醇、脂肪酸、膽酸、石膽酸、二烷基甘油酯、二醯基甘油酯、磷脂、鞘脂、萘普生、布洛芬、維生素E、生物素、結合血清蛋白的適體等。還已知包含多個硫代磷酸酯鍵的寡核苷酸與血清蛋白結合，因此主鏈中包含多個硫代磷酸酯鍵的短寡核苷酸（例如含有約5個鹼基、10個鹼基、15個鹼基或20個鹼基的寡核苷酸）也可在本發明的組合物和/或方法中用作配體。In some embodiments, the ligand linked to the TMPRSS6 dsRNA agent of the present invention acts as a pharmacokinetic (PK) modulator. Examples of PK modulators that can be used in the compositions and methods of the present invention include, but are not limited to, lipophilic groups, bile acid, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, cholesterol, fatty acids, bile acid, cholic acid, dialkylglycerides, diacylglycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, serum protein-binding aptamers, and the like. Oligonucleotides containing multiple phosphorothioate bonds are also known to bind to serum proteins, and thus short oligonucleotides containing multiple phosphorothioate bonds in their backbone (e.g., oligonucleotides containing about 5 bases, 10 bases, 15 bases, or 20 bases) can also be used as ligands in the compositions and/or methods of the invention.
TMPRSS6 dsRNATMPRSS6 dsRNA藥劑組合物Pharmaceutical composition
在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑存在於組合物中。本發明的組合物可包含一種或多種TMPRSS6 dsRNA藥劑和以下項中任選的一者或多者：藥學上可接受的載劑、遞送劑、靶向劑、可檢測標記等。根據本發明的方法的一些實施方案可使用的靶向劑的非限制性示例是將本發明的TMPRSS6 dsRNA藥劑引導至待治療的細胞以及/或進入待治療的細胞中的藥劑。對靶向劑的選擇將取決於以下因素：TMPRSS6相關疾病或病症的性質，以及所靶向的細胞類型。在一個非限制性示例中，在本發明的一些實施方案中，可能期望將TMPRSS6 dsRNA藥劑靶向肝臟細胞以及/或進入該肝臟細胞中。應當理解，在本發明的方法的一些實施方案中，治療劑包括僅含有遞送劑的TMPRSS6 dsRNA藥劑，諸如包含N-乙醯半乳糖胺（GalNAc）的遞送劑，而沒有任何另外的連接元件。例如，在本發明的一些方面，TMPRSS6 dsRNA藥劑可與包含GalNAc的遞送化合物連接並被包含在含有藥學上可接受的載劑的組合物中，並且在沒有任何與TMPRSS6 dsRNA藥劑連接的可檢測標記或靶向劑等的情況下施用於細胞或受試者。In some embodiments of the present invention, a TMPRSS6 dsRNA agent is present in a composition. The compositions of the present invention may comprise one or more TMPRSS6 dsRNA agents and one or more of the following: a pharmaceutically acceptable carrier, a delivery agent, a targeting agent, a detectable label, and the like. Non-limiting examples of targeting agents that can be used according to some embodiments of the methods of the present invention are agents that direct the TMPRSS6 dsRNA agent of the present invention to and/or into the cells to be treated. The choice of targeting agent will depend on the nature of the TMPRSS6-related disease or condition, as well as the cell type being targeted. In one non-limiting example, in some embodiments of the present invention, it may be desirable to target a TMPRSS6 dsRNA agent to liver cells and/or to enter the liver cells. It will be appreciated that in some embodiments of the methods of the present invention, the therapeutic agent comprises a TMPRSS6 dsRNA agent comprising only a delivery agent, such as one comprising N-acetylgalactosamine (GalNAc), without any additional linking elements. For example, in some aspects of the present invention, a TMPRSS6 dsRNA agent can be linked to a delivery compound comprising GalNAc and included in a composition comprising a pharmaceutically acceptable carrier and administered to a cell or subject without any detectable label, targeting agent, or the like linked to the TMPRSS6 dsRNA agent.
在本發明的TMPRSS6 dsRNA藥劑與一種或多種遞送劑、靶向劑、標記劑等一起施用以及/或與該一種或多種遞送劑、靶向劑、標記劑等連接的情況下，技術人員將知道並且能夠選擇和使用用於本發明的方法的合適藥劑。標記劑可在本發明的某些方法中用於測定TMPRSS6 dsRNA藥劑在細胞和組織中的位置，並且可用於測定已經在本發明的方法中施用的包含TMPRSS6 dsRNA藥劑的治療組合物在細胞、組織或器官中的位置。用於連接和利用標記劑諸如酶標記、染料、放射性標記等的方法是本領域熟知的。應當理解，在本發明的組合物和方法的一些實施方案中，標記劑與包含在TMPRSS6 dsRNA藥劑中的有義多核苷酸和反義多核苷酸中的一者或兩者連接。Where the TMPRSS6 dsRNA agents of the present invention are administered together with and/or linked to one or more delivery agents, targeting agents, labeling agents, etc., the skilled artisan will know and be able to select and use appropriate agents for use in the methods of the present invention. Labeling agents can be used in certain methods of the present invention to determine the location of TMPRSS6 dsRNA agents in cells and tissues, and can be used to determine the location of therapeutic compositions containing TMPRSS6 dsRNA agents that have been administered in the methods of the present invention in cells, tissues, or organs. Methods for attaching and utilizing labeling agents, such as enzyme labels, dyes, radioactive labels, and the like, are well known in the art. It will be understood that in some embodiments of the compositions and methods of the invention, the labeling agent is linked to one or both of the sense polynucleotide and the antisense polynucleotide contained in the TMPRSS6 dsRNA agent.
TMPRSS6 dsRNATMPRSS6 dsRNA藥劑和Medications andTMPRSS6TMPRSS6反義多核苷酸藥劑的遞送Delivery of antisense polynucleotide agents
本發明的方法的某些實施方案包括將TMPRSS6 dsRNA藥劑遞送至細胞中。如本文所用，術語“遞送”意指促進或實現細胞攝取或吸收。TMPRSS6 dsRNA藥劑的吸收或攝取可通過無輔助的擴散或主動細胞過程發生，或者通過使用可與本發明的TMPRSS6 dsRNA藥劑締合的遞送劑、靶向劑等發生。適於在本發明方法中使用的遞送方式包括但不限於：體內遞送，其中TMPRSS6 dsRNA藥劑被注射到組織部位或全身性施用。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑與遞送劑連接。Certain embodiments of the methods of the present invention include delivering a TMPRSS6 dsRNA agent into cells. As used herein, the term "delivery" means promoting or enabling cellular uptake or absorption. Absorption or uptake of the TMPRSS6 dsRNA agent can occur by unassisted diffusion or active cellular processes, or by using a delivery agent, targeting agent, or the like that can be conjugated to the TMPRSS6 dsRNA agent of the present invention. Suitable delivery methods for use in the methods of the present invention include, but are not limited to, intravital delivery, wherein the TMPRSS6 dsRNA agent is injected into a tissue site, or systemically administered. In some embodiments of the present invention, the TMPRSS6 dsRNA agent is linked to a delivery agent.
可用於將TMPRSS6 dsRNA藥劑遞送至細胞、組織和/或受試者的方法的非限制性示例包括：TMPRSS6 dsRNA-GalNAc綴合物、SAMiRNA技術、基於LNP的遞送方法，以及裸RNA遞送。這些遞送方法和其他遞送方法已在本領域成功用於遞送治療性RNAi藥劑以治療各種疾病和病症，諸如但不限於：肝病、急性間歇性卟啉症（AIP）、血友病、肺纖維化等。各種遞送方式的細節可參見出版物，諸如：Nikam, R.R. 和K. R. Gore (2018) Nucleic Acid Ther, 28 (4), 209-224 Aug 2018；Springer A.D.和S.F.Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118；Lee, K.等人，(2018) Arch Pharm Res, 41(9), 867-874；以及Nair, J.K.等人，(2014) J. Am. Chem.Soc.136:16958-16961，這些文獻中的每一篇的內容以引用方式併入本文。Non-limiting examples of methods that can be used to deliver TMPRSS6 dsRNA agents to cells, tissues, and/or subjects include TMPRSS6 dsRNA-GalNAc conjugates, SAMiRNA technology, LNP-based delivery methods, and naked RNA delivery. These and other delivery methods have been successfully used in the art to deliver therapeutic RNAi agents to treat various diseases and conditions, such as, but not limited to, liver disease, acute intermittent porphyria (AIP), hemophilia, and pulmonary fibrosis. Details of various delivery methods can be found in publications such as Nikam, R.R. and K. R. Gore (2018) Nucleic Acid Ther, 28 (4), 209-224 Aug 2018; Springer A.D. and S.F. Dowdy (2018) Nucleic Acid Ther. Jun 1; 28(3): 109–118; Lee, K. et al. (2018) Arch Pharm Res, 41(9), 867-874; and Nair, J.K. et al. (2014) J. Am. Chem. Soc. 136:16958-16961, each of which is incorporated herein by reference.
本發明的一些實施方案包括使用脂質納米顆粒（LNP）將本發明的TMPRSS6 dsRNA藥劑遞送至細胞、組織和/或受試者。LNP通常用於在體內遞送TMPRSS6 dsRNA藥劑，包括治療性TMPRSS6 dsRNA藥劑。使用LNP或其他遞送劑的一個益處是當使用該LNP或其他遞送劑將TMPRSS6 RNA藥劑遞送至受試者時，該TMPRSS6 RNA藥劑的穩定性增強。在本發明的一些實施方案中，LNP包括負載有一種或多種本發明的TMPRSS6 RNAi分子的陽離子LNP。將包含TMPRSS6 RNAi分子的LNP施用於受試者，該LNP及其所連接的TMPRSS6 RNAi分子經由胞吞作用被細胞攝取，它們的存在導致RNAi觸發分子的釋放，從而介導RNAi。Some embodiments of the present invention include the use of lipid nanoparticles (LNPs) to deliver TMPRSS6 dsRNA agents of the present invention to cells, tissues, and/or subjects. LNPs are generally used to deliver TMPRSS6 dsRNA agents, including therapeutic TMPRSS6 dsRNA agents, in vivo. One benefit of using LNPs or other delivery agents is the enhanced stability of the TMPRSS6 RNA agent when delivered to a subject using the LNPs or other delivery agents. In some embodiments of the present invention, the LNPs include cationic LNPs loaded with one or more TMPRSS6 RNAi molecules of the present invention. When LNPs containing TMPRSS6 RNAi molecules are administered to a subject, the LNPs and their linked TMPRSS6 RNAi molecules are taken up by cells via endocytosis. Their presence leads to the release of RNAi trigger molecules, thereby mediating RNAi.
可用於本發明的實施方案中以將本發明的TMPRSS6 dsRNA藥劑遞送至細胞、組織和/或受試者的遞送劑的另一個非限制性示例是包含GalNAc的藥劑，該遞送劑與本發明的TMPRSS6 dsRNA藥劑連接並將TMPRSS6 dsRNA藥劑遞送至細胞、組織和/或受試者。可用於本發明的方法和組合物的某些實施方案中的某些另外的包含GalNAc的遞送劑的示例公開於PCT申請：WO2020191183A1（其全文以引用方式併入本文）。可用於本發明的組合物和方法中以將TMPRSS6 dsRNA藥劑遞送至細胞的GalNAc靶向配體的非限制性示例是靶向配體簇。本文中呈現的靶向配體簇的示例被稱為：含有磷酸二酯鍵（GLO）的GalNAc配體和含有硫代磷酸酯鍵（GLS）的GalNAc配體。術語“GLX-n”在本文可用於表示所連接的含GalNAc的化合物是以下化合物中的任一者：GLS-1*、GLS-2*、GLS-3*、GLS-4*、GLS-5*、GLS-6*、GLS-7*、GLS-8*、GLS-9*、GLS-10*、GLS-11*、GLS-12*、GLS-13*、GLS-14*、GLS-15*、GLS-16*、GLO-1、GLO-2、GLO-3、GLO-4、GLO-5、GLO-6、GLO-7、GLO-8、GLO-9、GLO-10、GLO-11、GLO-12、GLO-13、GLO-14、GLO-15和GLO-16，這些化合物中的每一者的結構示於下文，並且下文具有GalNAc靶向配體與本發明的RNAi藥劑的連接位置，位於上述每一者的最右側（以“⸾”顯示）。應當理解，本發明的任何RNAi和dsRNA分子可與下文所示的GLS-1*、GLS-2*、GLS-3*、GLS-4*、GLS-5*、GLS-6*、GLS-7*、GLS-8*、GLS-9*、GLS-10*、GLS-11*、GLS-12*、GLS-13*、GLS-14*、GLS-15*、GLS-16*、GLO-1、GLO-2、GLO-3、GLO-4、GLO-5、GLO-6、GLO-7、GLO-8、GLO-9、GLO-10、GLO-11、GLO-12、GLO-13、GLO-14、GLO-15和GLO-16、GLO-1至GLO-16以及GLS-1*至GLS-16*連接。
在某些實施方案中，上述異甘露醇核苷酸可與一個或多個GalNAc靶向配體進一步綴合。與GalNAc靶向配體綴合的異甘露醇核苷酸的具體示例包括但不限於：In certain embodiments, the above-mentioned isomannose nucleotides may be further conjugated to one or more GalNAc targeting ligands. Specific examples of isomannose nucleotides conjugated to GalNAc targeting ligands include, but are not limited to:
、，其中短語“寡核苷酸”各自獨立地表示多核苷酸部分。、 , wherein the phrase "oligonucleotide" each independently refers to a polynucleotide portion.
在本發明的一些實施方案中，體內遞送還可以通過β-葡聚糖遞送系統進行，諸如描述於美國專利號5,032,401和5,607,677以及美國公開號2005/0281781中的那些，這些文獻據此以引用方式全文併入。還可使用本領域已知的方法，諸如電穿孔和脂質轉染法，在體外將TMPRSS6 RNAi藥劑引入細胞中。在本發明的方法的某些實施方案中，TMPRSS6 dsRNA可在沒有靶向劑的情況下遞送。這些RNA可作為“裸”RNA分子遞送。作為非限制性示例，本發明的TMPRSS6 dsRNA可以包含RNAi藥劑但不包含靶向劑（諸如GalNAc靶向化合物）的藥物組合物的形式施用於受試者，以治療受試者的TMPRSS6相關疾病或病症，諸如地中海貧血。In some embodiments of the present invention, in vivo delivery can also be performed using a β-glucan delivery system, such as those described in U.S. Patent Nos. 5,032,401 and 5,607,677 and U.S. Publication No. 2005/0281781, which are hereby incorporated by reference in their entirety. TMPRSS6 RNAi agents can also be introduced into cells in vitro using methods known in the art, such as electroporation and lipofection. In certain embodiments of the methods of the present invention, TMPRSS6 dsRNA can be delivered without a targeting agent. These RNAs can be delivered as "naked" RNA molecules. As a non-limiting example, the TMPRSS6 dsRNA of the present invention can be administered to a subject in the form of a pharmaceutical composition comprising an RNAi agent but not a targeting agent (such as a GalNAc targeting compound) to treat a TMPRSS6-related disease or condition, such as thalassemia, in the subject.
除了本文所述的某些遞送方式之外，應當理解，RNAi遞送方式（諸如但不限於本文所述的那些方式和本領域所使用的那些方式）可與本文所述的TMPRSS6 RNAi藥劑和治療方法的實施方案聯合使用。In addition to certain delivery methods described herein, it will be understood that RNAi delivery methods (such as, but not limited to, those described herein and those used in the art) can be used in conjunction with the embodiments of the TMPRSS6 RNAi agents and treatment methods described herein.
本發明的TMPRSS6 dsRNA藥劑可以有效降低細胞和/或受試者的TMPRSS6多肽水平和活性的量和方式施用於受試者。在本發明的方法的一些實施方案中，將一種或多種TMPRSS6 dsRNA藥劑施用於細胞和/或受試者以治療與TMPRSS6表達和活性相關的疾病或病症。在一些實施方案中，本發明的方法包括向需要此類治療的受試者施用一種或多種TMPRSS6 dsRNA藥劑，以減少該受試者的與TMPRSS6表達相關的疾病或病症。可施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑以降低體外細胞、離體細胞和體內細胞中的一者或多者中的TMPRSS6表達和/或活性。The TMPRSS6 dsRNA agents of the present invention can be administered to a subject in an amount and manner effective to reduce the level and activity of a TMPRSS6 polypeptide in cells and/or subjects. In some embodiments of the methods of the present invention, one or more TMPRSS6 dsRNA agents are administered to cells and/or subjects to treat a disease or condition associated with TMPRSS6 expression and activity. In some embodiments, the methods of the present invention comprise administering one or more TMPRSS6 dsRNA agents to a subject in need of such treatment to reduce a disease or condition associated with TMPRSS6 expression in the subject. The TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents of the present invention can be administered to reduce TMPRSS6 expression and/or activity in one or more of in vitro cells, ex vivo cells, and in vivo cells.
在本發明的一些實施方案中，通過將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑遞送（例如，引入）至細胞中，降低了細胞中的TMPRSS6多肽水平，並因此降低了該細胞中的TMPRSS6多肽活性。靶向劑和方法可用於輔助將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑遞送至受試者體內的特定細胞類型、細胞亞型、器官、空間區域，以及/或遞送至細胞內的亞細胞區域。TMPRSS6 dsRNA藥劑可在本發明的某些方法中單獨施用或者與一種或多種另外的TMPRSS6 dsRNA藥劑組合施用。在一些實施方案中，將2、3、4種或更多種獨立選擇的TMPRSS6 dsRNA藥劑施用於受試者。In some embodiments of the present invention, by delivering (e.g., introducing) a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent into a cell, the level of TMPRSS6 polypeptide in the cell is reduced, and thus the activity of the TMPRSS6 polypeptide in the cell is reduced. Targeting agents and methods can be used to facilitate delivery of TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents to specific cell types, cell subtypes, organs, spatial regions, and/or to subcellular regions within a subject. In certain methods of the present invention, a TMPRSS6 dsRNA agent can be administered alone or in combination with one or more additional TMPRSS6 dsRNA agents. In some embodiments, two, three, four, or more independently selected TMPRSS6 dsRNA agents are administered to a subject.
