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1. US20200362343 - METHODS AND MODIFICATIONS THAT PRODUCE ssRNAi COMPOUNDS WITH ENHANCED ACTIVITY, POTENCY AND DURATION OF EFFECT

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Claims

1- 14. (canceled)
15. A single strand modified oligoribonucleotide ags-siRNA or ags-IMiR compound for modulating the expression and/or function of at least one target nucleic acid sequence expressed in a cell, comprising:
(1) a nucleoside in position 1 and an associated linkage with the nucleoside in position 2 wherein
(i) the 5′ carbon of the 5′-end nucleoside sugar has a hydroxyl, a 5′-end phosphate group or a 5′-end phosphate analog and wherein the 5′-end phosphate analog is preferably 5′-(E)-vinylphosphonate (5′-VP), and
(ii) the nucleoside sugar is selected from the group consisting of ribose, 2′-fluoro, 2′-O-methyl, 2′-methoxyethyl, 2′-deoxyribose, LNA, FANA, 4'S-FANA, ALN, AENA, CENA, HM, HNA, EA, F-CeNA, CeNA, UNA, CRN R monomer and CRN Q monomer, and
(iii) the associated linkage is N3′ phosphoramidate or amide; and
(2) nucleosides in positions 2-19, said nucleosides comprising:
(i) modifications that increase basic nuclease resistance wherein said basic nuclease resistance is provided by
(a) 2′-fluoro alternating with 2′-O-methyl nucleosides with the 2′-fluoro being in the even numbered positions, where phosphorothioate linkages occur between nucleoside positions 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 15-16, 16-17, 17-18 and 18-19 and with phosphodiester linkages between the remaining nucleoside positions, or
(b) the alternating pattern of (a) wherein a 2′-O-methyl modification is used in position 2 and where two contiguous nucleosides in the region defined by positions 3-13 have a 2′-fluoro modified sugar so that when the alternating pattern of single 2′-O-methyl with single 2′fluoro modified sugar is continued, the 2′-fluoro modification falls on nucleoside positions 14 and 16, or
(c) the alternating pattern of (b) wherein the nucleoside in position 14 is ribose or HM and optionally the nucleoside in position 16 is independently ribose or HM,
wherein N3′-phosphoramidate linkages are optionally used in place of phosphorothioate and/or phosphodiester linkages in one or more positions; and
(ii) at least 4 accommodating guide strand designs (AGSD) modifications are present independently selected from the group of AGSD base modifications consisting of replacement of an U with 2-thiouracil, 5-methyluracil or pseudouracil base and replacement of a C with a 4-thiouracil, and/or from the group of AGSD sugar modifications consisting of LNA, HNA, ANA, CRN R monomer, CRN Q monomer, HM, FHNA, CeNA, F-CeNA and cET; and
(3) 1, 2, 3, or 4 optional overhang precursor units in positions 20-23 linked together and to the nucleoside in position 19 to form a 3′-end overhang precursor using a nuclease resistant linkage selected from the group consisting of phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylborane phosphine, carbamate, urea, thiourea, N3′phosphoramidate and amide; and
(4) a region of complementary base pairing with the target nucleic acid that is at least consecutive nucleosides in length beginning at position 2 and extending at least through position 16; and
wherein the compound exhibits a maximal plateau level of activity in a dose response curve against the target that constitutes at least a 50% change in expression and/or function of the target and said level of change in activity is at least 20 percentage points greater than the maximal level of activity obtained using an ssRNAi of the same sequence, same 5′-end phosphate group or a 5′-end phosphate analog, if any, and lacking the AGSD modifications found in the ags-siRNA or ags-IMiR compound and administered using the same dosage regimen where the compounds to be compared are delivered without an enveloping protective carrier.
