ANTISENSE COMPOUNDS TARGETING GENES ASSOCIATED WITH FIBRONECTIN
The present invention provides compounds comprising oligonucleotides complementary to a fibronectin transcript. Certain such compounds are useful for hybridizing to a fibronectin transcript, including but not limited to a fibronectin transcript in a cell. In certain embodiments, such hybridization results in modulation of splicing of the fibronectin transcript. In certain embodiments, such compounds are used to treat one or more symptoms associated with fibrosis. In certain embodiments, such compounds are used to treat one or more symptoms associated with renal fibrosis.
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The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0197USC1SEQ.txt, created Oct. 18, 2017, which is 264 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTIONFibronectin is a high-molecular weight glycoprotein of the extracellular matrix that binds to membrane-spanning receptor integrin proteins. Fibronectin has been implicated in a number of fibrotic disorders, including renal fibrosis. Alternative splicing of fibronectin pre-mRNA leads to the creation of fibronectin mRNA having a different combination of exons, which in turn leads to the creation of several isoforms of fibronectin protein. In certain instances, alternative splicing of the fibronectin gene results in a fibronectin protein isoform containing the extra type III domain A (EDA). Fibronectin containing extra type III domain A (EDA) is implicated in the formation of fibrosis. See, e.g., Muro et al., An Essential Role for Fibronectin Extra Type III Domain A in Pulmonary Fibrosis, American Journal of Respitory and Critical Care Medicine, Vol. 177, 638 (2008).
Antisense compounds have been used to modulate target nucleic acids. Antisense compounds comprising a variety of chemical modifications and motifs have been reported. In certain instances, such compounds are useful as research tools, diagnostic reagents, and as therapeutic agents. In certain instances antisense compounds have been shown to modulate protein expression by binding to a target messenger RNA (mRNA) encoding the protein. In certain instances, such binding of an antisense compound to its target mRNA results in cleavage of the mRNA. Antisense compounds that modulate processing of a pre-mRNA have also been reported. Such antisense compounds alter splicing, interfere with polyadenylation or prevent formation of the 5′-cap of a pre-mRNA.
Certain antisense compounds have been described previously. See for example U.S. Pat. No. 7,399,845 and published International Patent Application No. WO 2008/049085, which are hereby incorporated by reference herein in their entirety.
SUMMARY OF THE INVENTIONIn certain embodiments, the present invention provides compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides are complementary to a fibronectin transcript. In certain such embodiments, oligonucleotides are complementary to a target region of the fibronectin transcript comprising the EDA exon. In certain such embodiments, oligonucleotides are complementary to a target region of the fibronectin transcript comprising an intron adjacent to the EDA exon. In certain such embodiments, oligonucleotides are complementary to a target region of the fibronectin transcript comprising an intron adjacent to the EDA exon and downstream of the EDA exon. In certain such embodiments, oligonucleotides are complementary to a target region of the fibronectin transcript comprising an intron adjacent to the EDA exon and upstream of the EDA exon. In certain embodiments, the fibronectin transcript comprises an exonic splice enhancer for the EDA exon. In certain embodiments, the fibronectin transcript comprises an exonic splice silencer for the EDA exon. In certain embodiments, oligonucleotides inhibit inclusion of the EDA exon. In certain embodiments, oligonucleotides promote skipping of the of the EDA exon. In certain such embodiments, fibronectin mRNA without EDA mRNA is increased. In certain such embodiments, fibronectin protein without EDA is increased.
The present disclosure provides the following non-limiting numbered embodiments:
Embodiment 1A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a fibronectin transcript.
Embodiment 2The compound of embodiment 1, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
Embodiment 3The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide comprises at least 10 contiguous nucleobases.
Embodiment 4The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide comprises at least 12 contiguous nucleobases.
Embodiment 5The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide comprises at least 15 contiguous nucleobases.
Embodiment 6The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide comprises at least 18 contiguous nucleobases.
Embodiment 7The compound of embodiment 1 or 2, wherein the complementary region of the modified oligonucleotide comprises at least 20 contiguous nucleobases.
Embodiment 8The compound of any of embodiments 1-5, wherein the nucleobase sequence of the oligonucleotide is at least 80% complementary to an equal-length region of the fibronectin transcript, as measured over the entire length of the oligonucleotide.
Embodiment 9The compound of any of embodiments 1-5, wherein the nucleobase sequence of the oligonucleotide is at least 90% complementary to an equal-length region of the fibronectin transcript, as measured over the entire length of the oligonucleotide.
Embodiment 10The compound of any of embodiments 1-5, wherein the nucleobase sequence of the oligonucleotide is 100% complementary to an equal-length region of the fibronectin transcript, as measured over the entire length of the oligonucleotide.
Embodiment 11The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55469 and nucleobase 55790 of SEQ ID NO.: 1.
Embodiment 12The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55469 and nucleobase 55511 of SEQ ID NO.: 1.
Embodiment 13The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55511 and nucleobase 55732 of SEQ ID NO.: 1.
Embodiment 14The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55732 and nucleobase 55790 of SEQ ID NO.: 1.
Embodiment 15The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55491 and nucleobase 55511 of SEQ ID NO.: 1.
Embodiment 16The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55490 and nucleobase 55510 of SEQ ID NO.: 1.
Embodiment 17The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55491 and nucleobase 55513 of SEQ ID NO.: 1.
Embodiment 18The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55536 and nucleobase 55555 of SEQ ID NO.: 1.
Embodiment 19The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55576 and nucleobase 55600 of SEQ ID NO.: 1.
Embodiment 20The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55604 and nucleobase 55623 of SEQ ID NO.: 1.
Embodiment 21The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55610 and nucleobase 55697 of SEQ ID NO.: 1.
Embodiment 22The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55701 and nucleobase 55737 of SEQ ID NO.: 1.
Embodiment 23The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55738 and nucleobase 55757 of SEQ ID NO.: 1.
Embodiment 24The compound of any of embodiments 1-10, wherein the target region is within nucleobase 55753 and nucleobase 55781 of SEQ ID NO.: 1.
Embodiment 25The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 5.
Embodiment 26The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 9.
Embodiment 27The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 13.
Embodiment 28The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 14.
Embodiment 29The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 15.
Embodiment 30The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 18.
Embodiment 31The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 22.
Embodiment 32The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 66.
Embodiment 33The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 67.
Embodiment 34The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 5 to 24.
Embodiment 35The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 30 to 90.
Embodiment 36The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 413.
Embodiment 37The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises SEQ ID NO: 346.
Embodiment 38The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 104 to 176.
Embodiment 39The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 177 to 329.
Embodiment 40The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 403 to 435.
Embodiment 41The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises any of SEQ ID NOs: 105, 87, 126, 133, 134, 140, 141, 147, 149, 157, 159, 190, 223, 238, 244, 268, 285, 300, 302, 303, 308, 319, 327, 381, 382, 339, 346, 348, 364, 365, 367, 368, 369, 370, 268, 276, 280, 406, 407, 412, 413, and 324.
Embodiment 42The compound of any of embodiments 1-24, wherein the antisense oligonucleotide has a nucleobase sequence comprising CTTCTTCT.
Embodiment 43The compound of any of embodiments 1-24, wherein the antisense oligonucleotide has a nucleobase sequence comprising GTTCC.
Embodiment 44The compound of any of embodiments 1-24, wherein the antisense oligonucleotide has a nucleobase sequence comprising GTCCC.
Embodiment 45The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises a sugar motif described by Formula I as follows:
[(A)-(B)2-(A)]n
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 46The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises a sugar motif described by Formula II as follows:
(A)2-[(B)2-(A)]n-(A)
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 47The compound of any of embodiments 1-24, wherein the antisense oligonucleotide comprises a sugar motif described by Formula III as follows:
(A)2-[(B)2-(A)]n
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 48The compound of any of embodiments 45 to 47, wherein each A comprises a bicyclic nucleoside selected from among LNA and cEt.
Embodiment 49The compound of any of embodiments 45 to 47, wherein each A comprises a cEt modification.
Embodiment 50The compound of any of embodiments 45 to 47, wherein each A comprises an LNA modification.