在本發明的某些實施方案中，將TMPRSS6 dsRNA藥劑和與一種或多種治療TMPRSS6相關疾病或病症的另外的治療方案聯合施用於受試者以治療該TMPRSS6相關疾病或病症。另外的治療方案的非限制性示例是：施用一種或多種本發明的TMPRSS6反義多核苷酸，施用非TMPRSS6 dsRNA治療劑，以及行為矯正。可在以下時間中的一個或多個時間施用另外的治療方案：在施用本發明的TMPRSS6 dsRNA藥劑之前、同時和之後。應當理解，如本文所用的同時、在時間零的5分鐘內、在時間零的10分鐘內、在時間零的30分鐘內、在時間零的45分鐘內以及在時間零的60分鐘內，其中“時間零”是將本發明的TMPRSS6 dsRNA藥劑施用於受試者的時間。非TMPRSS6 dsRNA治療劑的非限制性示例是：鐵螯合劑（例如去鐵胺、地拉羅司（Exjade）、去鐵酮、維生素E、小麥胚芽油、生育酚和黃素）、葉酸、輸血、放血、止癢劑、收斂劑、局部麻醉劑或抗炎劑、治療潰瘍的藥劑、增加胎兒血紅蛋白水平的藥劑（例如羥基脲）、控制感染的藥劑（例如抗生素和抗病毒藥）、治療血栓形成狀態的藥劑。這些治療劑和其他治療劑以及行為矯正是本領域已知的，並且用於治療受試者的TMPRSS6相關疾病或病症，並且可與一種或多種本發明的TMPRSS6 dsRNA藥劑的施用組合而施用於受試者以治療TMPRSS6相關疾病或病症。施用於細胞或受試者以治療TMPRSS6相關疾病或病症的本發明的TMPRSS6 dsRNA藥劑可以協同方式與一種或多種其他治療劑或治療性活動作用，並增強該一種或多種治療劑或活性劑的有效性以及/或增強該TMPRSS6 dsRNA藥劑在治療該TMPRSS6相關疾病或病症方面的有效性。In certain embodiments of the present invention, a TMPRSS6 dsRNA agent is administered to a subject in combination with one or more additional therapeutic regimens for treating a TMPRSS6-associated disease or condition to treat the TMPRSS6-associated disease or condition. Non-limiting examples of additional therapeutic regimens include administration of one or more TMPRSS6 antisense polynucleotides of the present invention, administration of a non-TMPRSS6 dsRNA therapeutic agent, and behavioral modification. The additional therapeutic regimen can be administered at one or more of the following times: before, concurrently, or after administration of the TMPRSS6 dsRNA agent of the present invention. It should be understood that as used herein, simultaneously, within 5 minutes of time zero, within 10 minutes of time zero, within 30 minutes of time zero, within 45 minutes of time zero, and within 60 minutes of time zero, wherein "time zero" is the time at which the TMPRSS6 dsRNA agent of the present invention is administered to the subject. Non-limiting examples of non-TMPRSS6 dsRNA therapeutics are: iron chelators (e.g., deferoxamine, deferasirox (Exjade), deferoxone, vitamin E, wheat germ oil, tocopherols and flavins), folic acid, blood transfusions, phlebotomies, antipruritics, astringents, local anesthetics or anti-inflammatory agents, agents for treating ulcers, agents that increase fetal hemoglobin levels (e.g., hydroxyureas), agents that control infection (e.g., antibiotics and antivirals), agents for treating thrombotic states. These and other therapeutic agents and behavioral modifiers are known in the art and are used to treat TMPRSS6-associated diseases or conditions in subjects and can be administered to subjects in combination with administration of one or more TMPRSS6 dsRNA agents of the present invention to treat TMPRSS6-associated diseases or conditions. TMPRSS6 dsRNA agents of the present invention administered to cells or subjects to treat TMPRSS6-associated diseases or conditions can act synergistically with one or more other therapeutic agents or therapeutic activities and enhance the effectiveness of the one or more therapeutic agents or activities and/or enhance the effectiveness of the TMPRSS6 dsRNA agent in treating the TMPRSS6-associated disease or condition.
本發明的包括施用TMPRSS6 dsRNA藥劑的治療方法可在以下時間使用：在TMPRSS6相關疾病或病症發作之前以及/或當TMPRSS6相關疾病或病症存在時，包括該疾病或病症的早期、中期和晚期以及在這些階段中的任一者之前和之後的所有時間。本發明的方法還可用於治療先前已用一種或多種其他治療劑和/或治療性活動治療過的患有治療TMPRSS6相關疾病或病症的受試者，這些治療劑和/或治療性活動在治療該受試者的TMPRSS6相關疾病或病症方面不成功、最低限度地成功和/或不再成功。The therapeutic methods of the present invention comprising administering a TMPRSS6 dsRNA agent can be used before the onset of a TMPRSS6-related disease or condition and/or while a TMPRSS6-related disease or condition is present, including the early, middle, and late stages of the disease or condition, and all times before and after any of these stages. The methods of the present invention can also be used to treat a subject with a TMPRSS6-related disease or condition who has been previously treated with one or more other therapeutic agents and/or therapeutic activities that were unsuccessful, minimally successful, and/or no longer successful in treating the subject's TMPRSS6-related disease or condition.
載體編碼的Carrier EncodeddsRNAdsRNA
在本發明的某些實施方案中，可使用載體將TMPRSS6 dsRNA藥劑遞送至細胞中。TMPRSS6 dsRNA藥劑轉錄單元可包含在DNA或RNA載體中。製備和使用此類編碼轉基因的載體以將序列遞送至細胞和/或受試者中是本領域熟知的。載體可用於本發明的導致TMPRSS6 dsRNA瞬時表達的方法中，例如達至少1小時、2小時、3小時、4小時、5小時、6小時、7小時、8小時、9小時、10小時或更多小時，1周、2周、3周、4周、5周、6周、7周、8周、9周、10周或更多周。瞬時表達的時長可使用基於元件（諸如但不限於所選的特定載體構建體以及靶細胞和/或組織）的常規方法來測定。此類轉基因可作為線性構建體、環狀質粒或病毒載體引入，該病毒載體可以是整合載體或非整合載體。轉基因還可被構建成使其作為染色體外質粒遺傳（Gassmann等人，Proc.Natl.Acad.Sci.USA (1995) 92:1292）。In certain embodiments of the present invention, vectors can be used to deliver TMPRSS6 dsRNA agents to cells. The transcriptional unit of the TMPRSS6 dsRNA agent can be contained in a DNA or RNA vector. The preparation and use of such vectors encoding transgenes for delivery of sequences to cells and/or subjects is well known in the art. The vectors can be used in the methods of the present invention to cause transient expression of TMPRSS6 dsRNA, for example, for at least 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, or more hours, or for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, or more weeks. The duration of transient expression can be determined using conventional methods based on factors such as, but not limited to, the specific vector construct selected and the target cells and/or tissues. Such transgenes can be introduced as linear constructs, circular plasmids, or viral vectors, which can be integrating or non-integrating. Transgenes can also be constructed so that they are transmitted as extrachromosomal plasmids (Gassmann et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).
TMPRSS6 dsRNA藥劑的一條或多條單鏈可由表達載體上的啟動子轉錄。當表達兩條單獨的鏈以產生例如dsRNA時，可使用諸如轉染或感染的方式將兩個單獨的表達載體共同引入細胞中。在某些實施方案中，本發明的TMPRSS6 dsRNA藥劑的每條單獨的鏈可由均包含在相同表達載體上的啟動子轉錄。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑被表達為通過接頭多核苷酸序列接合的反向重複多核苷酸，使得TMPRSS6 dsRNA藥劑含有莖環結構。One or more individual strands of a TMPRSS6 dsRNA agent can be transcribed from a promoter on an expression vector. When expressing two separate strands to produce, for example, dsRNA, the two separate expression vectors can be co-introduced into cells using methods such as transfection or infection. In certain embodiments, each individual strand of a TMPRSS6 dsRNA agent of the present invention can be transcribed from a promoter contained on the same expression vector. In certain embodiments of the present invention, the TMPRSS6 dsRNA agent is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence, such that the TMPRSS6 dsRNA agent comprises a stem-loop structure.
RNA表達載體的非限制性示例是DNA質粒或病毒載體。可用於本發明實施方案的表達載體可與真核細胞相容。真核細胞表達載體是本領域常規使用的，並且可從多種商業來源獲得。對表達TMPRSS6 dsRNA的載體的遞送可以是全身性的，諸如通過靜脈內或肌內施用，通過施用於從受試者體內移植隨後重新引入受試者體內的靶細胞，或者通過任何其他允許引入期望的靶細胞中的方式。Non-limiting examples of RNA expression vectors are DNA plasmids or viral vectors. Expression vectors useful in embodiments of the present invention may be compatible with eukaryotic cells. Eukaryotic cell expression vectors are routinely used in the art and are available from a variety of commercial sources. Delivery of vectors expressing TMPRSS6 dsRNA can be systemic, such as by intravenous or intramuscular administration, by administration to target cells removed from a subject and subsequently reintroduced into the subject, or by any other means that allows for introduction into the desired target cells.
可包含在本發明的方法的實施方案中的病毒載體系統包括但不限於(a)腺病毒載體；(b)逆轉錄病毒載體，包括但不限於慢病毒載體、莫洛尼鼠白血病病毒等；(c)腺相關病毒載體；(d)單純皰疹病毒載體；(e)SV 40載體；(f)多瘤病毒載體；(g)乳頭狀瘤病毒載體；(h)小核糖核酸病毒載體；(i)痘病毒載體，諸如正痘病毒，例如牛痘病毒載體或禽痘，例如金絲雀痘或雞痘；和(j)輔助依賴性腺病毒或無宿主腺病毒。用於TMPRSS6 dsRNA藥劑的重組表達的構建體可包括調控元件，諸如啟動子、增強子等，其可被選擇來提供組成型或調控型/誘導型表達。病毒載體系統以及啟動子和增強子的使用等是本領域常規的，並且可與本文所述的方法和組合物聯合使用。Viral vector systems that may be included in embodiments of the methods of the present invention include, but are not limited to, (a) adenoviral vectors; (b) retroviral vectors, including, but not limited to, lentiviral vectors, Moloney murine leukemia virus, and the like; (c) adeno-associated viral vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) poxvirus vectors, such as orthopoxviruses, e.g., cowpox virus vectors, or avian pox, e.g., canarypox or chickenpox; and (j) helper-dependent adenoviruses or host-free adenoviruses. Constructs for recombinant expression of TMPRSS6 dsRNA agents can include regulatory elements, such as promoters and enhancers, which can be selected to provide constitutive or regulated/induced expression. Viral vector systems and the use of promoters and enhancers are routine in the art and can be used in conjunction with the methods and compositions described herein.
本發明的某些實施方案包括使用病毒載體將TMPRSS6 dsRNA藥劑遞送至細胞中。許多基於腺病毒的遞送系統在本領域中常規用於遞送至例如肺、肝、中樞神經系統、內皮細胞和肌肉中。可用於本發明的方法的病毒載體的非限制性示例是：AAV載體、痘病毒諸如牛痘病毒、改良的安卡拉病毒（MVA）、NYVAC、禽痘諸如雞痘或金絲雀痘。Certain embodiments of the present invention involve the use of viral vectors to deliver TMPRSS6 dsRNA agents to cells. Numerous adenovirus-based delivery systems are routinely used in the art for delivery to, for example, the lungs, liver, central nervous system, endothelial cells, and muscle. Non-limiting examples of viral vectors that can be used in the methods of the present invention include AAV vectors, poxviruses such as vaccinia virus, modified Ankara virus (MVA), NYVAC, and avian poxviruses such as chickenpox or canarypox.
本發明的某些實施方案包括使用載體將TMPRSS6 dsRNA藥劑遞送至細胞中的方法，並且此類載體可位於藥學上可接受的載劑中，該藥學上可接受的載劑可以但不一定包括其中包埋有基因遞送媒介物的緩釋基質。在一些實施方案中，用於遞送TMPRSS6 dsRNA的載體可由重組細胞產生，並且本發明的藥物組合物可包括一種或多種產生TMPRSS6 dsRNA遞送系統的細胞。Certain embodiments of the present invention include methods for delivering TMPRSS6 dsRNA agents to cells using vectors, and such vectors can be in a pharmaceutically acceptable carrier, which may, but need not, include a sustained-release matrix in which the gene delivery vehicle is embedded. In some embodiments, the vectors used to deliver TMPRSS6 dsRNA can be produced by recombinant cells, and the pharmaceutical compositions of the present invention can include one or more cells that produce the TMPRSS6 dsRNA delivery system.
含有containTMPRSS6 dsRNATMPRSS6 dsRNA或orssRNAssRNA藥劑的藥物組合物Drug combinations of pharmaceuticals
本發明的某些實施方案包括使用含有TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑和藥學上可接受的載劑的藥物組合物。含有TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的藥物組合物可用於本發明的方法中以降低細胞中的TMPRSS6基因表達和TMPRSS6活性，並且可用於治療TMPRSS6相關疾病或病症。此類藥物組合物可基於遞送模式來配製。用於遞送模式的製劑的非限制性示例是：被配製用於皮下遞送的組合物、被配製用於經由腸胃外遞送的全身性施用的組合物、被配製用於靜脈內（IV）遞送的組合物、被配製用於鞘內遞送的組合物、被配製用於直接遞送至大腦中的組合物等。施用本發明的藥物組合物以將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑遞送至細胞中可使用一種或多種方法進行，諸如：局部（例如通過經皮貼劑）、肺部（例如通過吸入或吹入粉末或氣溶膠，包括通過噴霧器）、氣管內、鼻內、表皮和經皮、口服或腸胃外施用。腸胃外施用包括靜脈內、動脈內、皮下、腹膜內或肌內注射或輸注；皮下施用，例如經由植入裝置；或顱內施用，例如通過實質內、鞘內或心室內施用。還可將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑直接遞送至靶組織中，例如直接遞送至肝臟中、直接遞送至腎臟中等。應當理解，“遞送TMPRSS6 dsRNA藥劑”或“遞送TMPRSS6反義多核苷酸藥劑”進入細胞涵蓋分別直接遞送TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑，以及在細胞中由遞送至細胞中的編碼載體表達TMPRSS6 dsRNA藥劑，或者通過任何合適的方式使TMPRSS6 dsRNA或TMPRSS6反義多核苷酸藥劑存在於細胞中。製劑的製備和使用以及用於遞送抑制性RNA的方式是本領域熟知的且常規使用的。Certain embodiments of the present invention involve the use of pharmaceutical compositions containing a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent and a pharmaceutically acceptable carrier. Pharmaceutical compositions containing a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can be used in the methods of the present invention to reduce TMPRSS6 gene expression and TMPRSS6 activity in cells and to treat TMPRSS6-related diseases or conditions. Such pharmaceutical compositions can be formulated based on the mode of delivery. Non-limiting examples of formulations for delivery modes are: compositions formulated for subcutaneous delivery, compositions formulated for systemic administration via parenteral delivery, compositions formulated for intravenous (IV) delivery, compositions formulated for intrathecal delivery, compositions formulated for direct delivery into the brain, etc. Administration of the pharmaceutical compositions of the present invention to deliver TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents to cells can be performed using one or more methods, such as: topical (e.g., via a transdermal patch), pulmonary (e.g., by inhalation or insufflation of a powder or aerosol, including via a nebulizer), intratracheal, intranasal, epidermal, and transdermal, oral, or parenteral administration. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; subcutaneous administration, for example, via an implant; or intracranial administration, for example, via intraparenchymal, intrathecal, or intraventricular administration. TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents can also be delivered directly to target tissues, for example, directly to the liver, directly to the kidneys, etc. It should be understood that "delivering a TMPRSS6 dsRNA agent" or "delivering a TMPRSS6 antisense polynucleotide agent" into a cell encompasses directly delivering a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent, respectively, as well as expressing a TMPRSS6 dsRNA agent in a cell from an encoding vector delivered to the cell, or causing the TMPRSS6 dsRNA or TMPRSS6 antisense polynucleotide agent to be present in the cell by any suitable means. The preparation and use of agents and methods for delivering inhibitory RNA are well known and routinely used in the art.
如本文所用，“藥物組合物”包含藥理學有效量的本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑和藥學上可接受的載劑。術語“藥學上可接受的載劑”是指用於施用治療劑的載劑。此類載劑包括但不限於鹽水、緩衝鹽水、右旋糖、水、甘油、乙醇以及它們的組合。該術語特別排除細胞培養基。對於口服施用的藥物，藥學上可接受的載劑包括但不限於藥學上可接受的賦形劑，諸如惰性稀釋劑、崩解劑、粘合劑、潤滑劑、甜味劑、調味劑、著色劑和防腐劑。合適的惰性稀釋劑包括碳酸鈉和碳酸鈣、磷酸鈉和磷酸鈣以及乳糖，同時玉米澱粉和海藻酸是合適的崩解劑。粘合劑可包括澱粉和明膠，同時潤滑劑（如果存在）通常是硬脂酸鎂、硬脂酸或滑石。如果需要，片劑可用諸如單硬脂酸甘油酯或二硬脂酸甘油酯的材料包衣，以延遲在胃腸道中的吸收。包含在藥物製劑中的藥劑在下文中進一步描述。As used herein, a "pharmaceutical composition" comprises a pharmacologically effective amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent of the present invention and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent. Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The term specifically excludes cell culture media. For drugs administered orally, pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweeteners, flavorings, coloring agents, and preservatives. Suitable inert diluents include sodium and calcium carbonates, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, if present, are typically magnesium stearate, stearic acid, or talc. If desired, tablets may be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. The agents included in the pharmaceutical formulations are further described below.
如本文所用，術語諸如：“藥理學有效量”、“治療有效量”和“有效量”是指產生預期的藥理學、治療性或預防性結果的本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。例如，如果給定的臨床治療在與疾病或障礙相關的可測量參數降低至少10%時被認為是有效的，則用於治療該疾病或障礙的藥物的治療有效量是使該參數降低至少10%所必需的量。例如，治療有效量的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可使TMPRSS6多肽水平降低至少10%。As used herein, terms such as "pharmacologically effective amount," "therapeutically effective amount," and "effective amount" refer to an amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention that produces a desired pharmacological, therapeutic, or preventive result. For example, if a given clinical treatment is considered effective when a measurable parameter associated with a disease or disorder is reduced by at least 10%, then a therapeutically effective amount of the agent for treating that disease or disorder is the amount necessary to reduce that parameter by at least 10%. For example, a therapeutically effective amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can reduce TMPRSS6 polypeptide levels by at least 10%.
有效量effective dose
本發明的方法在某些方面包括使細胞與有效量的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑接觸，以降低所接觸的細胞中的TMPRSS6基因表達。本發明的方法的某些實施方案包括將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑以有效降低受試者的TMPRSS6基因表達且治療TMPRSS6相關疾病或病症的量施用於該受試者。就降低TMPRSS6的表達以及/或治療TMPRSS6相關疾病或病症而言，所使用的“有效量”是實現期望的生物學效應所必需或足夠的量。例如，治療TMPRSS6相關疾病或病症的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量可以是(i)減緩或停止疾病或病症的進展；或(ii)逆轉、減輕或消除該疾病或病症的一種或多種症狀所必需的量。在本發明的一些方面，有效量是當施用於需要治療TMPRSS6相關疾病或病症的受試者時產生預防和/或治療該疾病或病症的治療反應的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。根據本發明的一些方面，有效量是當與用於TMPRSS6相關疾病或病症的另一種治療性處理組合或共同施用時產生預防和/或治療該疾病或病症的治療反應的本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。在本發明的一些實施方案中，用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑治療受試者的生物學效應可以是改善和/或完全消除由TMPRSS6相關疾病或病症引起的症狀。在本發明的一些實施方案中，生物學效應是指TMPRSS6相關疾病或病症的完全消除，例如通過指示受試者沒有TMPRSS6相關疾病或病症的診斷測試來證明。可檢測的生理症狀的非限制性示例包括在施用本發明的藥劑後受試者肝臟中TMPRSS6水平的降低。可使用本領域已知的其他評估TMPRSS6相關疾病或病症的狀態的方式來測定本發明的藥物和/或方法對TMPRSS6相關疾病或病症的效果。In certain aspects, the methods of the present invention comprise contacting a cell with an effective amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent to reduce TMPRSS6 gene expression in the contacted cell. Certain embodiments of the methods of the present invention comprise administering to a subject an amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent effective to reduce TMPRSS6 gene expression and treat a TMPRSS6-associated disease or condition in the subject. As used herein, an "effective amount" with respect to reducing TMPRSS6 expression and/or treating a TMPRSS6-associated disease or condition is an amount necessary or sufficient to achieve a desired biological effect. For example, an effective amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent for treating a TMPRSS6-associated disease or condition can be the amount necessary to (i) slow or halt the progression of the disease or condition; or (ii) reverse, alleviate, or eliminate one or more symptoms of the disease or condition. In some aspects of the present invention, an effective amount is an amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent that, when administered to a subject in need of treatment for a TMPRSS6-associated disease or condition, produces a therapeutic response that prevents and/or treats the disease or condition. According to some aspects of the invention, an effective amount is an amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention that, when combined or co-administered with another therapeutic treatment for a TMPRSS6-associated disease or condition, produces a therapeutic response that prevents and/or treats the disease or condition. In some embodiments of the invention, the biological effect of treating a subject with a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention can be improvement and/or complete elimination of symptoms caused by a TMPRSS6-associated disease or condition. In some embodiments of the invention, the biological effect refers to complete elimination of a TMPRSS6-associated disease or condition, as demonstrated, for example, by a diagnostic test indicating that the subject is free of the TMPRSS6-associated disease or condition. Non-limiting examples of detectable physiological symptoms include a decrease in TMPRSS6 levels in the liver of a subject after administration of an agent of the present invention. Other methods known in the art for assessing the status of a TMPRSS6-related disease or condition can be used to determine the effect of the agents and/or methods of the present invention on a TMPRSS6-related disease or condition.