16. The compound of claim 15, wherein AGSD modifications made to nucleoside positions 2-19 increase the probability that the modified nucleoside has its sugar in the C3′-endo conformation compared to the sugars (2′-fluoro, 2′-O-methyl and ribose) found in these positions in conventional ssRNAi compounds, wherein said ags-siRNA is directed to a target selected from the group consisting of pre-mRNA, mRNA, lncRNA, promoter associated RNA, enhancer RNA, snoRNA, piRNA, xiRNA, sdRNA, moRNA, MSY-RNA, tel-sRNA, crasiRNA, endogenous antisense RNA, a promoter, an enhancer and a suppressor.
17. A single strand modified oligoribonucleotide ags-siRNA or ags-IMiR compound for modulating the expression and/or function of at least one target nucleic acid sequence expressed in a cell, comprising:
(1) a nucleoside in position 1 and an associated linkage with the nucleoside in position 2 wherein
(i) the 5′ carbon of the 5′-end nucleoside sugar has a hydroxyl, a 5′-end phosphate group or a 5′-end phosphate analog and wherein the 5′-end phosphate analog is preferably 5′-(E)-vinylphosphonate (5′-VP), and
(ii) the nucleoside sugar is selected from the group consisting of ribose, 2′-fluoro, 2′-O-methyl, 2′-methoxyethyl, 2′-deoxyribose, LNA, FANA, 4'S-FANA, ALN, AENA, CENA, HM, HNA, EA, F-CeNA, CeNA, UNA, CRN R monomer and CRN Q monomer, and
(iii) the associated linkage is N3′ phosphoramidate or amide; and
(2) nucleosides in positions 2-19, said nucleosides comprising:
(i) modifications that increase basic nuclease resistance wherein said basic nuclease resistance is provided by
(a) 2′-fluoro alternating with 2′-O-methyl nucleosides with the 2′-fluoro being in the even numbered positions, where phosphorothioate linkages occur between nucleoside positions 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 15-16, 16-17, 17-18 and 18-19 and with phosphodiester linkages between the remaining nucleoside positions, or
(b) the alternating pattern of (a) wherein a 2′-O-methyl modification is used in position 2 and where two contiguous nucleosides in the region defined by positions 3-13 have a 2′-fluoro modified sugar so that when the alternating pattern of single 2′-O-methyl with single 2′fluoro modified sugar is continued, the 2′-fluoro modification falls on nucleoside positions 14 and 16, or
(c) the alternating pattern of (b) wherein the nucleoside in position 14 is ribose or HM and optionally the nucleoside in position 16 is independently ribose or HM,
wherein N3′-phosphoramidate linkages are used in place of phosphorothioate and/or phosphodiester linkages in one or more positions; and
(ii) at least 4 accommodating guide strand designs (AGSD) modifications are optionally present independently selected from the group of AGSD base modifications consisting of replacement of an U with 2-thiouracil, 5-methyluracil or pseudouracil base and replacement of a C with a 4-thiouracil, and/or from the group of AGSD sugar modifications consisting of LNA, HNA, ANA, CRN R monomer, CRN Q monomer, HM, FHNA, CeNA, F-CeNA and cET; and
wherein AGSD modifications increase the probability that the modified nucleoside having its sugar in the C3′-endo conformation compared to the sugars (2′-fluoro, 2′-O-methyl and ribose) found in these positions in conventional ssRNAi compounds; and
(3) 1, 2, 3, or 4 optional overhang precursor units in positions 20-23 linked together and to the nucleoside in position 19 to form a 3′-end overhang using a nuclease resistant linkage selected from the group consisting of phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylborane phosphine, carbamate, urea, thiourea, N3′phosphoramidate and amide; and
(4) a region of complementary base pairing with the target nucleic acid that is at least consecutive nucleosides in length beginning at position 2 and extending at least through position 16; and
wherein the compound exhibits a maximal plateau level of activity in a dose response curve against the target that constitutes at least a 50% change in expression and/or function of the target and said level of change in activity is at least 20 percentage points greater than the maximal level of activity obtained using an ssRNAi of the same sequence, same 5′-end phosphate group or a 5′-end phosphate analog, if any, and lacking the AGSD modifications found in the ags-siRNA or ags-IMiR compound and administered using the same dosage regimen where the compounds to be compared are delivered without an enveloping protective carrier, aid ags-siRNA is directed to a target selected from the group consisting of pre-mRNA, mRNA, lncRNA, promoter associated RNA, enhancer RNA, snoRNA, piRNA, xiRNA, sdRNA, moRNA, MSY-RNA, tel-sRNA, crasiRNA, endogenous antisense RNA, a promoter, an enhancer and a suppressor.