Embodiment 51The compound of any of embodiments 45 to 50, wherein each B comprises a 2′-substituted nucleoside having a 2′-modification selected from among 2′-OMe, 2′-F, and 2′-MOE.
Embodiment 52The compound embodiment 51, wherein the 2′-modification is a 2′-MOE modification.
Embodiment 53The compound of any of embodiments 45 to 50, wherein each B comprises a 2′-deoxynucleoside.
Embodiment 54The compound of any of embodiments 1-44, wherein the modified oligonucleotide comprises at least one modified nucleoside.
Embodiment 55The compound of embodiment 54, wherein at least one modified nucleoside comprises a modified sugar moiety.
Embodiment 56The compound of embodiment 55, wherein at least one modified sugar moiety is a 2′-substituted sugar moiety.
Embodiment 57The compound of embodiment 56, wherein the 2′-substituent of at least one 2′-substituted sugar moiety is selected from among: 2′-OMe, 2′-F, and 2′-MOE.
Embodiment 58The compound of embodiment 57, wherein the 2′-substituent of at least one 2′-substituted sugar moiety is a 2′-MOE.
Embodiment 59The compound of any of embodiments 54-56, wherein at least one modified sugar moiety is a bicyclic sugar moiety.
Embodiment 60The compound of embodiment 59, wherein at least one bicyclic sugar moiety is LNA or cEt.
Embodiment 61The compound of any of embodiments 54-60, wherein at least one sugar moiety is a sugar surrogate.
Embodiment 62The compound of embodiment 61, wherein at least one sugar surrogate is a morpholino.
Embodiment 63The compound of embodiment 61, wherein at least one sugar surrogate is a modified morpholino.
Embodiment 64The compound of any of embodiment 1-63, wherein the modified oligonucleotide comprises at least 5 modified nucleosides, each independently comprising a modified sugar moiety.
Embodiment 65The compound of embodiment 64, wherein the modified oligonucleotide comprises at least 10 modified nucleosides, each independently comprising a modified sugar moiety.
Embodiment 66The compound of embodiment 64, wherein the modified oligonucleotide comprises at least 15 modified nucleosides, each independently comprising a modified sugar moiety.
Embodiment 67The compound of any of embodiments 1 to 44 or 54 to 66, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside, each independently comprising a modified sugar moiety Embodiment 68. The compound of any of embodiments 1-67, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are the same as one another.
Embodiment 69The compound of any of embodiments 1-68, wherein the modified oligonucleotide comprises at least two modified nucleosides comprising modified sugar moieties that are different from one another.
Embodiment 70The compound of any of embodiments 1-69, wherein the modified oligonucleotide comprises a modified region of at least 5 contiguous modified nucleosides.
Embodiment 71The compound of any of embodiments 1 to 44 or 54 to 70, wherein the modified oligonucleotide comprises a modified region of at least 10 contiguous modified nucleosides.
Embodiment 72The compound of any of embodiments 1 to 44 or 54 to 70, wherein the modified oligonucleotide comprises a modified region of at least 15 contiguous modified nucleosides.
Embodiment 73The compound of any of embodiments 1 to 44 or 54 to 70, wherein the modified oligonucleotide comprises a modified region of at least 20 contiguous modified nucleosides.
Embodiment 74The compound of any of embodiments 70-73, wherein each modified nucleoside of the modified region has a modified sugar moiety independently selected from among: 2′-F, 2′-OMe, 2′-MOE, cEt, LNA, morpholino, and modified morpholino.
Embodiment 75The compound of any of embodiments 70-73, wherein the modified nucleosides of the modified region each comprise the same modification as one another.
Embodiment 76The compound of embodiment 75, wherein the modified nucleosides of the modified region each comprise the same 2′-substituted sugar moiety.
Embodiment 77The compound of embodiment 75, wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from 2′-F, 2′-OMe, and 2′-MOE.
Embodiment 78The compound of embodiment 77, wherein the 2′-substituted sugar moiety of the modified nucleosides of the region of modified nucleosides is 2′-MOE.
Embodiment 79The compound of embodiment 75, wherein the modified nucleosides of the region of modified nucleosides each comprise the same bicyclic sugar moiety.
Embodiment 80The compound of embodiment 79, wherein the bicyclic sugar moiety of the modified nucleosides of the region of modified nucleosides is selected from LNA and cEt.
Embodiment 81The compound of embodiment 75, wherein the modified nucleosides of the region of modified nucleosides each comprises a sugar surrogate.
Embodiment 82The compound of embodiment 81, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a morpholino.
Embodiment 83The compound of embodiment 81, wherein the sugar surrogate of the modified nucleosides of the region of modified nucleosides is a modified morpholino.
Embodiment 84The compound of any of embodiments 1-83, wherein the modified nucleotide comprises no more than 4 contiguous naturally occurring nucleosides.
Embodiment 85The compound of any of embodiments 1 to 44 or 54 to 85, wherein each nucleoside of the modified oligonucleotide is a modified nucleoside.
Embodiment 86The compound of embodiment 85 wherein each modified nucleoside comprises a modified sugar moiety.
Embodiment 87The compound of embodiment 86, wherein the modified nucleosides of the modified oligonucleotide comprise the same modification as one another.
Embodiment 88The compound of embodiment 87, wherein the modified nucleosides of the modified oligonucleotide each comprise the same 2′-substituted sugar moiety.
Embodiment 89The compound of embodiment 88, wherein the 2′-substituted sugar moiety of the modified oligonucleotide is selected from 2′-F, 2′-OMe, and 2′-MOE.
Embodiment 90The compound of embodiment 89, wherein the 2′-substituted sugar moiety of the modified oligonucleotide is 2′-MOE.
Embodiment 91The compound of embodiment 87, wherein the modified nucleosides of the modified oligonucleotide each comprise the same bicyclic sugar moiety.
Embodiment 92The compound of embodiment 91, wherein the bicyclic sugar moiety of the modified oligonucleotide is selected from LNA and cEt.
Embodiment 93The compound of embodiment 87, wherein the modified nucleosides of the modified oligonucleotide each comprises a sugar surrogate.
Embodiment 94The compound of embodiment 93, wherein the sugar surrogate of the modified oligonucleotide is a morpholino.
Embodiment 95The compound of embodiment 93, wherein the sugar surrogate of the modified oligonucleotide is a modified morpholino.
Embodiment 96The compound of any of embodiments 1-95, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
Embodiment 97The compound of embodiment 96, wherein each internucleoside linkage is a modified internucleoside linkage.
Embodiment 98The compound of embodiment 96 or 97, comprising at least one phosphorothioate internucleoside linkage.
Embodiment 99The compound of embodiment 77, wherein each internucleoside linkage is a modified internucleoside linkage and wherein each internucleoside linkage comprises the same modification.
Embodiment 100The compound of embodiment 99, wherein each internucleoside linkage is a phosphorothioate internucleoside linkage.
Embodiment 101The compound of any of embodiments 1-100 comprising at least one conjugate.
Embodiment 102The compound of any of embodiments 1-101 consisting of the modified oligonucleotide.
Embodiment 103The compound of any of embodiments 1-102, wherein the compound modulates splicing of the fibronectin transcript.
Embodiment 104The compound of any of embodiments 1-103, having a nucleobase sequence comprising any of the sequences as set forth in SEQ ID NOs. 5 to 25 or 30 to 90.
Embodiment 105The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 5.
Embodiment 106The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 9.
Embodiment 107The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 13.
Embodiment 108The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 14.
Embodiment 109The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 15.
Embodiment 110The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 18.
Embodiment 111The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 22.
Embodiment 112The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 66.
Embodiment 113The compound of any of embodiments 1-103, having a nucleobase sequence comprising the sequence as set forth in SEQ ID NO: 67.
Embodiment 114The compound of any of embodiments 1-103, wherein the antisense oligonucleotide has a nucleobase sequence comprising CTTCTTCT.
Embodiment 115The compound of any of embodiments 1-103, wherein the antisense oligonucleotide has a nucleobase sequence comprising GTTCC.