典型地，將在臨床試驗中測定使TMPRSS6多肽活性降低至治療TMPRSS6相關疾病或病症的水平的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量，從而在盲法研究中確立測試群體與對照群體的有效劑量。在一些實施方案中，有效量將是產生期望反應的量，例如，減輕患有TMPRSS6相關疾病或病症的細胞、組織和/或受試者的該疾病或病症的量。因此，用於治療可通過降低TMPRSS6多肽活性來治療的TMPRSS6相關疾病或病症的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量可以是這樣的量：在施用時使受試者的TMPRSS6多肽活性的量降低至低於在沒有施用TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的情況下存在於細胞、組織和/或受試者中的量的量。在本發明的某些方面，在未接觸或未施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的細胞、組織和/或受試者中存在的TMPRSS6多肽活性和/或TMPRSS6基因表達的水平被稱為“對照”量。在本發明的方法的一些實施方案中，受試者的對照量是受試者的治療前量，換句話說，在施用TMPRSS6藥劑之前受試者中的水平可以是該受試者的對照水平，並與在將siRNA施用於該受試者之後受試者的TMPRSS6多肽活性和/或TMPRSS6基因表達的水平進行比較。在治療TMPRSS6相關疾病或病症的情況下，期望反應可以是減輕或消除細胞、組織和/或受試者的疾病或病症的一種或多種症狀。鈣降低或消除可以是暫時的，或者可以是永久的。應當理解，TMPRSS6相關疾病或病症的狀態可使用測定TMPRSS6多肽活性、TMPRSS6基因表達、症狀評估、臨床試驗等的方法來監測。在本發明的一些方面，對與TMPRSS6相關的疾病或病症的治療的期望反應是延遲或者甚至預防該疾病或病症的發作。Typically, an effective amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent that reduces TMPRSS6 polypeptide activity to a level that treats a TMPRSS6-associated disease or condition will be determined in a clinical trial, thereby establishing effective doses for test and control populations in a blinded study. In some embodiments, an effective amount will be an amount that produces a desired response, for example, a reduction in the disease or condition in cells, tissues, and/or subjects suffering from a TMPRSS6-associated disease or condition. Thus, an effective amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent for treating a TMPRSS6-related disease or condition treatable by reducing TMPRSS6 polypeptide activity can be an amount that, when administered, reduces the amount of TMPRSS6 polypeptide activity in a subject to below the amount present in cells, tissues, and/or subjects in the absence of administration of the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent. In certain aspects of the invention, the level of TMPRSS6 polypeptide activity and/or TMPRSS6 gene expression present in cells, tissues, and/or subjects that have not been exposed to or administered a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention is referred to as a "control" amount. In some embodiments of the methods of the present invention, the control amount in a subject is the subject's pre-treatment amount. In other words, the level in a subject before administration of a TMPRSS6 agent can be the subject's control level and compared to the level of TMPRSS6 polypeptide activity and/or TMPRSS6 gene expression in the subject after administration of the siRNA to the subject. In the case of treating a TMPRSS6-related disease or condition, the desired response can be a reduction or elimination of one or more symptoms of the disease or condition in cells, tissues, and/or the subject. The reduction or elimination of calcium can be temporary or permanent. It should be understood that the status of a TMPRSS6-related disease or condition can be monitored using methods such as assays for TMPRSS6 polypeptide activity, TMPRSS6 gene expression, symptom assessment, clinical trials, and the like. In some aspects of the invention, the desired response to treatment of a disease or condition associated with TMPRSS6 is to delay or even prevent the onset of the disease or condition.
降低TMPRSS6多肽活性的化合物的有效量還可通過評估TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的施用對細胞或受試者的生理效應（諸如TMPRSS6相關疾病或病症在施用後的減輕）來測定。對受試者的測定和/或症狀監測可用於測定可以本發明的藥物化合物形式施用的本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的功效，並且用於測定存在或不存在對治療的反應。一個非限制性示例是，對血清鐵調素水平、鐵水平、轉鐵蛋白飽和水平的一種或多種本領域已知的測試。另一個非限制性示例是，在用本發明的TMPRSS6 dsRNA藥劑治療受試者之前和之後，可使用一種或多種本領域已知的肝功能測試來確定該受試者的TMPRSS6相關疾病的狀態。An effective amount of a compound that reduces the activity of a TMPRSS6 polypeptide can also be determined by assessing the physiological effects of administration of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent on cells or subjects (e.g., amelioration of a TMPRSS6-related disease or condition following administration). Assays and/or symptom monitoring in subjects can be used to determine the efficacy of the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention, which can be administered as a pharmaceutical compound of the present invention, and to determine the presence or absence of a response to treatment. A non-limiting example is one or more of the tests known in the art for serum hepcidin levels, iron levels, or transferrin saturation levels. As another non-limiting example, one or more liver function tests known in the art can be used to determine the status of a TMPRSS6-related disease in a subject before and after treatment with a TMPRSS6 dsRNA agent of the invention.
本發明的一些實施方案包括通過評估和/或監測受試者的TMPRSS6相關疾病或病症的一種或多種“生理特徵”來測定施用於該受試者的本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑治療該TMPRSS6相關疾病或病症的功效的方法。TMPRSS6相關疾病或病症的生理特徵的非限制性示例是TMPRSS6 mRNA水平、TMPRSS6蛋白水平或鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平、Hb A2水平等。測定此類生理特徵的標準方式是本領域已知的，包括但不限於血液測試、影像學研究、身體檢查等。Some embodiments of the present invention include methods for determining the efficacy of a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention in treating a TMPRSS6-related disease or condition administered to a subject by assessing and/or monitoring one or more "physiological characteristics" of the subject's TMPRSS6-related disease or condition. Non-limiting examples of physiological characteristics of a TMPRSS6-related disease or condition include TMPRSS6 mRNA levels, TMPRSS6 protein levels, or hepcidin levels, iron levels, transferrin saturation levels, and red blood cell (RBC) count measurements, hemoglobin levels, ferritin levels, Hb F levels, Hb A2 levels, and the like. Standard methods for measuring such physiological characteristics are known in the art and include, but are not limited to, blood tests, imaging studies, physical examinations, and the like.
應當理解，至少部分地基於針對受試者測定的疾病和/或病症狀態和/或生理特徵的此類測定結果，可改變施用於該受試者的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。治療量可有所變化，例如通過增加或減少TMPRSS6-dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量，通過改變其中分別施用TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的組合物，通過改變施用途徑，通過改變施用時間等。TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量將隨以下項而變化：所治療的特定病症、所治療的受試者的年齡和身體狀況、病症的嚴重程度、治療的持續時間、同步治療（如果有的話）的性質、具體的施用途徑以及醫療保健人員的知識和專業技能範圍的其他因素。例如，有效量可取決於有效治療TMPRSS6相關疾病或病症的TMPRSS6多肽活性和/或TMPRSS6基因表達的期望水平。技術人員可憑經驗確定用於本發明方法的本發明的特定TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量，而不需要進行過多的實驗。與本文所提供的教導內容結合，通過從本發明的各種TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑中進行選擇，以及權衡諸如效力、相對生物利用度、患者體重、不良副作用的嚴重程度和優選的施用模式等因素，可制定有效治療特定受試者的有效預防性或治療性處理方案。如本發明的實施方案中使用的，本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的有效量可以是當與細胞接觸時在細胞中產生期望的生物學效應的量。It will be appreciated that the amount of TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent administered to a subject can be varied, at least in part, based on the results of such determinations of the disease and/or condition state and/or physiological characteristics determined for the subject. The therapeutic amount can be varied, for example, by increasing or decreasing the amount of the TMPRSS6-dsRNA agent or TMPRSS6 antisense polynucleotide agent, by changing the composition in which the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent, respectively, is administered, by changing the route of administration, by changing the time of administration, and the like. The effective amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent will vary depending on the specific condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of treatment, the nature of concurrent treatment (if any), the specific route of administration, and other factors within the knowledge and expertise of a healthcare professional. For example, the effective amount may depend on the desired level of TMPRSS6 polypeptide activity and/or TMPRSS6 gene expression to effectively treat a TMPRSS6-associated disease or condition. The effective amount of a particular TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention for use in the methods of the present invention can be determined empirically by a skilled artisan without undue experimentation. In combination with the teachings provided herein, by selecting from the various TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents of the present invention and weighing factors such as potency, relative bioavailability, patient weight, severity of adverse side effects, and preferred mode of administration, an effective preventive or therapeutic treatment regimen can be formulated to effectively treat a particular subject. As used in the embodiments of the present invention, an effective amount of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention can be an amount that produces a desired biological effect in a cell when in contact with the cell.
應當認識到，TMPRSS6基因沉默可通過任何表達TMPRSS6的細胞（無論是組成型還是通過基因組工程型）和任何適當的測定法來測定。在本發明的一些實施方案中，通過施用本發明的TMPRSS6 dsRNA藥劑，TMPRSS6基因表達降低至少5%、6%、7%、8%、9%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或100%。在本發明的一些實施方案中，通過施用本發明的TMPRSS6 dsRNA藥劑，TMPRSS6基因表達降低5%至10%、5%至25%、10%至50%、10%至75%、25%至75%、25%至100%或50%至100%。It will be appreciated that TMPRSS6 gene silencing can be determined by any cell expressing TMPRSS6 (whether constitutively or genomically engineered) and by any appropriate assay. In some embodiments of the invention, TMPRSS6 gene expression is reduced by at least 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% by administering a TMPRSS6 dsRNA agent of the invention. In some embodiments of the invention, TMPRSS6 gene expression is reduced by 5% to 10%, 5% to 25%, 10% to 50%, 10% to 75%, 25% to 75%, 25% to 100%, or 50% to 100% by administering a TMPRSS6 dsRNA agent of the invention.
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TMPRSS6 dsRNA藥劑和TMPRSS6反義多核苷酸藥劑在藥物組合物中以足以抑制TMPRSS6基因表達的劑量遞送。在本發明的某些實施方案中，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的劑量在每天0.01毫克/千克接受者體重至200.0毫克/千克接受者體重的範圍內，通常在每天1mg/kg體重至50mg/kg體重、5mg/kg體重至40mg/kg體重、10mg/kg體重至30mg/kg體重、1mg/kg體重至20mg/kg體重、1mg/kg體重至10mg/kg體重、4mg/kg體重至15mg/kg體重的範圍內，包括端值。例如，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可以每次單劑量約0.01mg/kg、0.05mg/kg、0.1mg/kg、0.2mg/kg、0.3mg/kg、0.4mg/kg、0.5mg/kg、1mg/kg、1.1mg/kg、1.2mg/kg、1.3mg/kg、1.4mg/kg、1.5mg/kg、1.6mg/kg、1.7mg/kg、1.8mg/kg、1.9mg/kg、2mg/kg、2.1mg/kg、2.2mg/kg、2.3mg/kg、2.4mg/kg、2.5mg/kg、2.6mg/kg、2.7mg/kg、2.8mg/kg、2.9mg/kg、3.0mg/kg、3.1mg/kg、3.2mg/kg、3.3mg/kg、3.4mg/kg、3.5mg/kg、3.6mg/kg、3.7mg/kg、3.8mg/kg、3.9mg/kg、4mg/kg、4.1mg/kg、4.2mg/kg、4.3mg/kg、4.4mg/kg、4.5mg/kg、4.6mg/kg、4.7mg/kg、4.8mg/kg、4.9mg/kg、5mg/kg、5.1mg/kg、5.2mg/kg、5.3mg/kg、5.4mg/kg、5.5mg/kg、5.6mg/kg、5.7mg/kg、5.8mg/kg、5.9mg/kg、6mg/kg、6.1mg/kg、6.2mg/kg、6.3mg/kg、6.4mg/kg、6.5mg/kg、6.6mg/kg、6.7mg/kg、6.8mg/kg、6.9mg/kg、7mg/kg、7.1mg/kg、7.2mg/kg、7.3mg/kg、7.4mg/kg、7.5mg/kg、7.6mg/kg、7.7mg/kg、7.8mg/kg、7.9mg/kg、8mg/kg、8.1mg/kg、8.2mg/kg、8.3mg/kg、8.4mg/kg、8.5mg/kg、8.6mg/kg、8.7mg/kg、8.8mg/kg、8.9mg/kg、9mg/kg、9.1mg/kg、9.2mg/kg、9.3mg/kg、9.4mg/kg、9.5mg/kg、9.6mg/kg、9.7mg/kg、9.8mg/kg、9.9mg/kg、10mg/kg、11mg/kg、12mg/kg、13mg/kg、14mg/kg、15mg/kg、16mg/kg、17mg/kg、18mg/kg、19mg/kg、20mg/kg、21mg/kg、22mg/kg、23mg/kg、24mg/kg、25mg/kg、26mg/kg、27mg/kg、28mg/kg、29mg/kg、30mg/kg、31mg/kg、32mg/kg、33mg/kg、34mg/kg、35mg/kg、36mg/kg、37mg/kg、38mg/kg、39mg/kg、40mg/kg、41mg/kg、42mg/kg、43mg/kg、44mg/kg、45mg/kg、46mg/kg、47mg/kg、48mg/kg、49mg/kg至50mg/kg體重的量施用。TMPRSS6 dsRNA agents and TMPRSS6 antisense polynucleotide agents are delivered in pharmaceutical compositions in an amount sufficient to inhibit TMPRSS6 gene expression. In certain embodiments of the present invention, the dosage of TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents is in the range of 0.01 mg/kg to 200.0 mg/kg of recipient body weight per day, typically in the range of 1 mg/kg to 50 mg/kg, 5 mg/kg to 40 mg/kg, 10 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 4 mg/kg to 15 mg/kg, including end values. For example, TMPRSS6 The dsRNA agent or TMPRSS6 antisense polynucleotide agent can be administered at a single dose of about 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, 0.3 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.1 mg/kg, 1.2 mg/kg, 1.3 mg/kg, 1.4 mg/kg, 1.5 mg/kg, 1.6 mg/kg, 1.7 mg/kg, 1.8 mg/kg, 1.9 mg/kg, 2 mg/kg, 2.1 mg/kg, 2.2 mg/kg, 2.3 mg/kg, 2.4 mg/kg, 2.5 mg/kg, 2.6 mg/kg, 2.7 mg/kg, 2.8 mg/kg, 2.9 mg/kg, 3.0 mg/kg, 3.1 mg/kg, 3.2 mg/kg, 3.3 mg/kg. g, 3.4mg/kg, 3.5mg/kg, 3.6mg/kg, 3.7mg/kg, 3.8mg/kg, 3.9mg/kg, 4mg/kg, 4.1mg/kg, 4.2m g/kg, 4.3mg/kg, 4.4mg/kg, 4.5mg/kg, 4.6mg/kg, 4.7mg/kg, 4.8mg/kg, 4.9mg/kg, 5mg/kg, 5. 1mg/kg, 5.2mg/kg, 5.3mg/kg, 5.4mg/kg, 5.5mg/kg, 5.6mg/kg, 5.7mg/kg, 5.8mg/kg, 5.9mg/ kg, 6mg/kg, 6.1mg/kg, 6.2mg/kg, 6.3mg/kg, 6.4mg/kg, 6.5mg/kg, 6.6mg/kg, 6.7mg/kg, 6.8m g/kg, 6.9mg/kg, 7mg/kg, 7.1mg/kg, 7.2mg/kg, 7.3mg/kg, 7.4mg/kg, 7.5mg/kg, 7.6mg/kg, 7 .7mg/kg, 7.8mg/kg, 7.9mg/kg, 8mg/kg, 8.1mg/kg, 8.2mg/kg, 8.3mg/kg, 8.4mg/kg, 8.5mg/kg , 8.6mg/kg, 8.7mg/kg, 8.8mg/kg, 8.9mg/kg, 9mg/kg, 9.1mg/kg, 9.2mg/kg, 9.3mg/kg, 9.4mg /kg, 9.5mg/kg, 9.6mg/kg, 9.7mg/kg, 9.8mg/kg, 9.9mg/kg, 10mg/kg, 11mg/kg, 12mg/kg, 13mg /kg, 14mg/kg, 15mg/kg, 16mg/kg, 17mg/kg, 18mg/kg, 19mg/kg, 20mg/kg, 21mg/kg, 22mg/kg, 23mg/kg, 24mg/kg, 25mg/kg, 26mg/kg, 27mg/kg, 28mg/kg, 29mg/kg, 30mg/kg, 31mg/kg, 32mg/ kg, 33mg/kg, 34mg/kg, 35mg/kg, 36mg/kg, 37mg/kg, 38mg/kg, 39mg/kg, 40mg/kg, 41mg/kg, 42mg/kg, 43mg/kg, 44mg/kg, 45mg/kg, 46mg/kg, 47mg/kg, 48mg/kg, 49mg/kg to 50mg/kg body weight.
在確定本發明的TMPRSS6 dsRNA藥劑的劑量和遞送時間時，可考慮多種因素。所遞送的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的絕對量將取決於多種因素，包括同步治療、劑量數量和個體受試者參數，包括年齡、身體狀況、身高和體重。這些因素是本領域普通技術人員熟知的因素，並且僅通過常規實驗就可解決。在一些實施方案中，可使用最大劑量，即，根據合理醫學判斷的最高安全劑量。A variety of factors can be considered when determining the dosage and delivery schedule of the TMPRSS6 dsRNA agents of the present invention. The absolute amount of TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent delivered will depend on a variety of factors, including concurrent treatment, the number of doses, and individual subject parameters, including age, physical condition, height, and weight. These factors are well known to those of ordinary skill in the art and can be determined through routine experimentation. In some embodiments, a maximum dose can be used, i.e., the highest safe dose based on sound medical judgment.
在一些實施方案中，本發明的方法可包括向受試者施用1、2、3、4、5、6、7、8、9、10次或更多次劑量的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。在一些情況下，可至少每天、每隔一天、每週、每隔一周、每月等向受試者施用藥物化合物（例如，包含TMPRSS6 dsRNA藥劑或包含TMPRSS6反義多核苷酸藥劑）。劑量可每天施用一次或每天施用多次，例如，在一個24小時的時間段內施用2、3、4、5次或更多次。本發明的藥物組合物可每天施用一次，或者TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可以兩次、三次或更多次亞劑量的形式在全天以適當的間隔施用，或者甚至使用持續輸注或通過受控釋放製劑遞送。在本發明的方法的一些實施方案中，將本發明的藥物組合物以每天一次或多次、每週一次或多次、每月一次或多次或每年一次或多次施用於受試者。In some embodiments, the methods of the present invention can include administering 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent to a subject. In some cases, a drug compound (e.g., comprising a TMPRSS6 dsRNA agent or comprising a TMPRSS6 antisense polynucleotide agent) can be administered to a subject at least daily, every other day, weekly, every other week, monthly, etc. The dose can be administered once daily or multiple times daily, for example, 2, 3, 4, 5, or more times within a 24-hour period. The pharmaceutical composition of the present invention can be administered once daily, or the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent can be administered as two, three, or more sub-doses at appropriate intervals throughout the day, or even by continuous infusion or by controlled-release formulation. In some embodiments of the methods of the present invention, the pharmaceutical composition of the present invention is administered to the subject once or more daily, once or more weekly, once or more monthly, or once or more annually.