18. The compound of claim 15, for modulating expression and/or function of a target nucleic acid wherein said ags-siRNA is directed to a target selected from the group consisting of pre-mRNA, mRNA, lncRNA, promoter associated RNA, enhancer RNA, snoRNA, piRNA, xiRNA, sdRNA, moRNA, MSY-RNA, tel-sRNA, crasiRNA, endogenous antisense RNA, a promoter, an enhancer and a suppressor.
19. The compound of claim 15, for modulating expression and/or function of an miRNA target wherein said compound is an ags-IMiR.
20. The compound of claim 15, wherein said cell is selected from the group consisting of a cell line; a tissue sample from an organ, gland or neoplastic growth; an enriched sample of parenchymal cells from an organ, gland or neoplastic growth; epithelial tissue including simple, stratified, pseudostratified and transitional epithelium; connective tissues, reticular, adipose, blood and lymphoid tissues; of nervous tissue isolated from brain, spinal cord, ganglion, and nerve and, neuronal cells, glial cells, skeletal muscle cells, cardiac muscle cells and smooth muscle cells.
21. The compound of claim 15, wherein said overhang precursor units are selected from the group consisting of ˜G L˜U L˜U X, ˜U L˜U L˜U X, ˜C L˜U L˜U X, G L˜C L˜U X, ˜A L˜G L˜U X, A L˜G L˜C X, ˜A L˜A L˜A X, ˜A L˜G L˜A X, ˜G L˜A L˜A X, ˜C L˜G L˜C X, ˜G L˜G L˜C X and ˜G L˜G L˜U X where the symbol ˜ represents phosphorothioate linkages wherein one or more of the phosphorothiate linkages is optionally replaced by a nucleoside linkage selected from the group consisting of phosphorothioate, phosphonoacetate, (PACE), thiophosphonoacetate (thio-PACE), methylborane phosphine, amide, carbamate, urea, thiourea, N3′phosphoramidate and amide, the subscript L after a nucleoside represents that the nucleoside has an LNA sugar wherein said LNA sugar is optionally replaced with one or two of the sugars independently selected from the group consisting of 2′fluoro (F), ANA (J), CRN R monomer (W), CRN Q monomer (V), FHNA (Y), F-CeNA (T), thio-LNA (T L) and amino-LNA (I); and
the subscript X represents a nucleoside with a ribose, 2′-fluoro, or 2′-O-methyl sugar.
22. The compound of claim 21, wherein said overhang precursors are linked to the nucleoside in position 19 and to each other by a linkage selected from the group consisting of phosphorothioate, N3′ phosphoramidate and amide.
23. The compound of claim 18, wherein said compound and comparator ssRNAi are to be administered to a subject cell line or to primary parenchymal cells grown in vitro and are to be delivered to cells via a method selected from the group transfection, electroporation and gymnosis.