Embodiment 116The compound of any of embodiments 1-103, wherein the antisense oligonucleotide has a nucleobase sequence comprising GTCCC.
Embodiment 117A pharmaceutical composition comprising a compound according to any of embodiments 1-116 and a pharmaceutically acceptable carrier or diluent.
Embodiment 118The pharmaceutical composition of embodiment 117, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
Embodiment 119A method of decreasing the amount of EDA+ fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 120A method of increasing the amount of EDA− fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 121A method of reducing fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 122A method of reversing fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 123A method of reducing changes in cell phenotype due to fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 124A method of reversing changes in cell phenotype due to fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 125The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the modulation of cadherin expression.
Embodiment 126The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the induction of α Smooth Muscle Actin (αSMA).
Embodiment 127The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the alteration of cortical f-actin localization.
Embodiment 128The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the induction of connexin 43 (Cx 43) expression.
Embodiment 129The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the increased secretion of MMP2 & MMP9.
Embodiment 130The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the alteration of the amount vimentin or the arrangement of vimentin within a cell.
Embodiment 131The method of embodiments 123-124, wherein the change in cell phenotype due to fibrosis is the alteration of the amount tight junction protein ZO-1 or the arrangement of tight junction protein ZO-1 within a cell.
Embodiment 132A method of reducing loss of cell phenotype due to fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 133A method of reversing the loss of cell phenotype due to fibrosis, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 134A method of increasing the ratio of EDA+/EDA− fibronectin in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 135A method of decreasing the ratio of EDA+/EDA− fibronectin in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 136A method of increasing the ratio of EDA−/EDA+ fibronectin in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 137A method of decreasing the ratio of EDA−/EDA+ fibronectin in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 138A method of increasing the ratio of EDA+/EDA− fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 139A method of decreasing the ratio of EDA+/EDA− fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 140A method of increasing the ratio of EDA−/EDA+ fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 141A method of decreasing the ratio of EDA−/EDA+ fibronectin protein in a cell, comprising contacting the cell with a compound according to any of embodiments 1-117.
Embodiment 142The method of any of embodiments 119-142, wherein the cell is in vitro.
Embodiment 143The method of embodiments 119-142, wherein the cell is in an animal.
Embodiment 144The method of embodiments 119-142, wherein the animal is a mouse.
Embodiment 145The method of embodiments 119-142, wherein the animal is a human.
Embodiment 146The method of any of embodiments 119-145, wherein TGFβ1 is present in the cell.
Embodiment 147The method of any of embodiments 119-146, wherein the healing and/or restoration functions of the cell are not substantially affected.
Embodiment 148A pharmaceutical composition comprising a compound according to any of embodiments 1-117 and a pharmaceutically acceptable carrier or diluent.
Embodiment 149The pharmaceutical composition of embodiment 148, wherein the pharmaceutically acceptable carrier or diluent is sterile saline.
Embodiment 150A method comprising administering the pharmaceutical composition of embodiments 148 or 149 to an animal.
Embodiment 151The method of embodiment 150, wherein the animal is a mouse.
Embodiment 152The method of embodiment 150, wherein the animal is a human.
Embodiment 153The method of embodiment 150, wherein the administration is by injection.
Embodiment 154The method of embodiment 150, wherein the administration is systemic.
Embodiment 155The method of embodiment 150 wherein the administration is local.
Embodiment 156The method of any of embodiments 150-155, wherein the animal has one or more symptom associated with fibrosis.
Embodiment 157The method of embodiment 156, wherein the administration results in amelioration of at least one symptom associated with fibrosis.
Embodiment 158The method of embodiment 156-157, wherein the fibrosis is renal fibrosis.
Embodiment 159The method of embodiment 156-157, wherein the fibrosis is lung fibrosis.
Embodiment 160The method of embodiment 156-157, wherein the fibrosis is liver fibrosis.
Embodiment 161The method of embodiment 156-157, wherein the fibrosis is brain fibrosis.
Embodiment 162The method of embodiment 156-157, wherein the fibrosis is muscular fibrosis.
Embodiment 163The method of embodiment 156-157, wherein the fibrosis is cardiovascular fibrosis.
Embodiment 164The method of embodiment 156-157, wherein the fibrosis is in the bone or the bone marrow.
Embodiment 165The method of embodiment 156-157, wherein the fibrosis is intestinal fibrosis.
Embodiment 166The method of embodiment 156-157, wherein the fibrosis is epidural fibrosis.
Embodiment 167The method of any of embodiments 156-167, wherein the animal is a mouse.
Embodiment 168The method of any of embodiments 156-167, wherein the animal is a human.
Embodiment 169Use of the compound of any of embodiments 1 to 117 or the composition of embodiments 148-149 for the preparation of a medicament for use in the treatment of at least one symptom associated with fibrosis.
Embodiment 170Use of the compound of any of embodiments 1 to 117 or the composition of embodiments 148-149 for the preparation of a medicament for use in the amelioration of one or more symptoms associated with fibrosis.
Embodiment 171The use of any of embodiment 169-170, wherein the fibrosis is selected from among renal, lung, liver, brain, muscular, cardiovascular, bone or bone marrow, intestinal, and/or epidural fibrosis.
Embodiment 172A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a sugar motif described by Formula I as follows:
[(A)-(B)2-(A)]n
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 173A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a sugar motif described by Formula II as follows:
(A)2-[(B)2-(A)]n-(A)
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 174A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a sugar motif described by Formula III as follows:
(A)2-[(B)2-(A)]n
wherein:
each A is independently a bicyclic nucleoside;
each B is independently a 2′-substituted nucleoside or a 2′-deoxynucleoside; and
n is an integer from 3-6.
Embodiment 175The compound of any of embodiments 172 to 174, wherein each A comprises a bicyclic nucleoside selected from among LNA and cEt.
Embodiment 176The compound of any of embodiments 172 to 174, wherein each A comprises a cEt modification.
Embodiment 177The compound of any of embodiments 172 to 174, wherein each A comprises an LNA modification.
Embodiment 178The compound of any of embodiments 172 to 177, wherein each B comprises a 2′-substituted nucleoside having a 2′-modification selected from among 2′-OMe, 2′-F, and 2′-MOE.
Embodiment 179The compound embodiments 178, wherein the 2′-modification is a 2′-MOE modification.
Embodiment 180The compound of any of embodiments 172 to 179, wherein each B comprises a 2′-deoxynucleoside.
Embodiment 181A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kd2kd2kd2kd2kd2k motif, wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
Embodiment 182A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kkddkddkddkddkddkk motif, wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
Embodiment 183A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kkeekeekeekeekeeke motif, wherein each k comprises a cEt modification and each e comprises a 2′-MOE modification.
Embodiment 184A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kddkddkddkddkddk motif wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
Embodiment 185A compound comprising a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a keekeekeekeekeek motif, wherein each k comprises a cEt modification and each e comprises a 2′-MOE modification.
In certain embodiments, including, but not limited to any of the above numbered embodiments, the fibronectin transcript is in a human. In certain embodiments, including, but not limited to any of the above numbered embodiments, the fibronectin transcript is in a mouse.
Unless specific definitions are provided, the nomenclature used in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Certain such techniques and procedures may be found for example in “Carbohydrate Modifications in Antisense Research” Edited by Sangvi and Cook, American Chemical Society, Washington D.C., 1994; “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 21st edition, 2005; and “Antisense Drug Technology, Principles, Strategies, and Applications” Edited by Stanley T. Crooke, CRC Press, Boca Raton, Fla.; and Sambrook et al., “Molecular Cloning, A laboratory Manual,” 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, which are hereby incorporated by reference for any purpose. Where permitted, all patents, applications, published applications and other publications and other data referred to throughout in the disclosure are incorporated by reference herein in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
As used herein, “nucleoside” means a compound comprising a nucleobase moiety and a sugar moiety. Nucleosides include, but are not limited to, naturally occurring nucleosides (as found in DNA and RNA) and modified nucleosides. Nucleosides may be linked to a phosphate moiety.
As used herein, “chemical modification” means a chemical difference in a compound when compared to a naturally occurring counterpart. In reference to an oligonucleotide, chemical modification does not include differences only in nucleobase sequence. Chemical modifications of oligonucleotides include nucleoside modifications (including sugar moiety modifications and nucleobase modifications) and internucleoside linkage modifications.