在一些方面，本發明的方法包括單獨施用藥用化合物，與一種或多種其他TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑組合該施用藥用化合物，以及/或與施用於患有TMPRSS6相關疾病或病症的受試者的其他藥物療法或治療活動或方案組合施用該藥用化合物。藥物化合物可以藥物組合物的形式施用。本發明的方法中使用的藥物組合物可以是無菌的，並且含有一定量的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑，該TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑將使TMPRSS6多肽的活性降低至足以在適於施用於受試者的單位重量或體積內產生期望反應的水平。施用於受試者的包含降低TMPRSS6蛋白活性的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的藥物組合物的劑量可根據不同參數進行選擇，特別是根據所使用的施用模式和受試者的狀態進行選擇。其他因素包括期望的治療時間。如果在施加的初始劑量下受試者的反應不足，則可在患者耐受性允許的範圍內採用更高的劑量（或者通過不同的、更局部的遞送途徑來有效提高劑量）。In some aspects, the methods of the present invention include administering a pharmaceutical compound alone, in combination with one or more other TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents, and/or in combination with other drug therapies or therapeutic activities or regimens administered to a subject suffering from a TMPRSS6-related disease or condition. The pharmaceutical compound can be administered in the form of a pharmaceutical composition. The pharmaceutical composition used in the methods of the present invention can be sterile and contain an amount of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent that will reduce the activity of a TMPRSS6 polypeptide to a level sufficient to produce the desired response per unit weight or volume suitable for administration to a subject. The dose of a pharmaceutical composition comprising a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent that reduces TMPRSS6 protein activity administered to a subject can be selected based on various parameters, particularly the mode of administration used and the subject's condition. Other factors include the desired duration of treatment. If a subject's response to the initial dose is insufficient, a higher dose can be used (or the dose can be effectively increased through a different, more localized delivery route) as tolerated by the patient.
治療treatment
如本文所用，“TMPRSS6相關疾病”、“TMPRSS6相關疾病和病症”和“由TMPRSS6引起和/或調節的疾病和病症”包括與TMPRSS6基因或蛋白相關的任何疾病。此類疾病可能是由以下原因引起的：例如，TMPRSS6蛋白的過量產生、TMPRSS6基因的突變、TMPRSS6蛋白的異常切割、TMPRSS6與其他蛋白質或其他內源性或外源性物質之間的異常相互作用。示例性PCSK 9相關疾病包括但不限於：血色素沉著症（例如遺傳性血色素沉著症、特發性血色素沉著症、原發性血色素沉著症、繼發性血色素沉著症、嚴重的青少年血色素沉著症、新生兒血色素沉著症）、鐵粒幼細胞性貧血、溶血性貧血、紅細胞生成異常性貧血、鐮狀細胞性貧血、血紅蛋白病、地中海貧血（例如β地中海貧血和α地中海貧血）、真性紅細胞增多症、骨髓增生異常綜合征、先天性紅細胞生成異常性貧血、丙酮酸激酶缺乏症、慢性肝病、遲發性皮膚卟啉症、紅細胞生成性卟啉症、轉鐵蛋白缺乏症、遺傳性酪氨酸血症、腦肝腎綜合征、特發性肺含鐵血黃素沉著症、腎含鐵血黃素沉著症、帕金森病、阿爾茨海默病、弗裡德賴希共濟失調，或其他與鐵過載相關的障礙和/或無效紅細胞生成障礙。As used herein, "TMPRSS6-related diseases," "TMPRSS6-related diseases and conditions," and "diseases and conditions caused and/or regulated by TMPRSS6" include any disease associated with the TMPRSS6 gene or protein. Such diseases may be caused by, for example, overproduction of TMPRSS6 protein, mutations in the TMPRSS6 gene, abnormal cleavage of TMPRSS6 protein, abnormal interactions between TMPRSS6 and other proteins or other endogenous or exogenous substances. Exemplary PCSK 9 Related diseases include but are not limited to: hemochromatosis (e.g. hereditary hemochromatosis, idiopathic hemochromatosis, primary hemochromatosis, secondary hemochromatosis, severe juvenile hemochromatosis, neonatal hemochromatosis), sideroblastic anemia, hemolytic anemia, dyserythropoietic anemia, sickle cell anemia, hemoglobinopathy, thalassemia (e.g. beta thalassemia and alpha thalassemia), vera erythrocytosis Polymyalgia, myelodysplastic syndrome, congenital erythropoietic anemia, pyruvate kinase deficiency, chronic liver disease, porphyria cutanea, erythropoietic porphyria, transferrin deficiency, hereditary tyrosinemia, cerebrohepatorenal syndrome, idiopathic pulmonary hemosiderosis, renal hemosiderosis, Parkinson's disease, Alzheimer's disease, Friedreich's ataxia, or other disorders related to iron overload and/or ineffective erythropoiesis.
在本發明的某些方面，可在診斷TMPRSS6相關疾病或病症之前或之後的一個或多個時間，向受試者施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。在本發明的一些方面，受試者處於患有或發展TMPRSS6相關疾病或病症的風險中。處於發展TMPRSS6相關疾病或病症的風險中的受試者是與發展TMPRSS6相關疾病或病症的控制風險相比，發展TMPRSS6相關疾病或病症的可能性增加的受試者。在本發明的一些實施方案中，與控制風險水平相比的風險水平可以是統計學上顯著的。處於風險中的受試者可包括，例如，已經患有或者將患有預先存在的疾病和/或遺傳異常的受試者，該疾病和/或遺傳異常使該受試者比未患有該預先存在的疾病或遺傳異常的對照受試者更容易患上TMPRSS6相關疾病或病症；具有TMPRSS6相關疾病或病症的家族和/或個人病史的受試者；和先前已接受TMPRSS6相關疾病或病症治療的受試者。應當理解，使受試者更容易患上TMPRSS6相關疾病或病症的預先存在的疾病和/或遺傳異常，可以是當存在時先前已被鑒定為與發展TMPRSS6相關疾病或病症的較高可能性具有相關關係的疾病或遺傳異常。In certain aspects of the invention, a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention can be administered to a subject at one or more times before or after diagnosis of a TMPRSS6-related disease or condition. In some aspects of the invention, the subject is at risk of having or developing a TMPRSS6-related disease or condition. A subject at risk of developing a TMPRSS6-related disease or condition is one who has an increased likelihood of developing the TMPRSS6-related disease or condition compared to a control risk for developing the TMPRSS6-related disease or condition. In some embodiments of the invention, the risk level compared to the control risk level can be statistically significant. At-risk subjects may include, for example, subjects who have or will develop a pre-existing disease and/or genetic abnormality that makes the subject more susceptible to developing a TMPRSS6-related disease or condition than a control subject who does not have the pre-existing disease or genetic abnormality; subjects with a family and/or personal history of a TMPRSS6-related disease or condition; and subjects who have previously received treatment for a TMPRSS6-related disease or condition. It should be understood that a pre-existing disease and/or genetic abnormality that makes a subject more susceptible to developing a TMPRSS6-related disease or condition may be a disease or genetic abnormality that, when present, has been previously identified as being associated with a higher likelihood of developing a TMPRSS6-related disease or condition.
應當理解，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可基於個體受試者的醫療狀況而施用於受試者。例如，為受試者提供的醫療保健可評估從受試者獲得的樣本中測量的TMPRSS6水平，並確定通過施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑來降低受試者的TMPRSS6水平是合乎期望的。在該示例中，即使受試者未被診斷為患有TMPRSS6相關疾病，諸如本文公開的疾病，TMPRSS6水平也可被認為是TMPRSS6相關病症的生理特徵。醫療保健提供者可監測受試者TMPRSS6水平的變化，作為施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的功效的衡量標準。在一個非限制性示例中，可從受試者獲得生物樣本，諸如血液或血清樣本，並在該樣本中測定受試者的TMPRSS6水平。將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑施用於受試者，並在施用後從受試者獲得血液樣本，使用該樣本測定TMPRSS6水平，並將結果與受試者的施用前（之前）樣本中測定的結果進行比較。與施用前水平相比，受試者的TMPRSS6水平在施用後樣本中的降低表明所施用的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑在降低受試者的鐵調素水平、鐵水平或轉鐵蛋白飽和水平方面有效。It should be understood that a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can be administered to an individual subject based on the subject's medical condition. For example, a healthcare provider providing care to a subject may assess the TMPRSS6 level measured in a sample obtained from the subject and determine that it is desirable to lower the subject's TMPRSS6 level by administering a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent of the present invention. In this example, even if the subject has not been diagnosed with a TMPRSS6-associated disease, such as the diseases disclosed herein, the TMPRSS6 level can be considered a physiological characteristic of a TMPRSS6-associated condition. A healthcare provider can monitor changes in a subject's TMPRSS6 levels as a measure of the effectiveness of administering a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention. In one non-limiting example, a biological sample, such as a blood or serum sample, can be obtained from the subject and the subject's TMPRSS6 level measured in the sample. A TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent is administered to the subject, and after administration, a blood sample is obtained from the subject. The TMPRSS6 level is measured using the sample, and the result is compared to the result measured in a sample from the subject before administration. A decrease in the subject's TMPRSS6 level in a post-administration sample compared to the pre-administration level indicates that the administered TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent is effective in reducing the subject's hepcidin level, iron level, or transferrin saturation level.
本發明的方法的某些實施方案包括調整治療，包括至少部分地基於對由該治療引起的TMPRSS6相關疾病或病症的受試者的一種或多種生理特徵變化的評估，來向受試者施用本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。例如，在本發明的一些實施方案中，可為受試者測定所施用的本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的效果，並用於輔助調整隨後施用於受試者的本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。在一個非限制性示例中，向受試者施用本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑，在施用後測定受試者的TMPRSS6水平，並且至少部分地基於所測定的水平，確定需要更大量的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑以增加所施用的藥劑的生理效應，例如降低或進一步降低受試者的TMPRSS6水平。在另一個非限制性示例中，向受試者施用本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑，在施用後測定受試者的TMPRSS6水平，並且至少部分地基於所測定的水平，確定需要向受試者施用較低量的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。Certain embodiments of the methods of the present invention include adjusting treatment, including administering a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention to a subject based at least in part on an assessment of changes in one or more physiological characteristics of the subject resulting from the treatment for a TMPRSS6-related disease or condition. For example, in some embodiments of the present invention, the effect of an administered dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention can be determined for a subject and used to help adjust the amount of the dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention that is subsequently administered to the subject. In one non-limiting example, a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention is administered to a subject, the subject's TMPRSS6 level is measured after administration, and based at least in part on the measured level, it is determined that a larger amount of the dsRNA agent or TMPRSS6 antisense polynucleotide agent is needed to increase the physiological effect of the administered agent, such as to reduce or further reduce the subject's TMPRSS6 level. In another non-limiting example, a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the present invention is administered to a subject, the subject's TMPRSS6 level is measured after administration, and based at least in part on the measured level, it is determined that a lower amount of the dsRNA agent or TMPRSS6 antisense polynucleotide agent is needed to be administered to the subject.
因此，本發明的一些實施方案包括對由受試者的先前治療引起的一種或多種生理特徵變化的評估，以用於調整隨後施用於該受試者的本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的量。本發明的方法的一些實施方案包括對TMPRSS6相關疾病或病症的生理特徵的1、2、3、4、5、6個或更多個測定結果，以評估和/或監測所施用的本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的功效，並任選地使用這些測定結果來調整以下項中的一者或多者：本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的劑量、施用方案和/或施用頻率，從而治療受試者的TMPRSS6相關疾病或病症。在本發明的方法的一些實施方案中，將有效量的本發明的dsRNA藥劑或TMPRSS6反義多核苷酸藥劑施用於受試者的期望結果是，與受試者先前測定的水平或對照水平相比，受試者的TMPRSS6 mRNA水平、受試者的TMPRSS6蛋白水平或鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平、Hb A2水平等有所降低。Therefore, some embodiments of the invention include evaluating changes in one or more physiological characteristics resulting from a subject's prior treatment for use in adjusting the amount of a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention subsequently administered to the subject. Some embodiments of the methods of the invention include 1, 2, 3, 4, 5, 6 or more assay results for physiological characteristics of a TMPRSS6-associated disease or condition to assess and/or monitor the efficacy of an administered TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention, and optionally using these assay results to adjust one or more of the following: the dosage, administration regimen and/or frequency of administration of the dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention, thereby treating the TMPRSS6-associated disease or condition in the subject. In some embodiments of the methods of the invention, the desired outcome of administering an effective amount of a dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention to a subject is a decrease in the subject's TMPRSS6 mRNA level, the subject's TMPRSS6 protein level, or hepcidin level, iron level, transferrin saturation level, and red blood cell (RBC) count measurement, hemoglobin level, ferritin level, Hb F level, Hb A2 level, etc., compared to a previously determined level or a control level in the subject.
如本文所用，術語“治療”、“治療的”或“治療中”當用於TMPRSS6相關疾病或病症方面時，可以指降低受試者發展TMPRSS6相關疾病或病症的可能性的預防性治療，並且還可以指在受試者已發展TMPRSS6相關疾病或病症後的治療，以便消除或降低TMPRSS6相關疾病或病症的水平，防止TMPRSS6相關疾病或病症進一步惡化（例如，更嚴重），並且/或與缺乏使受試者的TMPRSS6多肽活性降低的治療的受試者相比，減緩受試者的TMPRSS6相關疾病或病症的進展。As used herein, the terms "treat," "therapeutic," or "treating," when used in relation to a TMPRSS6-related disease or condition, can refer to prophylactic treatment to reduce the likelihood that a subject will develop a TMPRSS6-related disease or condition, and can also refer to treatment after a subject has developed a TMPRSS6-related disease or condition in order to eliminate or reduce the level of the TMPRSS6-related disease or condition, prevent the TMPRSS6-related disease or condition from getting worse (e.g., getting more severe), and/or slow the progression of the TMPRSS6-related disease or condition in the subject compared to a subject in the absence of a treatment that reduces the activity of the subject's TMPRSS6 polypeptide.
本發明的藥劑、組合物和方法的某些實施方案可用於抑制TMPRSS6基因表達。如本文所用，關於TMPRSS6基因表達的術語“抑制”、“沉默”、“降低”、“下調”和“敲低”意指TMPRSS6基因的表達，如通過以下項中的一者或多者所測量的：當細胞、細胞組、組織、器官或受試者與本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑接觸（例如，用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑治療）時，與分別由TMPRSS6基因轉錄的RNA對照水平、所表達的TMPRSS6的活性水平或由mRNA翻譯的TMPRSS6水平相比，在轉錄TMPRSS6基因的細胞、細胞組、組織、器官或受試者中由基因轉錄的RNA水平、所表達的TMPRSS6的活性水平和由mRNA翻譯的TMPRSS6多肽、蛋白或蛋白亞基的水平均有所降低。在一些實施方案中，對照水平是在未接觸TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑（例如，未用該TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑治療）的細胞、組織、器官或受試者中的水平。Certain embodiments of the agents, compositions, and methods of the present invention can be used to inhibit TMPRSS6 gene expression. As used herein, the terms "inhibit," "silence," "reduce," "downregulate," and "knockdown" with respect to TMPRSS6 gene expression refer to expression of the TMPRSS6 gene as measured by one or more of the following: when a cell, cell group, tissue, organ, or subject is contacted with a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent of the present invention (e.g., with a TMPRSS6 antisense polynucleotide of the present invention); When a TMPRSS6 gene is transcribed, the level of RNA transcribed from the gene, the level of TMPRSS6 activity expressed, and the level of TMPRSS6 polypeptide, protein, or protein subunit translated from mRNA are reduced in a cell, cell group, tissue, organ, or subject that transcribes a TMPRSS6 gene, as compared to a control level of RNA transcribed from the TMPRSS6 gene, the level of TMPRSS6 activity expressed, or the level of TMPRSS6 translated from mRNA, respectively. In some embodiments, the control level is the level in a cell, tissue, organ, or subject that has not been exposed to (e.g., not treated with) the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent.
施用方法Application method
TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的多種施用途徑可用於本發明的方法。所選的特定遞送模式將至少部分地取決於所治療的特定病症和治療功效所需的劑量。一般來講，本發明的方法可採用醫學上可接受的任何施用模式來實踐，意指對TMPRSS6相關疾病或病症產生有效治療水平而不會引起臨床上無法接受的副作用的任何模式。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可經由口服、腸內、黏膜、皮下和/或腸胃外途徑施用。術語“腸胃外”包括皮下、靜脈內、鞘內、肌內、腹膜內和胸骨內注射或輸注技術。其他途徑包括但不限於鼻腔（例如，經由胃鼻管）、皮膚、陰道、直腸、舌下和吸入。本發明的遞送途徑可包括鞘內、心室內或顱內。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可置於緩釋基質內，並通過將該基質置於受試者體內而進行施用。在本發明的一些方面，可使用包被有靶向特定細胞或細胞器的遞送劑的納米顆粒將TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑遞送至受試者細胞。各種遞送方式、方法、藥劑是本領域已知的。遞送方法和遞送劑的非限制性示例另外提供於本文別處。在本發明的一些方面，關於TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的術語“遞送”可意指向細胞或受試者施用一種或多種“裸”TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑序列，並且在本發明的某些方面，“遞送”意指經由轉染方式向細胞或受試者施用，將包含TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的細胞遞送至受試者，將編碼TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的載體遞送至細胞和/或受試者等。使用轉染方式遞送TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可包括向細胞和/或受試者施用載體。A variety of routes of administration of TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents can be used in the methods of the present invention. The particular mode of delivery selected will depend, at least in part, on the particular condition being treated and the dosage required for therapeutic efficacy. Generally speaking, the methods of the present invention can be practiced using any medically acceptable mode of administration, meaning any mode that produces effective therapeutic levels for a TMPRSS6-related disease or condition without causing clinically unacceptable side effects. In some embodiments of the present invention, TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents can be administered via oral, enteral, mucosal, subcutaneous, and/or parenteral routes. The term "parenteral" includes subcutaneous, intravenous, intrathecal, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. Other routes include, but are not limited to, nasal (e.g., via a nasogastric tube), transdermal, vaginal, rectal, sublingual, and inhalation. The delivery routes of the present invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the present invention, a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent may be placed in a sustained-release matrix and administered by placing the matrix into a subject. In some aspects of the present invention, a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent may be delivered to a subject's cells using nanoparticles coated with a delivery agent that targets specific cells or organelles. Various delivery methods, methods, and agents are known in the art. Non-limiting examples of delivery methods and delivery agents are provided elsewhere herein. In some aspects of the invention, the term "delivery" with respect to a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can refer to administering one or more "naked" TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent sequences to a cell or subject, and in certain aspects of the invention, "delivery" means administering to a cell or subject via transfection, delivering cells comprising a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent to a subject, delivering a vector encoding a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent to a cell and/or subject, etc. Delivery of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent using transfection can include administering a vector to a cell and/or a subject.
在本發明的一些方法中，一種或多種TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可以製劑形式施用，這些製劑可以藥學上可接受的溶液形式施用，這些藥學上可接受的溶液通常可含有藥學上可接受濃度的鹽、緩衝劑、防腐劑、相容的載劑、佐劑和任選的其他治療成分。在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可與另一種治療劑被配製用於同時施用。根據本發明的方法，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可以藥物組合物的形式施用。一般來講，藥物組合物包含TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑和任選的藥學上可接受的載劑。藥學上可接受的載劑是本領域普通技術人員熟知的。如本文所用，藥學上可接受的載劑意指不干擾活性成分的生物活性的有效性的無毒材料，該生物活性例如TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑在細胞或受試者中抑制TMPRSS6基因表達的能力。施用和遞送dsRNA藥劑或TMPRSS6反義多核苷酸藥劑以用於治療用途的許多方法是本領域已知的，並且可用於本發明的方法中。In some methods of the present invention, one or more TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents can be administered in a formulation that can be administered in a pharmaceutically acceptable solution. These pharmaceutically acceptable solutions typically contain pharmaceutically acceptable concentrations of salt, a buffer, a preservative, a compatible carrier, an adjuvant, and optionally other therapeutic ingredients. In some embodiments of the present invention, a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can be formulated with another therapeutic agent for simultaneous administration. According to the methods of the present invention, a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can be administered in the form of a pharmaceutical composition. Generally speaking, pharmaceutical compositions comprise a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent and, optionally, a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those of ordinary skill in the art. As used herein, a pharmaceutically acceptable carrier refers to a nontoxic material that does not interfere with the effectiveness of the biological activity of the active ingredient, such as the ability of the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent to inhibit TMPRSS6 gene expression in a cell or subject. Many methods of administering and delivering dsRNA agents or TMPRSS6 antisense polynucleotide agents for therapeutic use are known in the art and can be used in the methods of the present invention.