24. The compound of claim 15, wherein said nucleoside in position 1 comprises a 5′VP.
25. The compound of claim 15, wherein the compound further provides one or more of the following advantages:
(a) an IC 50 or EC 50 against the target in said cell where the amount of the ags-siRNA or ags-IMiR compound is at least 2 fold lower than the amount of the ssRNAi of the same sequence, same 5′-end phosphate group or a 5′-end phosphate analog, if any, and lacking the AGSD modifications as well as administered using the same dosage regimen under the same conditions of administration;
(b) at least a 50% change in expression and/or function of the target in said cell at a time point after the last treatment where at that time point the level of change in expression and/or function of the target in said cell is equal to or greater than 40 percentage points higher than the level obtained at that time point using an ssRNAi of the same sequence, same 5′-phosphate or 5′-phosphate analog, if any, and lacking the AGSD modifications as well as administered using the same dosage regimen under the same conditions of administration; or
(c) at least a 50% change in expression and/or function of the target in said cell and said cell is not a liver organ sample or an enriched hepatocyte sample,
wherein the ags-siRNA or ags-IMiR compound is systemically administered to a subject without an enveloping protective carrier,
wherein the ratio of the weight in grams of the intact agsRNAi in said cell divided by a unit weight of said cell expressed in grams compared to the weight in grams of the intact agsRNAi in said sample of the liver organ or enriched hepatocyte population from the liver organ sample divided by a unit weight of sample of the liver organ or enriched hepatocyte population from the liver organ sample expressed in grams is less than 0.3, and wherein under the same conditions the level of change in expression and/or function of the target in said cells treated with said agsRNAi is at least 40 percentage points higher than the level obtained using an ssRNAi of the same sequence, same 5′-phosphate or 5′-phosphate analog, if any, and lacking AGSD modifications without an enveloping protective carrier.
26. The compound of claim 16, wherein off-target effects due to the seed region directing an ags-siRNA or ags-IMiR to have unintended miRNA mimicking activity are inhibited by using one or two of the following modifications:
(i) a 2′-O-methyl in position 2;
(ii) 1-3 CENA modified nucleosides inserted in the seed sequence with inclusion of position 3 from the 5′-end of the strand being preferred;
(iii) an UNA in position 7 from the 5′-end of the strand; or
(iv) the replacement of any adenine containing nucleoside with a modification selected from the group N2-propyl-2-aminopurine or N2-cyclopentyl-2-aminopurine and/or the replacement of a guanine containing nucleoside with a N2-cyclopentylguanine modification where one or more of these modifications can be used in position(s) 2 and/or 7 from the 5′-end of the strand.
27. A single strand modified oligoribonucleotide ags-siRNA or ags-IMiR compound for modulating the expression and/or function of at least one target nucleic acid sequence expressed in a cell, comprising:
(1) a nucleoside in position 1 and an associated linkage with the nucleoside in position 2 wherein
(i) the 5′ carbon of the 5′-end nucleoside sugar has a hydroxyl, a 5′-end phosphate group or a 5′-end phosphate analog and wherein the 5′-end phosphate analog is optionally 5′-(E)-vinylphosphonate (5′-VP), and
(ii) the nucleoside sugar is selected from the group consisting of ribose, 2′-fluoro, 2′-O-methyl, 2′-methoxyethyl, 2′-deoxyribose, LNA, FANA, 4'S-FANA, ALN, AENA, CENA, HM, HNA, EA, F-CeNA, CeNA, UNA, CRN R monomer and CRN Q monomer, and
(iii) the associated linkage is N3′ phosphoramidate or amide; and
(2) nucleosides in positions 2-19, said nucleosides comprising:
(i) modifications that increase basic nuclease resistance wherein said basic nuclease resistance is provided by
(a) 2′-fluoro alternating with 2′-O-methyl nucleosides with the 2′-fluoro being in the even numbered positions, where phosphorothioate linkages occur between nucleoside positions 2-3, 4-5, 6-7, 8-9, 10-11, 12-13, 14-15, 15-16, 16-17, 17-18 and 18-19 and with phosphodiester linkages between the remaining nucleoside positions, or
(b) the alternating pattern of (a) wherein a 2′-O-methyl modification is used in position 2 and where two contiguous nucleosides in the region defined by positions 3-13 have a 2′-fluoro modified sugar so that when the alternating pattern of single 2′-O-methyl with single 2′fluoro modified sugar is continued, the 2′-fluoro modification falls on nucleoside positions 14 and 16, or