As used herein, “furanosyl” means a structure comprising a 5-membered ring comprising four carbon atoms and one oxygen atom.
As used herein, “naturally occurring sugar moiety” means a ribofuranosyl as found in naturally occurring RNA or a deoxyribofuranosyl as found in naturally occurring DNA.
As used herein, “sugar moiety” means a naturally occurring sugar moiety or a modified sugar moiety of a nucleoside.
As used herein, “modified sugar moiety” means a substituted sugar moiety, a bicyclic or tricyclic sugar moiety, or a sugar surrogate.
As used herein, “substituted sugar moiety” means a furanosyl comprising at least one substituent group that differs from that of a naturally occurring sugar moiety. Substituted sugar moieties include, but are not limited to furanosyls comprising substituents at the 2′-position, the 3′-position, the 5′-position and/or the 4′-position.
As used herein, “2′-substituted sugar moiety” means a furanosyl comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted sugar moiety is not a bicyclic sugar moiety (i.e., the 2′-substituent of a 2′-substituted sugar moiety does not form a bridge to another atom of the furanosyl ring.
As used herein, “MOE” means —OCH2CH2OCH3.
As used herein, “bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
As used herein the term “sugar surrogate” means a structure that does not comprise a furanosyl and that is capable of replacing the naturally occurring sugar moiety of a nucleoside, such that the resulting nucleoside is capable of (1) incorporation into an oligonucleotide and (2) hybridization to a complementary nucleoside. Such structures include rings comprising a different number of atoms than furanosyl (e.g., 4, 6, or 7-membered rings); replacement of the oxygen of a furanosyl with a non-oxygen atom (e.g., carbon, sulfur, or nitrogen); or both a change in the number of atoms and a replacement of the oxygen. Such structures may also comprise substitutions corresponding to those described for substituted sugar moieties (e.g., 6-membered carbocyclic bicyclic sugar surrogates optionally comprising additional substituents). Sugar surrogates also include more complex sugar replacements (e.g., the non-ring systems of peptide nucleic acid). Sugar surrogates include without limitation morpholino, modified morpholinos, cyclohexenyls and cyclohexitols.
As used herein, “nucleotide” means a nucleoside further comprising a phosphate linking group. As used herein, “linked nucleosides” may or may not be linked by phosphate linkages and thus includes, but is not limited to “linked nucleotides.” As used herein, “linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e. no additional nucleosides are present between those that are linked).
As used herein, “nucleobase” means a group of atoms that can be linked to a sugar moiety to create a nucleoside that is capable of incorporation into an oligonucleotide, and wherein the group of atoms is capable of bonding with a complementary naturally occurring nucleobase of another oligonucleotide or nucleic acid. Nucleobases may be naturally occurring or may be modified.
As used herein, “heterocyclic base” or “heterocyclic nucleobase” means a nucleobase comprising a heterocyclic structure.
As used herein the terms, “unmodified nucleobase” or “naturally occurring nucleobase” means the naturally occurring heterocyclic nucleobases of RNA or DNA: the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) (including 5-methyl C), and uracil (U).
As used herein, “modified nucleobase” means any nucleobase that is not a naturally occurring nucleobase.
As used herein, “modified nucleoside” means a nucleoside comprising at least one chemical modification compared to naturally occurring RNA or DNA nucleosides. Modified nucleosides comprise a modified sugar moiety and/or a modified nucleobase.
As used herein, “bicyclic nucleoside” or “BNA” means a nucleoside comprising a bicyclic sugar moiety.
As used herein, “constrained ethyl nucleoside” or “cEt” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′ bridge.
As used herein, “locked nucleic acid nucleoside” or “LNA” means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH2—O-2′ bridge.
As used herein, “2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position other than H or OH. Unless otherwise indicated, a 2′-substituted nucleoside is not a bicyclic nucleoside.
As used herein, “2′-deoxynucleoside” means a nucleoside comprising 2′-H furanosyl sugar moiety, as found in naturally occurring deoxyribonucleosides (DNA). In certain embodiments, a 2′-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (e.g., uracil).
As used herein, “oligonucleotide” means a compound comprising a plurality of linked nucleosides. In certain embodiments, an oligonucleotide comprises one or more unmodified ribonucleosides (RNA) and/or unmodified deoxyribonucleosides (DNA) and/or one or more modified nucleosides.
As used herein “oligonucleoside” means an oligonucleotide in which none of the internucleoside linkages contains a phosphorus atom. As used herein, oligonucleotides include oligonucleosides.
As used herein, “modified oligonucleotide” means an oligonucleotide comprising at least one modified nucleoside and/or at least one modified internucleoside linkage.
As used herein “internucleoside linkage” means a covalent linkage between adjacent nucleosides in an oligonucleotide.
As used herein “naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a naturally occurring internucleoside linkage.
As used herein, “oligomeric compound” means a polymeric structure comprising two or more sub-structures. In certain embodiments, an oligomeric compound comprises an oligonucleotide. In certain embodiments, an oligomeric compound comprises one or more conjugate groups and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide.
As used herein, “terminal group” means one or more atom attached to either, or both, the 3′ end or the 5′ end of an oligonucleotide. In certain embodiments a terminal group is a conjugate group. In certain embodiments, a terminal group comprises one or more terminal group nucleosides.
As used herein, “conjugate” means an atom or group of atoms bound to an oligonucleotide or oligomeric compound. In general, conjugate groups modify one or more properties of the compound to which they are attached, including, but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and/or clearance properties.
As used herein, “conjugate linking group” means any atom or group of atoms used to attach a conjugate to an oligonucleotide or oligomeric compound.
As used herein, “antisense compound” means a compound comprising or consisting of an oligonucleotide at least a portion of which is complementary to a target nucleic acid to which it is capable of hybridizing, resulting in at least one antisense activity.
As used herein, “antisense activity” means any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid.
As used herein, “detecting” or “measuring” means that a test or assay for detecting or measuring is performed. Such detection and/or measuring may result in a value of zero. Thus, if a test for detection or measuring results in a finding of no activity (activity of zero), the step of detecting or measuring the activity has nevertheless been performed.
As used herein, “detectable and/or measurable activity” means a statistically significant activity that is not zero.
As used herein, “essentially unchanged” means little or no change in a particular parameter, particularly relative to another parameter which changes much more. In certain embodiments, a parameter is essentially unchanged when it changes less than 5%. In certain embodiments, a parameter is essentially unchanged if it changes less than two-fold while another parameter changes at least ten-fold. For example, in certain embodiments, an antisense activity is a change in the amount of a target nucleic acid. In certain such embodiments, the amount of a non-target nucleic acid is essentially unchanged if it changes much less than the target nucleic acid does, but the change need not be zero.
As used herein, “expression” means the process by which a gene ultimately results in a protein. Expression includes, but is not limited to, transcription, post-transcriptional modification (e.g., splicing, polyadenlyation, addition of 5′-cap), and translation.
As used herein, “target nucleic acid” means a nucleic acid molecule to which an antisense compound hybridizes.
As used herein, “mRNA” means an RNA molecule that encodes a protein.
As used herein, “pre-mRNA” means an RNA transcript that has not been fully processed into mRNA. Pre-RNA includes one or more intron.
As used herein, “transcript” means an RNA molecule transcribed from DNA. Transcripts include, but are not limited to mRNA, pre-mRNA, and partially processed RNA.
As used herein, “fibronectin transcript” means a transcript transcribed from a fibronectin gene. In certain embodiments, a fibronectin transcript comprises SEQ ID NO: 1: the complement of GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708.
As used herein, “fibronectin gene” means a gene that encodes a fibronectin protein and any fibronectin protein isoforms. In certain embodiments, a fibronectin gene is represented by GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708, or a variant thereof. In certain embodiments, a fibronectin gene is at least 95% identical to GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708. In certain embodiments, a fibronectin gene is at least 90% identical to GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708.
As used herein, “EDA− fibronectin protein” means a fibronectin protein isoform that does not contain extra type III domain A.