藥學上可接受的載劑包括稀釋劑、填充劑、鹽、緩衝劑、穩定劑、增溶劑和本領域熟知的其他物質。示例性的藥學上可接受的載劑描述於美國專利號5,211,657中，並且其他藥學上可接受的載劑是本領域技術人員已知的。此類製劑通常可含有鹽、緩衝劑、防腐劑、相容的載劑和任選的其他治療劑。當用於藥物時，鹽應當是藥學上可接受的鹽，但非藥學上可接受的鹽也可方便地用於製備其藥學上可接受的鹽並且不排除在本發明的範圍之外。此類藥理學和藥學上可接受的鹽包括但不限於由以下酸製備的那些鹽：鹽酸、氫溴酸、硫酸、硝酸、磷酸、馬來酸、乙酸、水楊酸、檸檬酸、甲酸、丙二酸、琥珀酸等。此外，藥學上可接受的鹽可被製備為鹼金屬鹽或鹼土金屬鹽，諸如鈉鹽、鉀鹽或鈣鹽。Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other substances well known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Patent No. 5,211,657, and other pharmaceutically acceptable carriers are known to those skilled in the art. Such formulations typically may contain salts, buffers, preservatives, compatible carriers, and optional other therapeutic agents. When used in pharmaceuticals, the salt should be a pharmaceutically acceptable salt, but non-pharmaceutically acceptable salts may also be conveniently used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. In addition, pharmaceutically acceptable salts can be prepared as alkaline metal salts or alkaline earth metal salts, such as sodium salts, potassium salts, or calcium salts.
本發明的方法的一些實施方案包括直接向組織施用一種或多種TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。在一些實施方案中，被施用化合物的組織是其中存在或可能出現TMPRSS6相關疾病或病症的組織，該組織的非限制性示例是心臟。直接的組織施用可通過直接注射或其他方式實現。許多口服遞送的化合物自然地移動到肝臟和腎臟並通過肝臟和腎臟，並且本發明的治療方法的一些實施方案包括向受試者口服施用一種或多種TMPRSS6 dsRNA藥劑。TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑單獨施用或與其他治療劑聯合施用，可施用一次，或者另選地，它們可多次施用。如果多次施用，則TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可經由不同途徑施用。例如，儘管並非旨在進行限制，但第一次（或前幾次）施用可經由皮下方式進行，並且一次或多次另外的施用可以是口服和/或全身性施用。Some embodiments of the methods of the present invention involve direct administration of one or more TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which a TMPRSS6-related disease or condition exists or may occur, a non-limiting example of which is the heart. Direct tissue administration can be achieved by direct injection or other means. Many orally delivered compounds naturally migrate to and pass through the liver and kidneys, and some embodiments of the treatment methods of the present invention involve orally administering one or more TMPRSS6 dsRNA agents to a subject. TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents can be administered alone or in combination with other therapeutic agents, and can be administered once, or alternatively, they can be administered multiple times. If administered multiple times, the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent can be administered via different routes. For example, although not intended to be limiting, the first (or first few) administrations can be administered subcutaneously, and one or more additional administrations can be oral and/or systemic.
對於本發明的期望全身性施用TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的實施方案，該TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可被配製用於通過注射的腸胃外施用，例如通過彈丸注射或持續輸注的腸胃外施用。注射用製劑可以單位劑型存在，例如在添加或未添加防腐劑的安瓿或多劑量容器中。TMPRSS6 dsRNA藥劑製劑（也稱為藥物組合物）可採取諸如在油性或水性媒介物中的懸浮液、溶液或乳液的形式，並且可含有配製劑諸如懸浮劑、穩定劑和/或分散劑。For embodiments of the present invention in which systemic administration of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent is desired, the TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent can be formulated for parenteral administration by injection, such as bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage form, such as in ampoules or multi-dose containers, with or without added preservatives. TMPRSS6 dsRNA agent formulations (also referred to as pharmaceutical compositions) can take the form of suspensions, solutions, or emulsions in oily or aqueous vehicles and can contain formulatory agents such as suspending agents, stabilizers, and/or dispersing agents.
用於腸胃外施用的製劑包括無菌水性溶液或非水性溶液、懸浮液和乳液。非水性溶劑的示例是丙二醇、聚乙二醇、植物油（諸如橄欖油）和可注射的有機酯（諸如油酸乙酯）。水性載劑包括水、醇/水性溶液、乳液或懸浮液，包括鹽水和緩衝介質。腸胃外媒介物包括氯化鈉溶液、林格氏右旋糖、右旋糖和氯化鈉、乳酸林格氏液或固定油。靜脈內媒介物包括液體和營養補充劑、電解質補充劑（諸如基於林格氏右旋糖的補充劑）等。還可存在防腐劑和其他添加劑，諸如例如抗微生物劑、抗氧化劑、螯合劑和惰性氣體等。其他施用形式（諸如靜脈內施用）將導致較低的劑量。如果在施加的初始劑量下受試者的反應不足，則可在患者耐受性允許的範圍內採用更高的劑量（或者通過不同的、更局部的遞送途徑來有效提高劑量）。每天可根據需要使用多次劑量，以達到一種或多種TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的適當全身性水平或局部水平並達到TMPRSS6蛋白活性的適當降低。Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil), and injectable organic esters (such as ethyl oleate). Aqueous vehicles include water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include liquid and nutrient supplements, electrolyte supplements (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases. Other forms of administration (e.g., intravenous administration) will result in lower doses. If the subject's response to the initial dose administered is insufficient, a higher dose may be used (or the dose may be effectively increased by a different, more localized delivery route) as tolerated by the patient. Multiple doses may be used daily as needed to achieve appropriate systemic or localized levels of one or more TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents and to achieve appropriate reductions in TMPRSS6 protein activity.
在又一些實施方案中，本發明的方法包括使用遞送媒介物，諸如生物相容的微粒、納米顆粒或適於植入接受者（例如受試者）體內的植入物。根據該方法可使用的示例性生物溶蝕植入物描述於PCT公開號WO 95/24929（以引用方式併入本文），該文獻描述了用於包含生物大分子的生物相容的且可生物降解的聚合物基質。In yet other embodiments, the methods of the present invention include the use of a delivery vehicle, such as a biocompatible microparticle, nanoparticle, or implant suitable for implantation into a recipient (e.g., a subject). Exemplary bioerodible implants that can be used according to this method are described in PCT Publication No. WO 95/24929 (incorporated herein by reference), which describes biocompatible and biodegradable polymer matrices for containing biomacromolecules.
可在本發明的方法中使用不可生物降解的聚合物基質和可生物降解的聚合物基質兩者，以將一種或多種TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑遞送至受試者。在一些實施方案中，基質可以是可生物降解的。基質聚合物可以是天然的或合成的聚合物。可基於期望釋放的時間段來選擇聚合物，通常為幾小時至一年或更長時間。通常，可使用在幾小時到三個月至十二個月範圍內的釋放時間。聚合物任選地呈水凝膠的形式，其可吸收高達其重量約90%的水，並且進一步任選地與多價離子或其他聚合物交聯。Both non-biodegradable and biodegradable polymer matrices can be used in the methods of the present invention to deliver one or more TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents to a subject. In some embodiments, the matrix can be biodegradable. The matrix polymer can be a natural or synthetic polymer. The polymer can be selected based on the time period over which release is desired, typically from a few hours to a year or longer. Typically, release times ranging from a few hours to three to twelve months can be used. The polymer is optionally in the form of a hydrogel that can absorb up to about 90% of its weight in water and is further optionally cross-linked with multivalent ions or other polymers.
一般來講，在本發明的一些實施方案中，TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑可使用生物溶蝕植入物通過擴散方式或通過聚合物基質的降解進行遞送。用於此類用途的示例性合成聚合物是本領域熟知的。根據本領域已知的方法，可使用可生物降解的聚合物和不可生物降解的聚合物來遞送TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑。生物粘附聚合物諸如生物溶蝕水凝膠（參見H. S. Sawhney、C. P. Pathak和J. A. Hubell in Macromolecules, 1993, 26, 581-587，該文獻的教導內容以引用方式併入本文）還可用於遞送TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑以用於治療TMPRSS6相關疾病或病症。另外的合適的遞送系統可包括時間釋放、延遲釋放或持續釋放遞送系統。此類系統可避免重複施用TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑，從而增加了對受試者和醫療保健專業人員的便利性。許多類型的釋放遞送系統是可用的並且是本領域普通技術人員已知的。（參見例如：美國專利號5,075,109、4,452,775、4,675,189、5,736,152、3,854,480、5,133,974和5,407,686（這些文獻中的每一篇的教導內容以引用方式併入本文））。此外，可使用基於泵的硬件遞送系統，其中一些泵適於植入。Generally speaking, in some embodiments of the present invention, a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent can be delivered using a bioerodible implant by diffusion or by degradation of a polymer matrix. Exemplary synthetic polymers for such uses are well known in the art. Biodegradable and non-biodegradable polymers can be used to deliver a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent according to methods known in the art. Bioadhesive polymers such as bioerodible hydrogels (see H. S. Sawhney, C. P. Pathak, and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein by reference) can also be used to deliver TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents for the treatment of TMPRSS6-related diseases or conditions. Additional suitable delivery systems may include time-release, delayed-release, or sustained-release delivery systems. Such systems can avoid repeated administration of TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents, thereby increasing convenience for subjects and healthcare professionals. Many types of release delivery systems are available and known to those of ordinary skill in the art. (See, for example, U.S. Patent Nos. 5,075,109, 4,452,775, 4,675,189, 5,736,152, 3,854,480, 5,133,974, and 5,407,686 (the teachings of each of which are incorporated herein by reference). In addition, hardware delivery systems based on pumps are available, some of which are suitable for implantation.
使用長期持續釋放植入物可能適用於受試者和處於發展復發性TMPRSS6相關疾病或病症的風險中的受試者的預防性治療。如本文所用，長期釋放意指植入物被構建和安排成遞送治療水平的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑至少長達10天、20天、30天、60天、90天、六個月、一年或更長時間。長期持續釋放植入物是本領域普通技術人員熟知的，包括上述一些釋放系統。The use of long-term sustained-release implants may be suitable for the prophylactic treatment of subjects and subjects at risk for developing recurrent TMPRSS6-related diseases or conditions. As used herein, long-term release means that the implant is constructed and arranged to deliver therapeutic levels of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent for at least 10 days, 20 days, 30 days, 60 days, 90 days, six months, a year, or longer. Long-term sustained-release implants are well known to those of ordinary skill in the art and include some of the release systems described above.
TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的呈凍幹製劑或水性溶液的形式的治療性製劑可通過以下方式製備以用於儲存：將具有期望純度的分子或化合物與任選的藥學上可接受的載劑、賦形劑或穩定劑混合[“雷氏藥學大全（Remington's Pharmaceutical Sciences）”，第21版，（2006年）]。可接受的載劑、賦形劑或穩定劑在所採用的劑量和濃度下對接受者無毒，包括緩衝劑，諸如磷酸鹽、檸檬酸鹽和其他有機酸；抗氧化劑，包括抗壞血酸和甲硫氨酸；防腐劑（諸如十八烷基二甲基苄基氯化銨、氯化六甲雙銨、苯紮氯銨、苄索氯銨、苯酚、丁醇或苄醇、烷基對羥基苯甲酸酯（諸如甲基或丙基對羥基苯甲酸酯）、鄰苯二酚、間苯二酚、環己醇、3-戊醇和間甲酚）；低分子量（少於約10個殘基）多肽；蛋白，諸如血清白蛋白、明膠或免疫球蛋白；親水性聚合物，諸如聚乙烯吡咯烷酮；氨基酸，諸如甘氨酸、穀氨醯胺、天冬醯胺、組氨酸、精氨酸或賴氨酸；單糖、二糖和其他碳水化合物，包括葡萄糖、甘露糖或糊精；螯合劑，諸如EDTA；糖類，諸如蔗糖、甘露糖醇、海藻糖或山梨糖醇；成鹽抗衡離子，諸如鈉；金屬絡合物（例如，Zn-蛋白絡合物）；和/或非離子型表面活性劑，諸如TWEEN®、PLURONICS®或聚乙二醇（PEG）。Therapeutic formulations of TMPRSS6 dsRNA agents or TMPRSS6 antisense polynucleotide agents in the form of lyophilized preparations or aqueous solutions can be prepared for storage by mixing the molecule or compound having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers ["Remington's Pharmaceutical Sciences," 21st edition, (2006)]. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, including buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzathonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parahydroxybenzoates (such as methyl or propyl parahydroxybenzoate), o-catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol); low molecular weight (less than about 10 residue groups) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such asTWEEN® ,PLURONICS® , or polyethylene glycol (PEG).
細胞、受試者和對照Cells, subjects, and controls
本發明的方法可與細胞、組織、器官和/或受試者聯合使用。在本發明的一些方面，受試者是人或脊椎動物哺乳動物，包括但不限於狗、貓、馬、牛、山羊、小鼠、大鼠和靈長類動物，例如猴。因此，本發明可用於治療人和非人受試者的TMPRSS6相關疾病或病症。在本發明的一些方面，受試者可以是農場動物、動物園動物、馴養動物或非馴養動物，並且本發明的方法可用於獸醫預防和治療方案。在本發明的一些實施方案中，受試者是人，並且本發明的方法可用於人預防和治療方案。The methods of the present invention can be used in conjunction with cells, tissues, organs, and/or subjects. In some aspects of the present invention, the subject is a human or a vertebrate mammal, including but not limited to dogs, cats, horses, cows, goats, mice, rats, and primates, such as monkeys. Thus, the present invention can be used to treat TMPRSS6-related diseases or conditions in both human and non-human subjects. In some aspects of the present invention, the subject can be a farm animal, a zoo animal, a domesticated animal, or a non-domesticated animal, and the methods of the present invention can be used in veterinary prevention and treatment protocols. In some embodiments of the present invention, the subject is a human, and the methods of the present invention can be used in human prevention and treatment protocols.
可應用本發明的受試者的非限制性示例是被診斷患有、懷疑患有或處於患有與高於期望的TMPRSS6表達和/或活性（也稱為“TMPRSS6表達水平升高”）相關的疾病或病症的風險中的受試者。與高於期望水平的TMPRSS6表達和/或活性相關的疾病和病症的非限制性示例描述於本文別處。可應用本發明的方法的受試者是，在治療時已被診斷為患有與高於期望的TMPRSS6表達和/或活性相關的疾病或病症的受試者，或被認為處於患有或發展與高於期望的TMPRSS6表達和/或活性相關的疾病或病症的風險中的受試者。在本發明的一些方面，與高於期望的TMPRSS6表達和/或活性水平相關的疾病或病症是急性疾病或病症，並且在本發明的某些方面，與高於期望的TMPRSS6表達和/或活性水平相關的疾病或病症是慢性疾病或病症。Non-limiting examples of subjects to which the present invention can be applied are subjects diagnosed with, suspected of having, or at risk of having a disease or condition associated with higher than desired TMPRSS6 expression and/or activity (also referred to as "elevated TMPRSS6 expression levels"). Non-limiting examples of diseases and conditions associated with higher than desired levels of TMPRSS6 expression and/or activity are described elsewhere herein. Subjects to whom the methods of the present invention can be applied are subjects who, at the time of treatment, have been diagnosed with, or are believed to be at risk of having or developing, a disease or condition associated with higher than desired TMPRSS6 expression and/or activity. In some aspects of the invention, the disease or condition associated with higher than desired TMPRSS6 expression and/or activity levels is an acute disease or condition, and in certain aspects of the invention, the disease or condition associated with higher than desired TMPRSS6 expression and/or activity levels is a chronic disease or condition.
在一個非限制性示例中，將本發明的TMPRSS6 dsRNA藥劑施用於被診斷患有、懷疑患有或處於患有地中海貧血的風險中的受試者，該地中海貧血是其中期望降低TMPRSS6表達的疾病。可應用本發明的方法的受試者是，在治療時已被診斷為患有該疾病或病症的受試者，或被認為處於患有或發展該疾病或病症的風險中的受試者。In one non-limiting example, a TMPRSS6 dsRNA agent of the present invention is administered to a subject diagnosed with, suspected of having, or at risk for thalassemia, a disease in which reduced TMPRSS6 expression is desirable. The methods of the present invention can be applied to subjects who have been diagnosed with, or are believed to be at risk for developing, developing, or developing the disease or condition at the time of treatment.
在另一個非限制性示例中，將本發明的TMPRSS6 dsRNA藥劑施用於被診斷患有、懷疑患有或處於患有地中海貧血的風險中的受試者，該地中海貧血是其中期望降低TMPRSS6表達的疾病。可應用本發明的方法的受試者是，在治療時已被診斷為患有該疾病或病症的受試者，或被認為處於患有或發展該疾病或病症的風險中的受試者。In another non-limiting example, a TMPRSS6 dsRNA agent of the present invention is administered to a subject diagnosed with, suspected of having, or at risk for thalassemia, a disease in which reduced TMPRSS6 expression is desirable. Subjects to whom the methods of the present invention can be applied are those who have been diagnosed with, or are believed to be at risk for developing, developing, or developing the disease or condition at the time of treatment.
可應用本發明的方法的細胞包括體外細胞、體內細胞、離體細胞。細胞可位於受試者體內、培養物中和/或懸浮液中，或者處於任何其他合適的狀態或條件下。可應用本發明的方法細胞可以是肝臟細胞、肝細胞、心臟細胞、胰腺細胞、心血管細胞、腎臟細胞或其他類型的脊椎動物細胞，包括人和非人哺乳動物細胞。在本發明的某些方面，可應用本發明的方法的細胞是未被確認為疾病細胞的健康正常細胞。在本發明的某些實施方案中，應用本發明的方法和組合物的細胞是肝臟細胞、肝細胞、心臟細胞、胰腺細胞、心血管細胞和/或腎臟細胞。在本發明的某些方面，對照細胞是正常細胞，但應當理解，患有疾病或病症的細胞在特定情況下也可用作對照細胞，例如用於比較患有疾病或病症的接受治療的細胞與患有該疾病或病症的未治療細胞的結果等。Cells to which the methods of the present invention can be applied include in vitro cells, in vivo cells, and ex vivo cells. The cells can be in vivo, in culture, and/or in suspension, or in any other suitable state or condition. Cells to which the methods of the present invention can be applied can be liver cells, hepatocytes, heart cells, pancreatic cells, cardiovascular cells, kidney cells, or other types of vertebrate cells, including human and non-human mammalian cells. In certain aspects of the present invention, the cells to which the methods of the present invention can be applied are healthy, normal cells that have not been identified as diseased cells. In certain embodiments of the present invention, the cells to which the methods and compositions of the present invention are applied are hepatocytes, liver cells, heart cells, pancreatic cells, cardiovascular cells, and/or kidney cells. In certain aspects of the present invention, the control cells are normal cells, but it should be understood that cells suffering from a disease or condition can also be used as control cells in certain circumstances, for example, to compare the results of cells treated for a disease or condition with those of untreated cells suffering from the disease or condition.