(c) the alternating pattern of (b) wherein the nucleoside in position 14 is ribose or HM and optionally the nucleoside in position 16 is independently ribose or HM,
wherein N3′-phosphoramidate linkages are used in place of phosphorothioate and/or phosphodiester linkages in one or more positions; and
(ii) at least 4 accommodating guide strand designs (AGSD) modifications are optionally present independently selected from the group of AGSD base modifications consisting of replacement of an U with 2-thiouracil, 5-methyluracil or pseudouracil base and replacement of a C with a 4-thiouracil, and/or from the group of AGSD sugar modifications consisting of LNA, HNA, ANA, CRN R monomer, CRN Q monomer, HM, FHNA, CeNA, F-CeNA and cET; and
wherein AGSD modifications increase the probability that the modified nucleoside having its sugar in the C3′-endo conformation compared to the sugars (2′-fluoro, 2′-O-methyl and ribose) found in these positions in conventional ssRNAi compounds; and
(3) 1, 2, 3, or 4 optional overhang precursor units in positions 20-23 linked together and to the nucleoside in position 19 to form a 3′-end overhang using a nuclease resistant linkage selected from the group consisting of phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylborane phosphine, carbamate, urea, thiourea, N3′phosphoramidate and amide; and
(4) a region of complementary base pairing with the target nucleic acid that is at least consecutive nucleosides in length beginning at position 2 and extending at least through position 16; and
wherein the compound exhibits a maximal plateau level of activity in a dose response curve against the target that constitutes at least a 50% change in expression and/or function of the target and said level of change in activity is at least 20 percentage points greater than the maximal level of activity obtained using an ssRNAi of the same sequence and, if any, with the same 5′-end phosphate group or a 5′-end phosphate analog and lacking the AGSD modifications found in the ags-siRNA or ags-IMiR compound and administered using the same dosage regimen where the compounds to be compared are delivered without an enveloping protective carrier.
28. The compound of claim 27 wherein the rules for applying AGSD sugar modifications to positions 2-19 are as follows:
(a) the sugar in position 2 is a 2′-O-methyl,
(b) in purine rich regions at least one nucleoside with an AGSD sugar modification per 3 nucleosides with a purine base is present,
(c) no more than 5 contiguous nucleosides with a pyrimidine base without at least one nucleoside with an AGSD sugar and/or AGSD base are present,
(d) at least one nucleoside with a non-AGSD sugar is present between nucleosides with AGSD sugars, and/or
(e) at least 4 nucleosides with an AGSD sugar are present among nucleosides 2-19 and at least 2 of these are in the 5′-end half of said composition and 2 in the 3′end half.
29. A method for modulating expression and or function of a target nucleic acid via contacting a cell with the compound of claim 27, wherein said ags-siRNA is directed to a target selected from the group consisting of pre-mRNA, mRNA, lncRNA, promoter associated RNA, enhancer RNA, snoRNA, piRNA, xiRNA, sdRNA, moRNA, MSY-RNA, tel-sRNA, crasiRNA, endogenous antisense RNA as well gene promoters, enhancers and suppressors.
30. The method of claim 27, for modulating expression and or function of an miRNA target wherein said compound is an ags-IMiR.
31. The method of claim 27, wherein said subject cells are of the same type and are selected from the group consisting of a cell line; a tissue sample from an organ, gland or neoplastic growth; an enriched sample of parenchymal cells from an organ, gland or neoplastic growth; epithelial tissue including simple, stratified, pseudostratified and transitional epithelium; connective tissues, reticular, adipose, blood and lymphoid tissues; of nervous tissue isolated from brain, spinal cord, ganglion, and nerve and, neuronal cells, glial cells, skeletal muscle cells, cardiac muscle cells and smooth muscle cells.
32. The compound of claim 27, wherein said the nucleoside in position 1 and the associated linkage with the nucleoside in position 2 promote binding of the compound to an argonaute protein and comprise one or more sugar modifications consisting of 2′-fluoro, 2′-O-methyl, 2′-methoxyethyl, 2′-deoxyribose, LNA, FANA, 4'S-FANA, ALN, AENA, CENA, HM, ETNA, EA, F-CeNA, CeNA, UNA, CRN R monomer and CRN Q monomer and one or more linkage modifications selected from phosphodiester, phosphorothioate, N3′ phosphoramidate and amide.