As used herein, “EDA+ fibronectin protein” means a fibronectin protein isoform that contains extra type III domain A.
As used herein, “EDA− fibronectin mRNA” means a fibronectin transcript that does not contain the extra type III domain A exon.
As used herein, “EDA+ fibronectin mRNA” means a fibronectin transcript that contains the extra type III domain A exon.
As used herein, “targeting” or “targeted to” means the association of an antisense compound to a particular target nucleic acid molecule or a particular region of a target nucleic acid molecule. An antisense compound targets a target nucleic acid if it is sufficiently complementary to the target nucleic acid to allow hybridization under physiological conditions.
As used herein, “nucleobase complementarity” or “complementarity” when in reference to nucleobases means a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase means a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair. Nucleobases comprising certain modifications may maintain the ability to pair with a counterpart nucleobase and thus, are still capable of nucleobase complementarity.
As used herein, “non-complementary” in reference to nucleobases means a pair of nucleobases that do not form hydrogen bonds with one another.
As used herein, “complementary” in reference to oligomeric compounds (e.g., linked nucleosides, oligonucleotides, or nucleic acids) means the capacity of such oligomeric compounds or regions thereof to hybridize to another oligomeric compound or region thereof through nucleobase complementarity under stringent conditions. Complementary oligomeric compounds need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated. In certain embodiments, complementary oligomeric compounds or regions are complementary at 70% of the nucleobases (70% complementary). In certain embodiments, complementary oligomeric compounds or regions are 80% complementary. In certain embodiments, complementary oligomeric compounds or regions are 90% complementary. In certain embodiments, complementary oligomeric compounds or regions are 95% complementary. In certain embodiments, complementary oligomeric compounds or regions are 100% complementary.
As used herein, “hybridization” means the pairing of complementary oligomeric compounds (e.g., an antisense compound and its target nucleic acid). While not limited to a particular mechanism, the most common mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
As used herein, “specifically hybridizes” means the ability of an oligomeric compound to hybridize to one nucleic acid site with greater affinity than it hybridizes to another nucleic acid site. In certain embodiments, an antisense oligonucleotide specifically hybridizes to more than one target site.
As used herein, “percent complementarity” means the percentage of nucleobases of an oligomeric compound that are complementary to an equal-length portion of a target nucleic acid. Percent complementarity is calculated by dividing the number of nucleobases of the oligomeric compound that are complementary to nucleobases at corresponding positions in the target nucleic acid by the total length of the oligomeric compound.
As used herein, “percent identity” means the number of nucleobases in a first nucleic acid that are the same type (independent of chemical modification) as nucleobases at corresponding positions in a second nucleic acid, divided by the total number of nucleobases in the first nucleic acid.
As used herein, “modulation” means a change of amount or quality of a molecule, function, or activity when compared to the amount or quality of a molecule, function, or activity prior to modulation. For example, modulation includes the change, either an increase (stimulation or induction) or a decrease (inhibition or reduction) in gene expression. As a further example, modulation of expression can include a change in splice site selection of pre-mRNA processing, resulting in a change in the absolute or relative amount of a particular splice-variant compared to the amount in the absence of modulation.
As used herein, “motif” means a pattern of chemical modifications in an oligomeric compound or a region thereof. Motifs may be defined by modifications at certain nucleosides and/or at certain linking groups of an oligomeric compound.
As used herein, “nucleoside motif” means a pattern of nucleoside modifications in an oligomeric compound or a region thereof. The linkages of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only nucleosides are intended to be nucleoside motifs. Thus, in such instances, the linkages are not limited.
As used herein, “sugar motif” means a pattern of sugar modifications in an oligomeric compound or a region thereof.
As used herein, “linkage motif” means a pattern of linkage modifications in an oligomeric compound or region thereof. The nucleosides of such an oligomeric compound may be modified or unmodified. Unless otherwise indicated, motifs herein describing only linkages are intended to be linkage motifs. Thus, in such instances, the nucleosides are not limited.
As used herein, “nucleobase modification motif” means a pattern of modifications to nucleobases along an oligonucleotide. Unless otherwise indicated, a nucleobase modification motif is independent of the nucleobase sequence.
As used herein, “sequence motif” means a pattern of nucleobases arranged along an oligonucleotide or portion thereof. Unless otherwise indicated, a sequence motif is independent of chemical modifications and thus may have any combination of chemical modifications, including no chemical modifications.
As used herein, “type of modification” in reference to a nucleoside or a nucleoside of a “type” means the chemical modification of a nucleoside and includes modified and unmodified nucleosides. Accordingly, unless otherwise indicated, a “nucleoside having a modification of a first type” may be an unmodified nucleoside.
As used herein, “differently modified” mean chemical modifications or chemical substituents that are different from one another, including absence of modifications. Thus, for example, a MOE nucleoside and an unmodified DNA nucleoside are “differently modified,” even though the DNA nucleoside is unmodified. Likewise, DNA and RNA are “differently modified,” even though both are naturally-occurring unmodified nucleosides. Nucleosides that are the same but for comprising different nucleobases are not differently modified. For example, a nucleoside comprising a 2′-OMe modified sugar and an unmodified adenine nucleobase and a nucleoside comprising a 2′-OMe modified sugar and an unmodified thymine nucleobase are not differently modified.
As used herein, “the same type of modifications” refers to modifications that are the same as one another, including absence of modifications. Thus, for example, two unmodified DNA nucleoside have “the same type of modification,” even though the DNA nucleoside is unmodified. Such nucleosides having the same type modification may comprise different nucleobases.
As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administering to an animal. In certain embodiments, a pharmaceutically acceptable carrier or diluent is sterile saline. In certain embodiments, such sterile saline is pharmaceutical grade saline.
As used herein, “substituent” and “substituent group,” means an atom or group that replaces the atom or group of a named parent compound. For example a substituent of a modified nucleoside is any atom or group that differs from the atom or group found in a naturally occurring nucleoside (e.g., a modified 2′-substituent is any atom or group at the 2′-position of a nucleoside other than H or OH). Substituent groups can be protected or unprotected. In certain embodiments, compounds of the present invention have substituents at one or at more than one position of the parent compound. Substituents may also be further substituted with other substituent groups and may be attached directly or via a linking group such as an alkyl or hydrocarbyl group to a parent compound.
Likewise, as used herein, “substituent” in reference to a chemical functional group means an atom or group of atoms differs from the atom or a group of atoms normally present in the named functional group. In certain embodiments, a substituent replaces a hydrogen atom of the functional group (e.g., in certain embodiments, the substituent of a substituted methyl group is an atom or group other than hydrogen which replaces one of the hydrogen atoms of an unsubstituted methyl group). Unless otherwise indicated, groups amenable for use as substituents include without limitation, halogen, hydroxyl, alkyl, alkenyl, alkynyl, acyl (—C(O)Raa), carboxyl (—C(O)O—Raa), aliphatic groups, alicyclic groups, alkoxy, substituted oxy (—O—Raa), aryl, aralkyl, heterocyclic radical, heteroaryl, heteroarylalkyl, amino (—N(Rbb)(Rcc)), imino(═NRbb), amido (—C(O)N(Rbb)(Rcc) or —N(Rbb)C(O)Raa), azido (—N3), nitro (—NO2), cyano (—CN), carbamido (—OC(O)N(Rbb)(Rcc) or —N(Rbb)C(O)ORaa), ureido (—N(Rbb)C(O)N(Rbb)(Rcc)), thioureido (—N(Rbb)C(S)N(Rbb)—(Rcc)), guanidinyl (—N(Rbb)C(═NRbb)N(Rbb)(Rcc)), amidinyl (—C(═NRbb)N(Rbb)(Rcc) or —N(Rbb)C(═NRbb)(Raa)), thiol (—SRbb), sulfinyl (—S(O)Rbb), sulfonyl (—S(O)2Rbb) and sulfonamidyl (—S(O)2N(Rbb)(Rcc) or —N(Rbb)S—(O)2Rbb). Wherein each Raa, Rbb and Rcc is, independently, H, an optionally linked chemical functional group or a further substituent group with a preferred list including without limitation, alkyl, alkenyl, alkynyl, aliphatic, alkoxy, acyl, aryl, aralkyl, heteroaryl, alicyclic, heterocyclic and heteroarylalkyl. Selected substituents within the compounds described herein are present to a recursive degree.