根據本發明的方法，可測定TMPRSS6多肽活性的水平並與TMPRSS6多肽活性的對照水平進行比較。對照可以是預先確定的值，其可採取多種形式。它可以是單個截止值，諸如中值或平均值。它可基於比較組來確立，諸如在具有正常水平的TMPRSS6多肽和/或TMPRSS6多肽活性的組中和具有增加水平的TMPRSS6多肽和/或TMPRSS6多肽活性的組中。比較組的另一個非限制性示例可以是具有TMPRSS6相關疾病或病症的一種或多種症狀或診斷的組；不具有該疾病或病症的一種或多種症狀或診斷的組；已經施用本發明的siRNA治療的受試者組；未施用本發明的siRNA治療的受試者組。通常，對照可基於處於適當年齡段的看起來健康的正常個體或看起來健康的細胞。應當理解，除了預先確定的值之外，根據本發明的對照還可以是與實驗材料同步測試的材料樣本。示例包括來自對照群體的樣本或通過製造產生的用於與實驗樣本同步測試的對照樣本。在本發明的一些實施方案中，對照可包括未接觸或未用本發明的TMPRSS6 dsRNA藥劑治療的細胞或受試者，並且在此類情況下，可將TMPRSS6多肽和/或TMPRSS6多肽活性的對照水平與接觸本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑的細胞或受試者的TMPRSS6多肽和/或TMPRSS6多肽活性的水平進行比較。According to the methods of the present invention, the level of TMPRSS6 polypeptide activity can be determined and compared to a control level of TMPRSS6 polypeptide activity. The control can be a predetermined value, which can take a variety of forms. It can be a single cutoff value, such as a median or mean. It can be established based on a comparison group, such as a group with normal levels of TMPRSS6 polypeptide and/or TMPRSS6 polypeptide activity and a group with increased levels of TMPRSS6 polypeptide and/or TMPRSS6 polypeptide activity. Another non-limiting example of a comparison group can be a group with one or more symptoms or diagnoses of a TMPRSS6-related disease or condition; a group without one or more symptoms or diagnoses of the disease or condition; a group of subjects who have been treated with an siRNA of the present invention; or a group of subjects who have not been treated with an siRNA of the present invention. Typically, the control can be based on a normal, apparently healthy individual of an appropriate age range or on apparently healthy cells. It should be understood that, in addition to predetermined values, a control according to the present invention can also be a sample of material tested concurrently with the experimental material. Examples include samples from a control population or control samples generated by manufacturing for concurrent testing with the experimental samples. In some embodiments of the invention, a control may include cells or subjects that have not been exposed to or treated with a TMPRSS6 dsRNA agent of the invention, and in such cases, the control level of TMPRSS6 polypeptide and/or TMPRSS6 polypeptide activity can be compared to the level of TMPRSS6 polypeptide and/or TMPRSS6 polypeptide activity in a cell or subject exposed to a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent of the invention.
在本發明的一些實施方案中，針對受試者測定的TMPRSS6多肽水平可以是對照水平，將同一受試者在不同時間測定的TMPRSS6多肽水平與之比較。在一個非限制性示例中，測定從未施用本發明的TMPRSS6治療的受試者獲得的生物樣本中的TMPRSS6水平。在一些實施方案中，該生物樣本是血清樣本。從受試者獲得的樣本中測定的TMPRSS6多肽水平可用作受試者的基線或對照值。在本發明的治療方法中，在向受試者施用一次或多次TMPRSS6 dsRNA藥劑後，可從受試者獲得一個或多個另外的血清樣本，並且可將後續一個或多個樣本中的TMPRSS6多肽水平與受試者的對照/基線水平進行比較。此類比較可用於評估受試者的TMPRSS6相關疾病或病症的發作、進展或消退。例如，從受試者獲得的基線樣本中的TMPRSS6多肽水平高於在向受試者施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑後從同一受試者獲得的水平，這表明TMPRSS6相關疾病或病症的消退，並且表明所施用的本發明的TMPRSS6 dsRNA藥劑對治療TMPRSS6相關疾病或病症的功效。In some embodiments of the present invention, the TMPRSS6 polypeptide level measured for a subject can be a control level to which TMPRSS6 polypeptide levels measured for the same subject at different times are compared. In one non-limiting example, the TMPRSS6 level is measured in a biological sample obtained from a subject who has not been administered a TMPRSS6 treatment according to the present invention. In some embodiments, the biological sample is a serum sample. The TMPRSS6 polypeptide level measured in the sample obtained from the subject can be used as a baseline or control value for the subject. In the treatment methods of the present invention, after one or more TMPRSS6 dsRNA doses are administered to a subject, one or more additional serum samples can be obtained from the subject, and the TMPRSS6 polypeptide level in the subsequent sample or samples can be compared to the subject's control/baseline level. Such comparisons can be used to assess the onset, progression, or regression of a TMPRSS6-associated disease or condition in a subject. For example, a baseline sample obtained from a subject with a TMPRSS6 polypeptide level that is higher than the level obtained from the same subject after administration of a TMPRSS6 dsRNA agent or TMPRSS6 antisense polynucleotide agent of the invention indicates regression of the TMPRSS6-associated disease or condition and indicates the efficacy of the administered TMPRSS6 dsRNA agent of the invention in treating the TMPRSS6-associated disease or condition.
在本發明的一些方面，針對受試者測定的TMPRSS6多肽水平和/或TMPRSS6多肽活性水平中的一者或多者的值可用作對照值，以用於對該同一受試者的TMPRSS6多肽水平和/或TMPRSS6活性水平進行後續比較，從而允許評估相對於受試者的“基線”TMPRSS6多肽活性的變化。因此，初始TMPRSS6多肽水平和/或初始TMPRSS6多肽活性水平可存在於受試者中以及/或針對該受試者進行測定，並且本發明的方法和化合物可用於降低該受試者的TMPRSS6多肽水平和/或TMPRSS6多肽活性水平，其中該初始水平用作該受試者的對照水平。In some aspects of the invention, a value for one or more of the TMPRSS6 polypeptide level and/or TMPRSS6 polypeptide activity level determined for a subject can be used as a control value for subsequent comparisons of the TMPRSS6 polypeptide level and/or TMPRSS6 activity level of the same subject, thereby allowing assessment of changes relative to the subject's "baseline" TMPRSS6 polypeptide activity. Thus, an initial TMPRSS6 polypeptide level and/or initial TMPRSS6 polypeptide activity level can be present in a subject and/or determined for the subject, and the methods and compounds of the invention can be used to reduce the TMPRSS6 polypeptide level and/or TMPRSS6 polypeptide activity level in the subject, wherein the initial level serves as the control level for the subject.
使用本發明的方法，可將本發明的TMPRSS6 dsRNA藥劑和/或TMPRSS6反義多核苷酸藥劑施用於受試者。當與在先前時間點從受試者獲得的血清樣本中的TMPRSS6多肽的施用前水平相比，或與非接觸對照水平（例如對照血清樣本中的TMPRSS6多肽水平）相比，從受試者獲得的血清樣本中的TMPRSS6多肽水平降低至少0.5%、1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更多時，可評估本發明的施用和治療的功效。應當理解，TMPRSS6多肽水平和TMPRSS6多肽活性水平兩者均與TMPRSS6基因表達水平相關。本發明的方法的某些實施方案包括將本發明的TMPRSS6 dsRNA和/或TMPRSS6反義藥劑以有效抑制TMPRSS6基因表達的量施用於受試者，從而降低該受試者的TMPRSS6多肽水平和TMPRSS6多肽活性水平。Using the methods of the present invention, a TMPRSS6 dsRNA agent and/or TMPRSS6 antisense polynucleotide agent of the present invention can be administered to a subject. The efficacy of the administration and treatment of the present invention can be assessed when the level of TMPRSS6 polypeptide in a serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more compared to the pre-administration level of TMPRSS6 polypeptide in a serum sample obtained from the subject at a previous time point, or compared to a non-contact control level (e.g., the level of TMPRSS6 polypeptide in a control serum sample). It should be understood that both TMPRSS6 polypeptide levels and TMPRSS6 polypeptide activity levels correlate with TMPRSS6 gene expression levels. Certain embodiments of the methods of the present invention include administering to a subject a TMPRSS6 dsRNA and/or TMPRSS6 antisense agent of the present invention in an amount effective to inhibit TMPRSS6 gene expression, thereby reducing the level of TMPRSS6 polypeptide and TMPRSS6 polypeptide activity in the subject.
本發明的一些實施方案包括測定從一個或多個受試者獲得的一個或多個生物樣本中的TMPRSS6多肽的存在、不存在和/或量（本文也稱為水平）。該測定結果可用於評估本發明的治療方法的功效。例如，本發明的方法和組合物可用於測定從先前已施用本發明的TMPRSS6 dsRNA藥劑和/或TMPRSS6反義藥劑治療的受試者獲得的生物樣本中的TMPRSS6多肽水平。與針對受試者測定的TMPRSS6多肽的預處理水平相比，或與非接觸對照生物樣本水平相比，從接受治療的受試者獲得的血清樣本中測定的TMPRSS6多肽水平降低至少0.5%、1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更多，這表明施用於受試者的治療的功效水平。Some embodiments of the present invention include determining the presence, absence, and/or amount (also referred to herein as the level) of a TMPRSS6 polypeptide in one or more biological samples obtained from one or more subjects. The results of this determination can be used to assess the efficacy of the treatment methods of the present invention. For example, the methods and compositions of the present invention can be used to determine the level of a TMPRSS6 polypeptide in a biological sample obtained from a subject who has previously been treated with a TMPRSS6 dsRNA agent and/or a TMPRSS6 antisense agent of the present invention. A reduction in the level of TMPRSS6 polypeptide as measured in a serum sample obtained from a treated subject of at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more, compared to the pre-treatment level of TMPRSS6 polypeptide measured for the subject, or compared to the level in an unexposed control biological sample, indicates a level of efficacy of the treatment administered to the subject.
在本發明的一些實施方案中，針對受試者測定的TMPRSS6相關疾病或病症的生理特徵可以是對照測定結果，將同一受試者在不同時間的生理特徵測定結果與之比較。在一個非限制性示例中，在從未施用本發明的TMPRSS6治療的受試者獲得的生物樣本（諸如血清樣本）中測定生理特徵，諸如受試者的TMPRSS6 mRNA水平、TMPRSS6蛋白水平，或血漿或組織樣本中的鐵調素水平、鐵水平、轉鐵蛋白飽和水平。從受試者獲得的樣本中測定的TMPRSS6 mRNA水平（和/或TMPRSS6疾病或病症的其他生理特徵）可用作受試者的基線或對照值。在本發明的治療方法中，在向受試者施用一次或多次TMPRSS6 dsRNA藥劑後，可從受試者獲得一個或多個另外的血清樣本，並將後續一個或多個樣本中的TMPRSS6 mRNA水平和/或TMPRSS6蛋白水平分別與受試者的對照/基線水平和/或比率進行比較。此類比較可用於評估受試者的TMPRSS6相關疾病或病症的發作、進展或消退。例如，從受試者獲得的基線樣本中的TMPRSS6 mRNA水平高於在向受試者施用本發明的TMPRSS6 dsRNA藥劑或TMPRSS6反義多核苷酸藥劑後從同一受試者獲得的樣本中測定的TMPRSS6 mRNA水平，這表明TMPRSS6相關疾病或病症的消退，並且表明施用本發明的TMPRSS6 dsRNA藥劑對治療TMPRSS6相關疾病或病症的功效。In some embodiments of the present invention, a physiological characteristic of a TMPRSS6-related disease or condition measured for a subject can be a control measurement result, to which a physiological characteristic measurement result of the same subject at a different time is compared. In one non-limiting example, a physiological characteristic, such as the subject's TMPRSS6 mRNA level, TMPRSS6 protein level, or hepcidin level, iron level, or transferrin saturation level in a plasma or tissue sample, is measured in a biological sample (e.g., a serum sample) obtained from a subject who has not been treated with a TMPRSS6 therapy according to the present invention. The TMPRSS6 mRNA level (and/or other physiological characteristic of a TMPRSS6 disease or condition) measured in the sample obtained from the subject can be used as a baseline or control value for the subject. In the treatment methods of the present invention, after one or more doses of TMPRSS6 dsRNA are administered to a subject, one or more additional serum samples can be obtained from the subject, and the TMPRSS6 mRNA level and/or TMPRSS6 protein level in the subsequent one or more samples can be compared to the subject's control/baseline level and/or ratio, respectively. Such comparisons can be used to assess the onset, progression, or regression of a TMPRSS6-related disease or condition in the subject. For example, a baseline TMPRSS6 mRNA level in a sample obtained from a subject that is higher than the TMPRSS6 mRNA level measured in a sample obtained from the same subject after administration of a TMPRSS6 dsRNA agent or a TMPRSS6 antisense polynucleotide agent of the invention to the subject indicates regression of a TMPRSS6-associated disease or disorder and indicates efficacy of administration of a TMPRSS6 dsRNA agent of the invention for treating a TMPRSS6-associated disease or disorder.
在本發明的一些方面，針對受試者測定的TMPRSS6相關疾病或病症的生理特徵中的一者或多者的值可用作對照值，以用於對該同一受試者的生理特徵進行後續比較，從而允許評估相對於受試者的“基線”生理特徵的變化。因此，初始生理特徵可存在於受試者中以及/或針對該受試者進行測定，並且本發明的方法和化合物可用於降低該受試者的TMPRSS6多肽水平和/或TMPRSS6多肽活性水平，其中該初始生理特徵測定結果用作該受試者的對照。In some aspects of the invention, the value of one or more of the physiological characteristics of a TMPRSS6-related disease or condition measured for a subject can be used as a control value for subsequent comparisons of the physiological characteristic of the same subject, thereby allowing assessment of changes relative to the subject's "baseline" physiological characteristic. Thus, an initial physiological characteristic may be present in a subject and/or measured for the subject, and the methods and compounds of the invention can be used to reduce the level of TMPRSS6 polypeptide and/or the level of TMPRSS6 polypeptide activity in the subject, wherein the initial physiological characteristic measurement result serves as a control for the subject.
使用本發明的方法，可將本發明的TMPRSS6 dsRNA藥劑和/或TMPRSS6反義多核苷酸藥劑以有效量施用於受試者，以用於治療TMPRSS6疾病或病症。本發明的施用和治療功效可通過測定TMPRSS6疾病或病症的一種或多種生理特徵的變化進行評估。在一個非限制性示例中，與在先前時間點從受試者獲得的血清樣本中的施用前水平相比，或與非接觸對照水平（例如對照血清樣本中的TMPRSS6 mRNA水平）相比，從受試者獲得的血清樣本中的TMPRSS6 mRNA水平降低至少0.5%、1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%、95%或更多。應當理解，受試者的TMPRSS6 mRNA水平、TMPRSS6蛋白水平，或血漿或組織樣本中的鐵調素水平、鐵水平、轉鐵蛋白飽和水平和紅細胞（RBC）計數測定值、血紅蛋白水平、鐵蛋白水平、Hb F水平、Hb A2水平各自與TMPRSS6基因表達的水平相關。本發明的方法的某些實施方案包括將本發明的TMPRSS6 dsRNA和/或TMPRSS6反義藥劑以有效抑制TMPRSS6基因表達的量施用於受試者，從而降低該受試者的TMPRSS6 mRNA水平、TMPRSS6蛋白水平，或者以其他方式積極影響該受試者的TMPRSS6相關疾病或病症的生理特徵。Using the methods of the present invention, a TMPRSS6 dsRNA agent and/or TMPRSS6 antisense polynucleotide agent of the present invention can be administered to a subject in an effective amount for treating a TMPRSS6 disease or condition. The efficacy of administration and treatment of the present invention can be assessed by measuring changes in one or more physiological characteristics of a TMPRSS6 disease or condition. In one non-limiting example, the level of TMPRSS6 mRNA in a serum sample obtained from the subject is reduced by at least 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more compared to pre-administration levels in a serum sample obtained from the subject at a previous time point, or compared to untreated control levels (e.g., TMPRSS6 mRNA levels in a control serum sample). It should be understood that the level of TMPRSS6 mRNA, TMPRSS6 protein, or hepcidin, iron, transferrin saturation, and red blood cell (RBC) count, hemoglobin, ferritin, Hb F, and Hb A2 in a subject's plasma or tissue sample are each correlated with the level of TMPRSS6 gene expression. Certain embodiments of the methods of the present invention include administering to a subject an amount of a TMPRSS6 dsRNA and/or TMPRSS6 antisense agent of the present invention effective to inhibit TMPRSS6 gene expression, thereby reducing the subject's TMPRSS6 mRNA or TMPRSS6 protein levels, or otherwise positively affecting the physiological characteristics of a TMPRSS6-related disease or condition in the subject.
本發明的一些實施方案包括使用諸如但不限於以下項的方法來測定TMPRSS6相關疾病或病症的生理特徵的存在、不存在和/或變化：(1)評估從一個或多個受試者獲得的一個或多個生物樣本的生理特徵；(2)對受試者進行影像學檢查（例如但不限於獲得肝臟圖像）；和/或(3)對受試者進行身體檢查。該測定結果可用於評估本發明的治療方法的功效。Some embodiments of the present invention include determining the presence, absence, and/or changes in physiological characteristics of a TMPRSS6-associated disease or condition using methods such as, but not limited to, the following: (1) assessing physiological characteristics of one or more biological samples obtained from one or more subjects; (2) performing an imaging study on the subject (e.g., but not limited to, obtaining liver images); and/or (3) performing a physical examination on the subject. The results of such determinations can be used to assess the efficacy of the treatment methods of the present invention.
試劑盒Test kit
還在本發明的範圍內的是包含一種或多種TMPRSS6 dsRNA藥劑和/或TMPRSS6反義多核苷酸藥劑及其用於本發明方法的使用說明書的試劑盒。本發明的試劑盒可包含可用於治療TMPRSS6相關疾病或病症的TMPRSS6 dsRNA藥劑、TMPRSS6有義多核苷酸和TMPRSS6反義多核苷酸藥劑中的一者或多者。可製備含有一種或多種TMPRSS6 dsRNA藥劑、TMPRSS6有義多核苷酸和TMPRSS6反義多核苷酸藥劑的試劑盒，以用於本發明的治療方法。本發明的試劑盒的組分可以水性介質或凍幹形式進行包裝。本發明的試劑盒可包含被區室化的載劑，以在其中封閉地容納一個或多個容器裝置或一系列容器裝置，諸如試管、小瓶、燒瓶、瓶子、注射器等。第一容器裝置或第一系列容器裝置可容納一種或多種化合物，諸如TMPRSS6 dsRNA藥劑和/或TMPRSS6有義或反義多核苷酸藥劑。第二容器裝置或第二系列容器裝置可容納靶向劑、標記劑、遞送劑等，它們可作為TMPRSS6 dsRNA藥劑和/或TMPRSS6反義多核苷酸的一部分被包括在內以在本發明的治療方法的實施方案中施用。Also within the scope of the present invention are kits containing one or more TMPRSS6 dsRNA agents and/or TMPRSS6 antisense polynucleotide agents and instructions for use in the methods of the present invention. The kits of the present invention may contain one or more of a TMPRSS6 dsRNA agent, a TMPRSS6 sense polynucleotide, and a TMPRSS6 antisense polynucleotide agent that can be used to treat a TMPRSS6-related disease or condition. Kits containing one or more TMPRSS6 dsRNA agents, TMPRSS6 sense polynucleotides, and TMPRSS6 antisense polynucleotide agents can be prepared for use in the treatment methods of the present invention. The components of the kits of the present invention can be packaged in aqueous media or in lyophilized form. The kits of the present invention may comprise a compartmentalized carrier to enclose one or more container means or a series of container means, such as test tubes, vials, flasks, bottles, syringes, etc. A first container means or a first series of container means may contain one or more compounds, such as a TMPRSS6 dsRNA agent and/or a TMPRSS6 sense or antisense polynucleotide agent. A second container means or a second series of container means may contain a targeting agent, a labeling agent, a delivery agent, etc., which may be included as part of a TMPRSS6 dsRNA agent and/or a TMPRSS6 antisense polynucleotide for administration in embodiments of the treatment methods of the present invention.