As used herein, “alkyl,” as used herein, means a saturated straight or branched hydrocarbon radical containing up to twenty four carbon atoms. Examples of alkyl groups include without limitation, methyl, ethyl, propyl, butyl, isopropyl, n-hexyl, octyl, decyl, dodecyl and the like. Alkyl groups typically include from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms (C1-C12 alkyl) with from 1 to about 6 carbon atoms being more preferred.
As used herein, “alkenyl,” means a straight or branched hydrocarbon chain radical containing up to twenty four carbon atoms and having at least one carbon-carbon double bond. Examples of alkenyl groups include without limitation, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, dienes such as 1,3-butadiene and the like. Alkenyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkenyl groups as used herein may optionally include one or more further substituent groups.
As used herein, “alkynyl,” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms and having at least one carbon-carbon triple bond. Examples of alkynyl groups include, without limitation, ethynyl, 1-propynyl, 1-butynyl, and the like. Alkynyl groups typically include from 2 to about 24 carbon atoms, more typically from 2 to about 12 carbon atoms with from 2 to about 6 carbon atoms being more preferred. Alkynyl groups as used herein may optionally include one or more further substituent groups.
As used herein, “acyl,” means a radical formed by removal of a hydroxyl group from an organic acid and has the general Formula —C(O)—X where X is typically aliphatic, alicyclic or aromatic. Examples include aliphatic carbonyls, aromatic carbonyls, aliphatic sulfonyls, aromatic sulfinyls, aliphatic sulfinyls, aromatic phosphates, aliphatic phosphates and the like. Acyl groups as used herein may optionally include further substituent groups.
As used herein, “alicyclic” means a cyclic ring system wherein the ring is aliphatic. The ring system can comprise one or more rings wherein at least one ring is aliphatic. Preferred alicyclics include rings having from about 5 to about 9 carbon atoms in the ring. Alicyclic as used herein may optionally include further substituent groups.
As used herein, “aliphatic” means a straight or branched hydrocarbon radical containing up to twenty four carbon atoms wherein the saturation between any two carbon atoms is a single, double or triple bond. An aliphatic group preferably contains from 1 to about 24 carbon atoms, more typically from 1 to about 12 carbon atoms with from 1 to about 6 carbon atoms being more preferred. The straight or branched chain of an aliphatic group may be interrupted with one or more heteroatoms that include nitrogen, oxygen, sulfur and phosphorus. Such aliphatic groups interrupted by heteroatoms include without limitation, polyalkoxys, such as polyalkylene glycols, polyamines, and polyimines. Aliphatic groups as used herein may optionally include further substituent groups.
As used herein, “alkoxy” means a radical formed between an alkyl group and an oxygen atom wherein the oxygen atom is used to attach the alkoxy group to a parent molecule. Examples of alkoxy groups include without limitation, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy, neopentoxy, n-hexoxy and the like. Alkoxy groups as used herein may optionally include further substituent groups.
As used herein, “aminoalkyl” means an amino substituted C1-C12 alkyl radical. The alkyl portion of the radical forms a covalent bond with a parent molecule. The amino group can be located at any position and the aminoalkyl group can be substituted with a further substituent group at the alkyl and/or amino portions.
As used herein, “aralkyl” and “arylalkyl” mean an aromatic group that is covalently linked to a C1-C12 alkyl radical. The alkyl radical portion of the resulting aralkyl (or arylalkyl) group forms a covalent bond with a parent molecule. Examples include without limitation, benzyl, phenethyl and the like. Aralkyl groups as used herein may optionally include further substituent groups attached to the alkyl, the aryl or both groups that form the radical group.
As used herein, “aryl” and “aromatic” mean a mono- or polycyclic carbocyclic ring system radicals having one or more aromatic rings. Examples of aryl groups include without limitation, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl and the like. Preferred aryl ring systems have from about 5 to about 20 carbon atoms in one or more rings. Aryl groups as used herein may optionally include further substituent groups.
As used herein, “halo” and “halogen,” mean an atom selected from fluorine, chlorine, bromine and iodine.
As used herein, “heteroaryl,” and “heteroaromatic,” mean a radical comprising a mono- or polycyclic aromatic ring, ring system or fused ring system wherein at least one of the rings is aromatic and includes one or more heteroatoms. Heteroaryl is also meant to include fused ring systems including systems where one or more of the fused rings contain no heteroatoms. Heteroaryl groups typically include one ring atom selected from sulfur, nitrogen or oxygen. Examples of heteroaryl groups include without limitation, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl and the like. Heteroaryl radicals can be attached to a parent molecule directly or through a linking moiety such as an aliphatic group or hetero atom. Heteroaryl groups as used herein may optionally include further substituent groups.
Oligomeric CompoundsIn certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, such oligomeric compounds comprise oligonucleotides optionally comprising one or more conjugate and/or terminal groups. In certain embodiments, an oligomeric compound consists of an oligonucleotide. In certain embodiments, oligonucleotides comprise one or more chemical modifications. Such chemical modifications include modifications one or more nucleoside (including modifications to the sugar moiety and/or the nucleobase) and/or modifications to one or more internucleoside linkage.
Certain Sugar Moieties
In certain embodiments, oligomeric compounds of the invention comprise one or more modified nucleosides comprising a modified sugar moiety. Such oligomeric compounds comprising one or more sugar-modified nucleosides may have desirable properties, such as enhanced nuclease stability or increased binding affinity with a target nucleic acid relative to oligomeric compounds comprising only nucleosides comprising naturally occurring sugar moieties. In certain embodiments, modified sugar moieties are substituted sugar moieties. In certain embodiments, modified sugar moieties are bicyclic or tricyclic sugar moieties. In certain embodiments, modified sugar moieties are sugar surrogates. Such sugar surrogates may comprise one or more substitutions corresponding to those of substituted sugar moieties.
In certain embodiments, modified sugar moieties are substituted sugar moieties comprising one or more substituent, including but not limited to substituents at the 2′ and/or 5′ positions. Examples of sugar substituents suitable for the 2′-position, include, but are not limited to: 2′-F, 2′-OCH3 (“OMe” or “O-methyl”), and 2′-O(CH2)2OCH3 (“MOE”). In certain embodiments, sugar substituents at the 2′ position is selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, O—C1-C10 substituted alkyl; I—C1-C10 alkoxy; O—C1-C10 substituted alkoxy, OCF3, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), and O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. Examples of sugar substituents at the 5′-position, include, but are not limited to: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In certain embodiments, substituted sugars comprise more than one non-bridging sugar substituent, for example, 2′-F-5′-methyl sugar moieties (see, e.g., PCT International Application WO 2008/101157, for additional 5′, 2′-bis substituted sugar moieties and nucleosides).
Nucleosides comprising 2′-substituted sugar moieties are referred to as 2′-substituted nucleosides. In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from halo, allyl, amino, azido, O—C1-C10 alkoxy; O—C1-C10 substituted alkoxy, SH, CN, OCN, CF3, OCF3, O-alkyl, S-alkyl, N(Rm)-alkyl; O-alkenyl, S-alkenyl, or N(Rm)-alkenyl; O-alkynyl, S-alkynyl, N(Rm)-alkynyl; O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn) or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl. These 2′-substituent groups can be further substituted with one or more substituent groups independently selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro (NO2), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl, alkenyl and alkynyl.
In certain embodiments, a 2′-substituted nucleoside comprises a 2′-substituent group selected from F, NH2, N3, OCF3, O—CH3, O(CH2)3NH2, CH2—CH═CH2, O—CH2—CH═CH2, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (O—CH2—C(═O)—N(Rm)(Rn) where each Rm and Rn is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.
In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, OCF3, O—CH3, OCH2CH2OCH3, O(CH2)2SCH3, O—(CH2)2—O—N(CH3)2, —O(CH2)2O(CH2)2N(CH3)2, and O—CH2—C(═O)—N(H)CH3.