本發明的試劑盒還可包含說明書。說明書通常為書面形式，並且將為實施試劑盒所包含的治療和基於該治療做出的決定提供指導。The kits of the present invention may also include instructions. The instructions are typically in written form and will provide guidance for implementing the treatment contained in the kit and making decisions based on the treatment.
所提供的以下實施例用於說明本發明實踐的具體實例，並非旨在對本發明的範圍進行限制。對於本領域普通技術人員來說將顯而易見的是，本發明將應用於各種組合物和方法中。 實施例The following examples are provided to illustrate specific examples of the practice of the present invention and are not intended to limit the scope of the present invention. It will be apparent to one of ordinary skill in the art that the present invention can be applied to a variety of compositions and methods.Examples
實施例1.亞磷醯胺化合物1Example 1. Phosphamide Compound 1
在N2氣氛和0℃至5℃下，向化合物B（500mg，1.11mmol，1.0當量）的DCM（5.0mL）溶液中添加化合物D（607mg，3.34mmol，3.0當量）和DIEA（432mg，3.34mmol，582μL，3.0當量），將混合物在25℃下攪拌1.0小時。LC-MS顯示化合物B完全消耗，LC-MS上顯示出幾個新的峰並檢測到約70.9%的期望化合物。將所得反應混合物冷卻至-20℃並倒入冷（0℃至5℃）的飽和NaHCO3（5.0mL）溶液中，用DCM（5.0mL×2）萃取，將合併的有機層用冷（0℃至5℃）的飽和NaHCO3/鹽水=1:1（5.0mL/5.0mL）洗滌，經Na2SO4乾燥並真空濃縮，得到殘餘物（約5mL）。將殘餘物通過柱色譜（鹼性Al2O3，石油醚/乙酸乙酯=10/1至5/1，0.1% Et3N）純化，得到化合物1（280mg，471μmol，42.3%產率），為白色固體。To a solution of compound B (500 mg, 1.11 mmol, 1.0 equiv) in DCM (5.0 mL) were added compound D (607 mg, 3.34 mmol, 3.0 equiv) and DIEA (432 mg, 3.34 mmol, 582 μL, 3.0 equiv) under an N atmosphere at 0°C to 5°C, and the mixture was stirred at 25°C for 1.0 hour. LC-MS analysis showed complete consumption of compound B, with several new peaks revealed, and approximately 70.9% of the desired compound was detected. The resulting reaction mixture was cooled to -20°C and poured into a cold (0°C to 5°C) saturatedNaHCO₃ solution (5.0 mL). The mixture was extracted with DCM (5.0 mL x 2). The combined organic layers were washed with cold (0°C to 5°C) saturatedNaHCO₃ /brine water (1:1) (5.0 mL/5.0mL ), dried overNa₂SO₄ , and concentrated in vacuo to yield a residue (approximately 5 mL). The residue was purified by column chromatography (basicAl₂O₃ ,petroleum ether/ethyl acetate = 10/1 to 5/1, 0.1%Et₃N ) to yield compound 1 (280 mg, 471 μmol, 42.3% yield) as a white solid.
1H NMR: EC10615-49-P1N (400 MHz, DMSO-d6) δ ppm 7.44 (br d, J=7.63 Hz, 2 H), 7.31 (br t, J=7.94 Hz, 6 H), 7.18 - 7.26 (m, 1 H), 6.89 (brd, J=8.00 Hz, 4 H), 4.08 - 4.13 (m, 1 H), 3.95 - 4.03 (m, 1 H), 3.84 - 3.93 (m, 1 H), 3.77 - 3.83 (m, 1 H), 3.74 (s, 6 H), 3.43 - 3.53 (m, 3 H), 3.38 (br d, J=6.75 Hz, 1 H), 2.94 - 3.04 (m, 1 H), 2.70 - 2.85 (m, 1 H), 1.09 - 1.15 (m, 12 H), 1.07 (br s, 3 H)。1H NMR: EC10615-49-P1N (400 MHz, DMSO-d6) δ ppm 7.44 (br d, J=7.63 Hz, 2 H), 7.31 (br t, J=7.94 Hz, 6 H), 7.18 - 7.26 (m, 1 H), 6.89 (brd, J=8.00 Hz, 4 H), 4.08 - 4.13 (m, 1 H), 3.95 - 4.03 (m, 1 H), 3.84 - 3.93 (m, 1 H), 3.77 - 3.83 (m, 1 H), 3.74 (s, 6 H), 3.43 - 3.53 (m, 3 H), 3.38 (br d, J=6.75 Hz, 1 H), 2.94 - 3.04 (m, 1 H), 2.70 - 2.85 (m, 1 H), 1.09 - 1.15 (m, 12 H), 1.07 (br s, 3 H).
實施例2.亞磷醯胺化合物2Example 2. Phosphamide Compound 2
將DMTrCl（232g，684mmol，1.0當量）的吡啶（400mL）溶液添加到異甘露醇化合物A（100g，684mmol，1.0當量）的吡啶（600mL）溶液中，並將混合物在25℃下攪拌12小時。LC-MS顯示化合物A完全消耗並檢測到一個具有期望質量的主峰。將所得反應混合物用水（500mL）稀釋，用DCM（500mL×2）萃取，並將合併的有機相用鹽水（500mL）洗滌，經Na2SO4乾燥並真空濃縮，得到殘餘物。將殘餘物通過柱色譜（DCM/MeOH=100/1至50/1，0.1% Et3N）純化，得到化合物B（150g，48.9%產率），為黃色固體。A solution of DMTrCl (232 g, 684 mmol, 1.0 eq) in pyridine (400 mL) was added to a solution of isomannitol Compound A (100 g, 684 mmol, 1.0 eq) in pyridine (600 mL), and the mixture was stirred at 25°C for 12 hours. LC-MS indicated complete consumption of Compound A, with the detection of a major peak with the desired mass. The resulting reaction mixture was diluted with water (500 mL) and extracted with DCM( 500 mL x 2). The combined organic phases were washed with brine (500 mL), dried overNa₂SO₄ , and concentrated in vacuo to yield a residue. The residue was purified by column chromatography (DCM/MeOH = 100/1 to 50/1, 0.1% Et3 N) to give compound B (150 g, 48.9% yield) as a yellow solid.
1H NMR: EC4783-404-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.46 (br d, J=7.63 Hz, 2 H) 7.28 - 7.37 (m, 6 H) 7.19 - 7.25 (m, 1 H) 6.90 (br d, J=7.88 Hz, 4 H) 4.70 (d, J=6.50 Hz, 1 H) 3.99 - 4.09 (m, 6 H) 3.88 - 3.96 (m, 2 H) 3.83 (br dd, J=7.82, 6.94 Hz, 1 H) 3.74 (s, 6 H) 3.41 (br t, J=8.13 Hz, 1 H) 3.05 (t, J=8.44 Hz, 1 H) 2.85 (br t, J=7.50 Hz, 1 H)。1H NMR: EC4783-404-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.46 (br d, J=7.63 Hz, 2 H) 7.28 - 7.37 (m, 6 H) 7.19 - 7.25 (m, 1 H) 6.90 (br d, J=7.88 Hz, 4 H) 4.70 (d, J=6.50 Hz, 1 H) 3.99 - 4.09 (m, 6 H) 3.88 - 3.96 (m, 2 H) 3.83 (br dd, J=7.82, 6.94 Hz, 1 H) 3.74 (s, 6 H) 3.41 (br t, J=8.13 Hz, 1H) 3.05 (t, J=8.44 Hz, 1 H) 2.85 (br t, J=7.50 Hz, 1 H).
在25℃和N2氣氛下，向化合物B（80.0g，178mmol，1.0當量）的DCM（800mL）溶液中滴加2H-四唑（0.45M，436mL，1.1當量），然後向混合物中滴加化合物C（80.6g，267mmol，85.0mL，1.5當量）的DCM（200mL）溶液。將反應混合物在25℃下攪拌1.0小時。LC-MS顯示化合物B完全消耗並檢測到一個具有期望質量的主峰。將所得反應混合物冷卻至-20℃並倒入冰冷的飽和NaHCO3（500mL）中，用DCM（500mL×3）萃取，將合併的有機層用飽和NaHCO3/鹽水=1:1（300mL/300mL）洗滌，經Na2SO4乾燥並真空濃縮（35℃），得到殘餘物（100mL）。將殘餘物通過柱色譜（Al2O3，DCM/MeOH=100/1至50/1，0.1% Et3N）純化，得到化合物2（77g，119mmol，66.5%產率），為白色固體。To a solution of compound B (80.0 g, 178 mmol, 1.0 equiv) in DCM (800 mL) was added dropwise 2H-tetrazole (0.45 M, 436 mL, 1.1 equiv) at 25°C underN₂ atmosphere. A solution of compound C (80.6 g, 267 mmol, 85.0 mL, 1.5 equiv) in DCM (200 mL) was then added dropwise. The reaction mixture was stirred at 25°C for 1.0 hour. LC-MS analysis indicated complete consumption of compound B, with a major peak of the expected mass detected. The resulting reaction mixture was cooled to -20°C and poured into ice-cold saturatedNaHCO₃ (500 mL). The mixture was extracted with DCM (500 mL x 3). The combined organic layers were washed with saturatedNaHCO₃ /brine water (1:1) (300 mL/300 mL), dried overNa₂SO₄ , and concentrated in vacuo (35°C) to obtain a residue (100mL ). The residue was purified by column chromatography (Al₂O₃ , DCM/MeOH = 100/1 to 50/1, 0.1%Et₃N ) to obtain compound 2 (77 g, 119 mmol, 66.5% yield) as a white solid.
1H NMR: EC4783-423-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.22 (br d, J=7.50 Hz, 2 H) 7.05 - 7.14 (m, 6 H) 6.96 - 7.02 (m, 1 H) 6.67 (br dd, J=8.82, 1.81 Hz, 4 H) 3.95 - 4.07 (m, 2 H) 3.73 - 3.83 (m, 1 H) 3.62 - 3.72 (m, 2 H) 3.48 - 3.53 (m, 6 H) 3.27 - 3.37 (m, 3 H) 3.11 (s, 6 H) 2.82 (td, J=8.54, 2.31 Hz, 1 H) 2.47 - 2.63 (m, 3 H) 2.28 (br d, J=1.63 Hz, 3 H) 0.82 - 1.00 (m, 13 H)。1H NMR: EC4783-423-P1B1_C (400 MHz, DMSO-d6) δ ppm 7.22 (br d, J=7.50 Hz, 2 H) 7.05 - 7.14 (m, 6 H) 6.96 - 7.02 (m, 1 H) 6.67 (br dd, J=8.82, 1.81 Hz, 4 H) 3.95 - 4.07 (m, 2 H) 3.73 - 3.83 (m, 1 H) 3.62 - 3.72 (m, 2 H) 3.48 - 3.53 (m, 6 H) 3.27 - 3.37 (m, 3 H) 3.11 (s, 6 H) 2.82 (td, J=8.54, 2.31 Hz, 1 H) 2.47 - 2.63 (m, 3 H) 2.28 (br d, J=1.63 Hz, 3 H) 0.82 - 1.00 (m, 13 H).
其他亞磷醯胺可根據本文和/或現有技術所述的方法（諸如但不限於US426,220和WO02/36743）進行製備。Other phosphoramidites can be prepared according to methods described herein and/or in the prior art (such as, but not limited to, US 426,220 and WO 02/36743).
實施例3.包含本發明的亞磷醯胺單體的固體支持物的製備Example 3. Preparation of a solid support containing the phosphoramidite monomer of the present invention
表示胺甲基聚乙烯大孔樹脂載劑部分Indicates the aminomethyl polyethylene macroporous resin carrier part
在氮氣保護下，將二氯甲烷（19.50kg）添加到50L玻璃釜中並開始攪拌。將溫度控制在20℃至30℃，並將DMTr異甘露醇殘基（1.47kg）、三乙胺（1.50kg）、4-二甲基氨基吡啶（0.164kg）和琥珀酸酐（1.34kg）添加到該玻璃釜中。將體系在20℃至30℃下保持18小時，取樣並結束反應。將飽和碳酸氫鈉溶液（22.50kg）添加到反應體系中，攪拌10分鐘至20分鐘並使其分層。將有機相分離並將水相用二氯甲烷萃取兩次，將有機相合併，並經無水硫酸鈉乾燥，過濾並真空濃縮，得到殘餘物，形成1.83kg灰色至灰白色的固體。Under nitrogen, dichloromethane (19.50 kg) was added to a 50 L glass kettle and stirring was initiated. The temperature was controlled between 20°C and 30°C, and DMTr isomannitol residue (1.47 kg), triethylamine (1.50 kg), 4-dimethylaminopyridine (0.164 kg), and succinic anhydride (1.34 kg) were added to the kettle. The system was maintained at 20°C to 30°C for 18 hours, after which a sample was taken and the reaction was terminated. A saturated sodium bicarbonate solution (22.50 kg) was added to the reaction system, stirred for 10 to 20 minutes, and the layers were allowed to separate. The organic phase was separated and the aqueous phase was extracted twice with dichloromethane. The organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give a residue, forming 1.83 kg of a gray to off-white solid.
將N,N-二甲基甲醯胺（23.50kg）添加到100L玻璃釜中並攪拌。將溫度控制在20℃至30℃。在氮氣保護下，將上一步驟產物O-苯並三唑四甲基脲六氟磷酸鹽（0.33kg）和N,N-二異丙基乙胺（0.13kg）通過固體進料漏斗添加到上述100L玻璃釜中並攪拌10分鐘至30分鐘，然後倒入50L鋅桶中備用。將大孔胺甲基樹脂（3.25kg）（購自天津南開和成科學技術有限公司，批號為HA2X1209，負荷容量為0.48mmol/g）通過固體進料漏斗添加到上述100L固相合成反應器中，將溫度控制在20℃至30℃，將N,N-二甲基甲醯胺（21.00kg+21.00kg）和上一步驟的鋅桶中的反應溶液添加到該固相合成反應器中。該體系進行保溫反應，並對固體負荷跟蹤至≥250μmol/g，並且負荷檢測方法為紫外光（UV）。在氮氣壓力下過濾該體系，將濾餅用N,N-二甲基甲醯胺洗滌三次（26.00kg+26.10kg+26.00kg），並將濾餅留在該釜中。將CAP.A（50%乙腈和50%乙酸酐，4.40kg+4.42kg+4.30kg）和CAP.B（20%吡啶和30% N-甲基咪唑和50%乙腈，4.40kg+4.40kg+4.47kg）添加到80L玻璃釜中，並在使用前攪拌3分鐘至8分鐘。重複該操作三次以加帽，並將乙腈（18.00kg+18.00kg+18.00kg+17.50kg+17.50kg）添加到固相合成釜中。在氮氣鼓泡達10分鐘至30分鐘後進行壓濾。重複該操作四次，在固相合成釜中將濾餅用氮氣吹掃2小時至4小時，然後轉移到50L壓濾罐中。將溫度控制在15℃至30℃，繼續乾燥，並在乾燥後得到黃色至白色固體產物，重量：3.516 kg。Add N,N-dimethylformamide (23.50 kg) to a 100-L glass kettle and stir. Maintain the temperature between 20°C and 30°C. Under nitrogen, add O-benzotriazole tetramethyluronium hexafluorophosphate (0.33 kg) and N,N-diisopropylethylamine (0.13 kg), the product from the previous step, to the 100-L glass kettle via a solid addition funnel. Stir for 10 to 30 minutes, then pour into a 50-L zinc drum for later use. Macroporous amine methyl resin (3.25 kg) (purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd., batch number HA2X1209, loading capacity 0.48 mmol/g) was added to the above-mentioned 100-L solid-phase synthesis reactor via a solid feeding funnel. The temperature was controlled between 20°C and 30°C. N,N-dimethylformamide (21.00 kg + 21.00 kg) and the reaction solution from the zinc drum from the previous step were added to the solid-phase synthesis reactor. The system was kept warm, and the solid loading was monitored to ≥250 μmol/g. The loading was monitored using ultraviolet light (UV). The system was filtered under nitrogen pressure, and the filter cake was washed three times with N,N-dimethylformamide (26.00 kg + 26.10 kg + 26.00 kg), leaving the filter cake in the kettle. CAP.A (50% acetonitrile and 50% acetic anhydride, 4.40 kg + 4.42 kg + 4.30 kg) and CAP.B (20% pyridine and 30% N-methylimidazole and 50% acetonitrile, 4.40 kg + 4.40 kg + 4.47 kg) were added to an 80 L glass kettle and stirred for 3 to 8 minutes before use. This operation was repeated three times to cap the reaction, and acetonitrile (18.00 kg + 18.00 kg + 18.00 kg + 17.50 kg + 17.50 kg) was added to the solid-phase synthesis kettle. After bubbling nitrogen for 10 to 30 minutes, pressure filtration was performed. This operation was repeated four times. The filter cake was purged with nitrogen for 2 to 4 hours in the solid-phase synthesis reactor and then transferred to a 50L autoclave. Drying was continued while controlling the temperature between 15°C and 30°C. After drying, a yellow to white solid product weighing 3.516 kg was obtained.
實施例4.TMPRSS6 RNAi藥劑的合成。Example 4. Synthesis of TMPRSS6 RNAi Agent.
根據以下通用方法合成上文表2至表3所示的TMPRSS6 RNAi藥劑雙鏈體：The TMPRSS6 RNAi agent duplexes shown in Tables 2 and 3 above were synthesized according to the following general method:
使用基於亞磷醯胺化學的成熟的固相合成方法，在寡核苷酸合成儀上合成siRNA的有義鏈序列和反義鏈序列。寡核苷酸鏈的增長通過4步循環實現：脫保護、偶聯、加帽以及氧化或硫化步驟，以用於添加每個核苷酸。合成是在由可控孔徑玻璃（CPG，1000Å）製成的固體支持物上進行。單體亞磷醯胺購自商業來源，或者可以是實施例1至實施例2或WO2023/045995中的亞磷醯胺化合物。本文的亞磷醯胺化合物可作為單體核苷酸與CPG或聚苯乙烯固體支持物的3'-端連接。在連接在5'-端處的情況下，亞磷醯胺化合物可用於最終的偶聯反應，並且可進一步與靶配體綴合（如果需要）。The sense and antisense strands of siRNA are synthesized on an oligonucleotide synthesizer using a well-established solid-phase synthesis method based on phosphoramidite chemistry. The growth of the oligonucleotide chain is achieved through a four-step cycle: deprotection, coupling, capping, and oxidation or sulfurization steps for the addition of each nucleotide. The synthesis is carried out on a solid support made of controlled pore glass (CPG, 1000Å). Monomeric phosphoramidites are purchased from commercial sources or can be the phosphoramidite compounds described in Examples 1 to 2 or WO2023/045995. The phosphoramidite compounds herein can be linked as monomeric nucleotides to the 3'-end of a CPG or polystyrene solid support. When attached at the 5'-end, the phosphoramidite compound can be used for the final coupling reaction and can be further conjugated to the target ligand if desired.