In certain embodiments, a 2′-substituted nucleoside comprises a sugar moiety comprising a 2′-substituent group selected from F, O—CH3, and OCH2CH2OCH3.
Certain modified sugar moieties comprise a bridging sugar substituent that forms a second ring resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugar substituents, include, but are not limited to: —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or, —C(RaRb)—O—N(R)—; 4′- CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′; 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2′; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (cEt) and 4′-CH(CH2OCH3)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′ and analogs thereof, (see, e.g., WO2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ and analogs thereof (see, e.g., WO2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see, e.g., US2004/0171570, published Sep. 2, 2004); 4′-CH2—O—N(R)-2′, and 4′-CH2—N(R)-0-2′-, wherein each R is, independently, H, a protecting group, or C1-C12 alkyl; 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C(H)(CH3)-2′ (see, e.g., Chattopadhyaya, et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ and analogs thereof (see, published PCT International Application WO 2008/154401, published on Dec. 8, 2008).
In certain embodiments, such 4′ to 2′ bridges independently comprise from 1 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═NRa)—, —C(═O)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl, or a protecting group.
Nucleosides comprising bicyclic sugar moieties are referred to as bicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are not limited to, (A) α-L-Methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C) Ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) Oxyamino (4′-CH2—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA (also referred to as constrained ethyl or cEt), (G) methylene-thio (4′-CH2—S-2′) BNA, (H) methylene-amino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH2—CH(CH3)-2′) BNA, and (J) propylene carbocyclic (4′-(CH2)3-2′) BNA as depicted below.
wherein Bx is a nucleobase moiety and R is, independently, H, a protecting group, or C1-C12 alkyl.
Additional bicyclic sugar moieties are known in the art, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J Am. Chem. Soc., 129(26) 8362-8379 (Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207, 6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and 7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO 2007/134181; U.S. Patent Publication Nos. US2004/0171570, US2007/0287831, and US2008/0039618; U.S. patent Ser. Nos. 12/129,154, 60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844; and PCT International Applications Nos. PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.
In certain embodiments, bicyclic sugar moieties and nucleosides incorporating such bicyclic sugar moieties are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) bicyclic nucleosides have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, substituted sugar moieties comprise one or more non-bridging sugar substituent and one or more bridging sugar substituent (e.g., 5′-substituted and 4′-2′ bridged sugars). (see, PCT International Application WO 2007/134181, published on Nov. 22, 2007, wherein LNA is substituted with, for example, a 5′-methyl or a 5′-vinyl group).
In certain embodiments, modified sugar moieties are sugar surrogates. In certain such embodiments, the oxygen atom of the naturally occurring sugar is substituted, e.g., with a sulfur, carbon or nitrogen atom. In certain such embodiments, such modified sugar moiety also comprises bridging and/or non-bridging substituents as described above. For example, certain sugar surrogates comprise a 4′-sulfur atom and a substitution at the 2′-position (see, e.g., published U.S. Patent Application US2005/0130923, published on Jun. 16, 2005) and/or the 5′ position. By way of additional example, carbocyclic bicyclic nucleosides having a 4′-2′ bridge have been described (see, e.g., Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740).
In certain embodiments, sugar surrogates comprise rings having other than 5-atoms. For example, in certain embodiments, a sugar surrogate comprises a six-membered tetrahydropyran. Such tetrahydropyrans may be further modified or substituted. Nucleosides comprising such modified tetrahydropyrans include, but are not limited to, hexitol nucleic acid (HNA), annitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), fluoro HNA (F-HNA), and those compounds having Formula VII:
wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:
Bx is a nucleobase moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′ or 3′-terminal group;
q1, q2, q3, q4, q5, q6 and q7 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, or substituted C2-C6 alkynyl; and each of R1 and R2 is independently selected from among: hydrogen, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2, and CN, wherein X is O, S or NJ1, and each J1, J2, and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is F. In certain embodiments, R1 is fluoro and R2 is H, R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
Many other bicyclic and tricyclic sugar and sugar surrogate ring systems are known in the art that can be used to modify nucleosides (see, e.g., review article: Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following structure:
In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”
Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′, 2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH2—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J Am. Chem. Soc. 2007, 129(26), 8362-8379).
Certain Nucleobases
In certain embodiments, nucleosides of the present invention comprise one or more unmodified nucleobases. In certain embodiments, nucleosides of the present invention comprise one or more modified nucleobases.
In certain embodiments, modified nucleobases are selected from: universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine; 5-hydroxymethyl cytosine, 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-halouracil and cytosine, 5-propynyl (—C≡C≡CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, 3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases as defined herein. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include without limitation, U.S. Pat. Nos. 3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.
Certain Internucleoside Linkages
In certain embodiments, the present invention provides oligomeric compounds comprising linked nucleosides. In such embodiments, nucleosides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters (P═O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P═S). Representative non-phosphorus containing internucleoside linking groups include, but are not limited to, methylenemethylimino (—CH2—N(CH3)—O—CH2—), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane (—O—Si(H)2—O—); and N,N′-dimethylhydrazine (—CH2—N(CH3)—N(CH3)—). Modified linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligomeric compound. In certain embodiments, internucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages are well known to those skilled in the art.
The oligonucleotides described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations that may be defined, in terms of absolute stereochemistry, as (R) or (S), α or β such as for sugar anomers, or as (D) or (L) such as for amino acids etc. Included in the antisense compounds provided herein are all such possible isomers, as well as their racemic and optically pure forms.
Neutral internucleoside linkages include without limitation, phosphotriesters, methylphosphonates, MMI (3′-CH2—N(CH3)—O-5′), amide-3 (3′-CH2—C(═O)—N(H)-5′), amide-4 (3′-CH2—N(H)—C(═O)-5′), formacetal (3′-O—CH2—O-5′), and thioformacetal (3′-S—CH2—O-5′). Further neutral internucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutral internucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
Certain Motifs
In certain embodiments, the present invention provides oligomeric compounds comprising oligonucleotides. In certain embodiments, such oligonucleotides comprise one or more chemical modification. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising modified sugars. In certain embodiments, chemically modified oligonucleotides comprise one or more modified nucleosides comprising one or more modified nucleobases. In certain embodiments, chemically modified oligonucleotides comprise one or more modified internucleoside linkages. In certain embodiments, the chemically modifications (sugar modifications, nucleobase modifications, and/or linkage modifications) define a pattern or motif. In certain embodiments, the patterns of chemical modifications of sugar moieties, internucleoside linkages, and nucleobases are each independent of one another. Thus, an oligonucleotide may be described by its sugar modification motif, internucleoside linkage motif and/or nucleobase modification motif (as used herein, nucleobase modification motif describes the chemical modifications to the nucleobases independent of the sequence of nucleobases).
Certain Sugar Motifs
In certain embodiments, oligonucleotides comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the oligonucleotides comprise or consist of a region having a gapmer sugar modification motif, which comprises two external regions or “wings” and an internal region or “gap.” The three regions of a gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside of the 3′-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap. In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar modification motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar modification motifs of the 5′-wing differs from the sugar modification motif of the 3′-wing (asymmetric gapmer). In certain embodiments, oligonucleotides comprise 2′-MOE modified nucleosides in the wings and 2′-F modified nucleosides in the gap.
In certain embodiments, oligonucleotides are fully modified. In certain such embodiments, oligonucleotides are uniformly modified. In certain embodiments, oligonucleotides are uniform 2′-MOE. In certain embodiments, oligonucleotides are uniform 2′-F. In certain embodiments, oligonucleotides are uniform morpholino. In certain embodiments, oligonucleotides are uniform BNA. In certain embodiments, oligonucleotides are uniform LNA. In certain embodiments, oligonucleotides are uniform cEt.
In certain embodiments, oligonucleotides comprise a uniformly modified region and additional nucleosides that are unmodified or differently modified. In certain embodiments, the uniformly modified region is at least 5, 10, 15, or 20 nucleosides in length. In certain embodiments, the uniform region is a 2′-MOE region. In certain embodiments, the uniform region is a 2′-F region. In certain embodiments, the uniform region is a morpholino region. In certain embodiments, the uniform region is a BNA region. In certain embodiments, the uniform region is a LNA region. In certain embodiments, the uniform region is a cEt region.