具有GalNAc配體簇（GLPA1、GLPA2和GLPA15作為非限制性示例）的亞磷醯胺根據方案1和方案2所示的方法或根據WO2023/045995中的程序進行合成。在GalNAc配體（GLO-0作為非限制性示例，其中GLO-0是指Jayaprakash等人，(2014) J. Am. Chem.Soc., 136, 16958−16961中的化合物GalNAc3。）連接在有義鏈的3'-端的情況下，使用連接GalNAc配體的CPG固體支持物。在GalNAc配體（GLS-5*或GLS-15*作為非限制性示例）連接在有義鏈的5'-端的情況下，針對最終的偶聯反應使用GalNAc亞磷醯胺（GLPA1、GLPA2或GLPA15作為非限制性示例）。使用3%三氯乙酸（TCA）的二氯甲烷溶液對4,4'-二甲氧基三苯甲基保護基團（DMT）進行脫保護。5-乙硫基-1H-四唑用作活化劑。I2的THF/Py/H2O溶液和苯乙醯基二硫化物（PADS）的吡啶/MeCN溶液分別用於氧化反應和硫化反應。在最終的固相合成步驟之後，裂解結合固體支持物的低聚物，並通過用40重量%甲胺的水溶液和28%氫氧化銨溶液的1:1體積溶液處理來除去保護基團。為了合成用於體外篩選的siRNA，將粗混合物濃縮。將其餘固體溶解在1.0M NaOAc中，並添加冰冷的EtOH以沉澱出單鏈產物，為鈉鹽，其無需進一步純化即可用於退火。為了合成用於體內測試的多靶點分子，將單鏈粗產物通過離子配對反相HPLC（IP-RP-HPLC）進一步純化。通過溶解在1.0M NaOAc中並通過添加冰冷的EtOH沉澱進行沉澱，將來自IP-RP-HPLC的純化單鏈寡核苷酸產物轉化為鈉鹽。將等莫耳互補的有義鏈和反義鏈寡核苷酸在水中退火，形成雙鏈siRNA產物，將其凍幹，得到蓬鬆的白色固體。Phosphoamidites bearing GalNAc ligand clusters (GLPA1, GLPA2, and GLPA15 as non-limiting examples) were synthesized according to the methods shown in Schemes 1 and 2 or the procedures in WO2023/045995. A GalNAc ligand (GLO-0 as a non-limiting example, where GLO-0 refers to the compound GalNAc3 described in Jayaprakash et al., (2014) J. Am. Chem. Soc., 136, 16958−16961)) was attached to the 3'-end of the sense chain, and a CPG solid support with the GalNAc ligand was used. With a GalNAc ligand (GLS-5* or GLS-15* as non-limiting examples) attached to the 5'-end of the sense chain, a GalNAc phosphoamidite (GLPA1, GLPA2, or GLPA15 as non-limiting examples) was used for the final coupling reaction. The 4,4'-dimethoxytrityl protecting group (DMT) was deprotected using 3% trichloroacetic acid (TCA) in dichloromethane. 5-Ethylthio-1H-tetrazole was used as an activating agent.I2 in THF/Py/H2O and phenylacetyl disulfide (PADS) in pyridine/MeCN were used for oxidation and sulfidation, respectively. After the final solid-phase synthesis step, the oligomers bound to the solid support were cleaved and the protecting groups removed by treatment with a 1:1 volume ratio of 40 wt% methylamine in water and 28% ammonium hydroxide solution. To synthesize siRNA for in vitro screening, the crude mixture was concentrated. The remaining solid was dissolved in 1.0 M NaOAc, and ice-cold EtOH was added to precipitate the single-chain product as the sodium salt, which was used for annealing without further purification. To synthesize multitarget molecules for in vivo testing, the single-chain crude product was further purified by ion-pairing reversed-phase HPLC (IP-RP-HPLC). The purified single-stranded oligonucleotide product from IP-RP-HPLC was converted to the sodium salt by dissolving in 1.0 M NaOAc and precipitating by adding ice-cold EtOH. Equimolar complementary sense and antisense oligonucleotides were annealed in water to form a double-stranded siRNA product, which was freeze-dried to yield a fluffy white solid.
方案1Solution 1
方案2
實施例5.TMPRSS6 siRNA雙鏈體的體外篩選Example 5. In vitro screening of TMPRSS6 siRNA duplexes
將Huh7細胞用胰蛋白酶消化並調節至適當的密度，與psiCHECK(TM)-2載體質粒和Lipofectamine 2000（Invitrogen-11668-019）的複合物混合並接種到96孔板中。根據製造商建議的方案，在第2天，使用Lipofectamine RNAiMax（Invitrogen -13778-150）用測試siRNA或對照siRNA轉染細胞。將兩種濃度（1nM和0.1nM）的siRNA重複測試三次。Huh7 cells were trypsinized and adjusted to an appropriate density. They were mixed with a complex of the psiCHECK(TM)-2 vector plasmid and Lipofectamine 2000 (Invitrogen 11668-019) and seeded into 96-well plates. On day 2, cells were transfected with test or control siRNA using Lipofectamine RNAiMax (Invitrogen 13778-150) according to the manufacturer's recommended protocol. Two siRNA concentrations (1 nM and 0.1 nM) were tested in triplicate.
第1天，TMPRSS6-psiCHECK(TM)-2載體轉染（一塊板）Day 1, TMPRSS6-psiCHECK(TM)-2 vector transfection (one plate)
(1)將適當產量的TMPRSS6-psiCHECK(TM)-2載體質粒轉移到不含核糖核酸酶的離心管（溶液混合物#1）中(1) Transfer the appropriate yield of TMPRSS6-psiCHECK(TM) -2 vector plasmid to an RNase-free centrifuge tube (solution mixture #1).
(2)在一個燒瓶中添加胰蛋白酶以分離Huh7細胞，並使用Vi-Cell計數儀對細胞進行計數，將細胞密度調節至適當的濃度。(2) Add trypsin to a flask to detach Huh7 cells and count the cells using a Vi-Cell counter to adjust the cell density to an appropriate concentration.
(3)將適當體積的Lipofectamine 2000（Invitrogen-11668-019）轉移到溶液混合物#1管中，進行混合。(3) Transfer an appropriate volume of Lipofectamine 2000 (Invitrogen-11668-019) to the solution mixture #1 tube and mix.
(4)將步驟3中的溶液添加到細胞懸浮液中，進行混合，並將懸浮液分配到96孔板中（100μl/孔）(4) Add the solution from step 3 to the cell suspension, mix, and dispense the suspension into a 96-well plate (100 μl/well).
第2天，siRNA轉染Day 2, siRNA transfection
(1)用Opti-MEM®培養基稀釋Lipofectamine®RNAiMAX試劑。(1) Dilute Lipofectamine® RNAiMAX reagent in Opti-MEM® medium.
(2)用不含RNA的水稀釋siRNA，以製備儲備溶液。(2) Dilute siRNA with RNA-free water to prepare a stock solution.
(3)將等體積稀釋的RNAiMax和siRNA混合。將混合物在室溫下孵育15分鐘，形成複合物。(3) Mix equal volumes of diluted RNAiMax and siRNA. Incubate the mixture at room temperature for 15 minutes to allow complex formation.
(4)將適當體積/孔的化合物Lipofectamine® RNAiMAX(Opti-MEM)混合物添加到適當體積/孔的DMEM新鮮培養基中，以製備工作溶液，然後棄去測定板中的上清液，將適當體積/孔的化合物混合物添加到96孔板中。(4) Add an appropriate volume/well of the compound Lipofectamine® RNAiMAX (Opti-MEM) mixture to an appropriate volume/well of fresh DMEM medium to prepare a working solution. Discard the supernatant in the assay plate and add an appropriate volume/well of the compound mixture to a 96-well plate.
(5)無化合物對照孔被定義為用psiCHECK(TM)-2載體轉染且未用siRNA處理的細胞；空白對照是僅含細胞的孔。(5) No compound control wells were defined as cells transfected with psiCHECK(TM)-2 vector and not treated with siRNA; blank controls were wells containing cells alone.
第3天，Dual-Glo®螢光素酶測定Day 3, Dual-Glo® Luciferase Assay
(1)將試劑添加到測定板中，等待一定時間以使細胞發生裂解。(1) Add the reagent to the assay plate and wait for a certain period of time to allow the cells to lyse.
(2)將適當體積的細胞裂解物轉移到板中，然後測量firefly發光。(2) Transfer an appropriate volume of cell lysate to a plate and measure firefly luminescence.
(3)將適當體積的Dual-Glo® Stop & Glo®試劑添加到測定板中並混合，等待一定時間，然後測量Renilla發光。(3) Add an appropriate volume of Dual-Glo® Stop & Glo® reagent to the assay plate and mix, wait for a specified time, and then measure Renilla luminescence.
(4)計算相對表達(4) Calculate relative expression
數據分析Data Analysis
樣本孔的比率=(樣本Renilla發光-背景Renilla發光)/(樣本Firefly發光-背景Firefly發光)Sample well ratio = (sample Renilla luminescence - background Renilla luminescence) / (sample Firefly luminescence - background Firefly luminescence)
無化合物對照孔的比率=(對照Renilla發光-背景Renilla發光)/(對照樣本Firefly發光-背景Firefly發光)Ratio of no compound control wells = (control Renilla luminescence - background Renilla luminescence) / (control sample Firefly luminescence - background Firefly luminescence)
抑制%=100-(樣本孔的比率/無化合物對照的平均比率)×100%Inhibition % = 100-(ratio of sample well/average ratio of no compound control) × 100%
表5提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
實施例6.TMPRSS6 siRNA雙鏈體的體外篩選Example 6. In vitro screening of TMPRSS6 siRNA duplexes
將Huh7細胞用胰蛋白酶消化並調節至適當的密度，接種到96孔板中。根據製造商建議的方案，在接種的第二天，使用Lipofectamine 2000（Invitrogen-11668-019）用psiCHECK(TM)-2載體質粒、空白載體PCNDA3.0、siRNA或對照siRNA的複合物轉染細胞。將不同濃度（1nM和0.1nM）的siRNA重複測試三次。Huh7 cells were trypsinized, adjusted to an appropriate density, and seeded into 96-well plates. The day after seeding, cells were transfected with a complex of the psiCHECK(TM)-2 vector plasmid, the blank vector pCNDA3.0, siRNA, or a control siRNA using Lipofectamine 2000 (Invitrogen 11668-019) according to the manufacturer's recommended protocol. Different siRNA concentrations (1 nM and 0.1 nM) were tested in triplicate.
第1天，在一個燒瓶中添加胰蛋白酶以分離Huh7細胞，並使用Vi-Cell計數儀對細胞進行計數，將細胞密度調節至1×10^5/ml並用DMEM培養基培養。On day 1, trypsin was added to a flask to detach Huh7 cells, and the cells were counted using a Vi-Cell counter. The cell density was adjusted to 1 × 10^5/ml and cultured in DMEM medium.
第2天，TMPSSR6-psiCHECK(TM)-2載體/空白載體pCDNA3.0/siRNA/Lipofectamine 2000混合物轉染On the second day, the mixture of TMPSSR6-psiCHECK(TM)-2 vector/blank vector pCDNA3.0/siRNA/Lipofectamine 2000 was transfected
(1)將適當的Lipofectamine 2000（Invitrogen-11668-019）與Opti-MEM®培養基混合（溶液混合物#1）。最終相當於每孔0.3μl Lipofectamine 2000和4.7μl Opti-MEM®培養基。(1) Mix the appropriate amount of Lipofectamine 2000 (Invitrogen-11668-019) with Opti-MEM® medium (Solution Mixture #1). The final volume is equivalent to 0.3 μl Lipofectamine 2000 and 4.7 μl Opti-MEM® medium per well.
(2)將適當的TMPSSR6--psiCHECK(TM)-2載體、空白pCNDNA 3.0載體和siRNA與Opti-MEM®培養基混合於每孔中（溶液混合物#2）。(2) Mix the appropriate TMPSSR6-psiCHECK(TM)-2 vector, blank pCNDNA 3.0 vector, and siRNA with Opti-MEM® medium in each well (Solution Mixture #2).
(3)將等體積的溶液混合物#1和混合物#2混合，最終每孔10μl。將混合物在室溫下孵育15分鐘，形成複合物。(3) Combine equal volumes of solution mixture #1 and solution mixture #2, resulting in a final volume of 10 μl per well. Incubate the mixture at room temperature for 15 minutes to allow complex formation.
(4)棄去DMEM培養基，添加10μl混合的溶液混合物#1和混合物#2以及90μl新鮮DMEM培養基。(4) Discard the DMEM medium and add 10 μl of the mixed solution mixture #1 and mixture #2 and 90 μl of fresh DMEM medium.
(5)無化合物對照孔被定義為用TMPSSR6-psiCHECK(TM)-2載體和空白載體pCNDNA 3.0轉染且未用siRNA處理的細胞；空白對照是僅含細胞的孔。(5) No compound control wells were defined as cells transfected with TMPSSR6-psiCHECK(TM)-2 vector and blank vector pCNDNA 3.0 and not treated with siRNA; blank controls were wells containing cells alone.
第3天，根據Dual Glo螢光素酶報告基因測定試劑盒（YEASEN、11405ES80）說明書進行Dual Glo螢光素酶測定。結果示於表6至表10中。On day 3, the Dual Glo Luciferase Assay was performed according to the instructions for the Dual Glo Luciferase Reporter Gene Assay Kit (YEASEN, 11405ES80). The results are shown in Tables 6 to 10.
表6提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
表7提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
表8提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
表9提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
表10提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體外研究的實驗結果。所使用的雙鏈體序列對應於表2所示的序列。
實施例7.TMPRSS6 siRNA雙鏈體的體內測試Example 7. In vivo testing of TMPRSS6 siRNA duplexes
為了評估TMPRSS6 siRNA的體內活性，使用了用編碼人TMPRSS6基因的AAV感染的小鼠（每組3只小鼠）。在第1天，通過靜脈內施用編碼人TMPRSS6基因的腺相關病毒8（AAV8）載體的溶液來感染雌性C57BL/6J小鼠。在第8天，向小鼠皮下施用0.5mg/kg、1mg/kg、2mg/kg、4mg/kg或6mg/kg的單一TMPRSS6 siRNA藥劑或PBS。在第15天或第22天，處死小鼠，收集肝臟組織樣本，根據製造商建議的方案，對人TMPRSS6的mRNA表達進行QPCR分析。通過比較給藥後第15天的人TMPRSS6 mRNA水平與PBS處理組中的人TMPRSS6 mRNA水平來計算敲低百分比。結果匯總於表11至表19中。所使用的雙鏈體序列對應於表3所示的序列。To evaluate the in vivo activity of TMPRSS6 siRNA, mice infected with AAV encoding the human TMPRSS6 gene (n=3 mice per group) were used. On day 1, female C57BL/6J mice were infected intravenously with a solution of an adeno-associated virus 8 (AAV8) vector encoding the human TMPRSS6 gene. On day 8, mice were subcutaneously administered with 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 4 mg/kg, or 6 mg/kg of a single TMPRSS6 siRNA dose or PBS. On day 15 or 22, mice were sacrificed, and liver tissue samples were collected for quantitative polymerase chain reaction (QPCR) analysis of human TMPRSS6 mRNA expression according to the manufacturer's recommended protocol. Percent knockdown was calculated by comparing human TMPRSS6 mRNA levels on day 15 post-administration with those in the PBS-treated group. The results are summarized in Tables 11 to 19. The duplex sequences used correspond to those shown in Table 3.
表11提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表12提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表13提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表14提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表15提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表16提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表17提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表18提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表19提供了使用各種TMPRSS6 RNAi藥劑抑制TMPRSS6表達的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
實施例8.TMPRSS6 siRNA雙鏈體的體內測試Example 8. In vivo testing of TMPRSS6 siRNA duplexes
食蟹猴（3歲至5歲，體重3kg至6kg，每組3只）入組該研究。在第1天（在siRNA給藥前），向每只猴皮下施用3mg/kg的單一TMPRSSR6 siRNA藥劑或PBS。通過QPCR方法測定肝臟中的TMPRSS6和鐵調素mRNA水平。結果示於表20至表21中。Cynomolgus monkeys (3 to 5 years old, weighing 3 to 6 kg, 3 per group) were enrolled in this study. On day 1 (before siRNA administration), each monkey received a subcutaneous dose of 3 mg/kg of a single TMPRSSR6 siRNA or PBS. Liver mRNA levels of TMPRSS6 and hepcidin were measured by qPCR. The results are shown in Tables 20 and 21.
表20提供了使用各種TMPRSS6 RNAi藥劑的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
表21提供了使用各種TMPRSS6 RNAi藥劑的體內研究的實驗結果。所使用的雙鏈體序列對應於表3所示的序列。
等同物equivalent
儘管本文已經描述並示出了本發明的若干實施方案，但是本領域普通技術人員將容易地設想用於執行本文所述的功能和/或獲得本文所述的結果和/或一個或多個優點的各種其他裝置和/或結構，並且此類變化和/或修改中的每一者均被認為在本發明的範圍內。更一般地，本領域技術人員將容易地理解，本文所述的所有參數、尺寸、材料和構型都是示例性的，並且實際的參數、尺寸、材料和/或構型將取決於使用本發明的教導內容的一個或多個具體應用。本領域技術人員將認識到或能夠僅使用常規實驗來確定本文描述的本發明的具體實施方案的許多等同物。因此，應當理解，前述實施方案僅以舉例的方式呈現，並且在所附申請專利範圍及其等同物的範圍內；本發明可以不同於具體描述和要求保護的方式來實踐。本發明涉及本文所述的每個單獨的特徵、系統、製品、材料和/或方法。此外，如果此類特徵、系統、製品、材料和/或方法不是相互矛盾的，則兩個或更多個此類特徵、系統、製品、材料和/或方法的任何組合均包括在本發明的範圍內。Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily conceive of various other devices and/or structures for performing the functions described herein and/or obtaining the results and/or one or more advantages described herein, and each of such variations and/or modifications is considered to be within the scope of the present invention. More generally, a person of ordinary skill in the art will readily understand that all parameters, dimensions, materials, and configurations described herein are exemplary, and that actual parameters, dimensions, materials, and/or configurations will depend on one or more specific applications in which the teachings of the present invention are used. A person of ordinary skill in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the present invention described herein. It should be understood, therefore, that the foregoing embodiments are presented by way of example only and are within the scope of the appended claims and their equivalents; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. Furthermore, any combination of two or more such features, systems, articles, materials, and/or methods is within the scope of the present invention if such features, systems, articles, materials, and/or methods are not mutually inconsistent.
如本文所定義和使用的所有定義應當理解為受控於字典的定義、以引用方式併入的文獻中的定義和/或所定義術語的普通含義。All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
除非明確地相反指明，否則如本說明書和申請專利範圍中所使用的不定冠詞“一”和“一個”應當理解為意指“至少一個”。Unless expressly indicated to the contrary, the indefinite articles "a" and "an" as used in this specification and claims should be understood to mean "at least one."
如本說明書和申請專利範圍中所使用的短語“和/或”應當理解為意指如此結合的要素：即，在一些情況下聯合存在並且在其他情況下分離存在的要素中的“任一者或兩者”。除了由“和/或”從句明確標識的元件之外，還可任選地存在其他要素，無論與明確標識的那些要素是否相關，除非清楚地指出相反情況。As used in this specification and claims, the phrase "and/or" should be understood to mean elements conjoined: that is, "either or both" of the elements that are present in some cases conjoined and in other cases separate. Other elements may optionally be present other than the elements expressly identified by the "and/or" clause, whether related or unrelated to those elements expressly identified, unless clearly stated to the contrary.
本申請中引用或提及的所有參考文獻、專利和專利申請以及出版物均以引用方式全文併入本文。All references, patents and patent applications, and publications cited or referred to in this application are incorporated herein by reference in their entirety.
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| EP3587578A1 (en)* | 2013-05-22 | 2020-01-01 | Alnylam Pharmaceuticals, Inc. | Tmprss6 irna compositions and methods of use thereof |
| WO2016085852A1 (en)* | 2014-11-24 | 2016-06-02 | Alnylam Pharmaceuticals, Inc. | Tmprss6 irna compositions and methods of use thereof |
| EP3277817A4 (en)* | 2015-04-03 | 2018-12-05 | Ionis Pharmaceuticals, Inc. | Compounds and methods for modulating tmprss6 expression |
| EP4330392A1 (en)* | 2021-04-26 | 2024-03-06 | Alnylam Pharmaceuticals, Inc. | Transmembrane protease, serine 6 (tmprss6) irna compositions and methods of use thereof |
| TWI868755B (en)* | 2022-06-24 | 2025-01-01 | 丹麥商諾佛 儂迪克股份有限公司 | Compositions and methods for inhibiting transmembrane serine protease 6 (tmprss6) expression |
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