In certain embodiments, the oligonucleotide does not comprise more than 4 contiguous unmodified 2′-deoxynucleosides. In certain circumstances, antisense oligonucleotides comprising more than 4 contiguous 2′-deoxynucleosides activate RNase H, resulting in cleavage of the target RNA. In certain embodiments, such cleavage is avoided by not having more than 4 contiguous 2′-deoxynucleosides, for example, where alteration of splicing and not cleavage of a target RNA is desired.
Certain Splicing Motifs
In certain embodiments, oligonucleotides have a certain modification pattern and/or motif designed to alter the splicing of certain nucleic acid transcripts. In certain embodiments, oligonucleotides have a certain modification pattern and/or motif designed to alter the splicing of certain pre-mRNA transcripts. In certain embodiments, oligonucleotides have a certain modification pattern and/or motif designed in such a fashion that the oligonucleotide will not recruit RNase H once bound to a target nucleic acid transcript. For example, in certain such embodiments, an oligonucleotide may have one or more sugar modifications placed throughout the oligonucleotide so as to have no segment comprising more than 4 contiguous 2′-deoxynucleosides. In certain such embodiments, an oligonucleotide may have one or more sugar modifications placed throughout the oligonucleotide so as to have no segment comprising more than 3 contiguous 2′-deoxynucleosides. In certain such embodiments, an oligonucleotide may have one or more sugar modifications placed throughout the oligonucleotide so as to have no segment comprising more than 2 contiguous 2′-deoxynucleosides.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a Ad2Ad2Ad2Ad2Ad2A motif, wherein each A independently comprises a bicyclic modification selected from among LNA and cEt and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a AAddAddAddAddAddAA motif, wherein each A independently comprises a bicyclic modification selected from among LNA and cEt and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a AABBABBABBABBABBAB motif, wherein each A independently comprises a bicyclic modification selected from among LNA and cEt and each B independently comprises a 2′-modification selected from among a 2′-OMe, 2′-F, or 2′-MOE modification.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a AddAddAddAddAddA motif wherein each A independently comprises a bicyclic modification selected from among LNA and cEt and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a keekeekeekeekeek motif, wherein each k comprises a cEt modification and each e comprises a 2′-MOE modification.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kd2kd2kd2kd2kd2k motif, wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kkddkddkddkddkddkk motif, wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kkeekeekeekeekeeke motif, wherein each k comprises a cEt modification and each e comprises a 2′-MOE modification.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a kddkddkddkddkddk motif wherein each k comprises a cEt modification and each d comprises a 2′-deoxynucleoside.
In certain embodiments, the oligonucleotide compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a target nucleic acid, wherein the modified oligonucleotide comprises a keekeekeekeekeek motif, wherein each k comprises a cEt modification and each e comprises a 2′-MOE modification.
Certain Internucleoside Linkage Motifs
In certain embodiments, oligonucleotides comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, internucleoside linkages are arranged in a gapped motif, as described above for sugar modification motif. In such embodiments, the internucleoside linkages in each of two wing regions are different from the internucleoside linkages in the gap region. In certain embodiments the internucleoside linkages in the wings are phosphodiester and the internucleoside linkages in the gap are phosphorothioate. The sugar modification motif is independently selected, so such oligonucleotides having a gapped internucleoside linkage motif may or may not have a gapped sugar modification motif and if it does have a gapped sugar motif, the wing and gap lengths may or may not be the same.
In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides of the present invention comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
Certain Nucleobase Modification Motifs
In certain embodiments, oligonucleotides comprise chemical modifications to nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or nucleobases modification motif. In certain such embodiments, nucleobase modifications are arranged in a gapped motif. In certain embodiments, nucleobase modifications are arranged in an alternating motif. In certain embodiments, each nucleobase is modified. In certain embodiments, none of the nucleobases is chemically modified.
In certain embodiments, oligonucleotides comprise a block of modified nucleobases. In certain such embodiments, the block is at the 3′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 3′-end of the oligonucleotide. In certain such embodiments, the block is at the 5′-end of the oligonucleotide. In certain embodiments the block is within 3 nucleotides of the 5′-end of the oligonucleotide.
In certain embodiments, nucleobase modifications are a function of the natural base at a particular position of an oligonucleotide. For example, in certain embodiments each purine or each pyrimidine in an oligonucleotide is modified. In certain embodiments, each adenine is modified. In certain embodiments, each guanine is modified. In certain embodiments, each thymine is modified. In certain embodiments, each cytosine is modified. In certain embodiments, each uracil is modified.
In certain embodiments, some, all, or none of the cytosine moieties in an oligonucleotide are 5-methyl cytosine moieties. Herein, 5-methyl cytosine is not a “modified nucleobase.” Accordingly, unless otherwise indicated, unmodified nucleobases include both cytosine residues having a 5-methyl and those lacking a 5 methyl. In certain embodiments, the methylation state of all or some cytosine nucleobases is specified.
Certain Overall Lengths
In certain embodiments, the present invention provides oligomeric compounds including oligonucleotides of any of a variety of ranges of lengths. In certain embodiments, the invention provides oligomeric compounds or oligonucleotides consisting of X to Y linked nucleosides, where X represents the fewest number of nucleosides in the range and Y represents the largest number of nucleosides in the range. In certain such embodiments, X and Y are each independently selected from 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, in certain embodiments, the invention provides oligomeric compounds which comprise oligonucleotides consisting of 8 to 9, 8 to 10, 8 to 11, 8 to 12, 8 to 13, 8 to 14, 8 to 15, 8 to 16, 8 to 17, 8 to 18, 8 to 19, 8 to 20, 8 to 21, 8 to 22, 8 to 23, 8 to 24, 8 to 25, 8 to 26, 8 to 27, 8 to 28, 8 to 29, 8 to 30, 9 to 10, 9 to 11, 9 to 12, 9 to 13, 9 to 14, 9 to 15, 9 to 16, 9 to 17, 9 to 18, 9 to 19, 9 to 20, 9 to 21, 9 to 22, 9 to 23, 9 to 24, 9 to 25, 9 to 26, 9 to 27, 9 to 28, 9 to 29, 9 to 30, 10 to 11, 10 to 12, 10 to 13, 10 to 14, 10 to 15, 10 to 16, 10 to 17, 10 to 18, 10 to 19, 10 to 20, 10 to 21, 10 to 22, 10 to 23, 10 to 24, 10 to 25, 10 to 26, 10 to 27, 10 to 28, 10 to 29, 10 to 30, 11 to 12, 11 to 13, 11 to 14, 11 to 15, 11 to 16, 11 to 17, 11 to 18, 11 to 19, 11 to 20, 11 to 21, 11 to 22, 11 to 23, 11 to 24, 11 to 25, 11 to 26, 11 to 27, 11 to 28, 11 to 29, 11 to 30, 12 to 13, 12 to 14, 12 to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to 22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to 29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to 19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to 26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to 17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to 24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to 16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to 23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to 30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to 23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to 30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to 24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to 19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to 26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to 22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to 29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to 26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to 24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to 23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to 30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to 30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to 26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to 29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to 30 linked nucleosides. In embodiments where the number of nucleosides of an oligomeric compound or oligonucleotide is limited, whether to a range or to a specific number, the oligomeric compound or oligonucleotide may, nonetheless further comprise additional other substituents. For example, an oligonucleotide comprising 8-30 nucleosides excludes oligonucleotides having 31 nucleosides, but, unless otherwise indicated, such an oligonucleotide may further comprise, for example one or more conjugates, terminal groups, or other substituents. In certain embodiments, a gapmer oligonucleotide has any of the above lengths. In certain embodiments, an antisense oligonucleotide has any of the above lengths.
One of skill in the art will appreciate that certain lengths may not be possible for certain motifs. For example: a gapmer having a 5′-wing region consisting of four nucleotides, a gap consisting of at least six nucleotides, and a 3′-wing region consisting of three nucleotides cannot have an overall length less than 13 nucleotides. Thus, one would understand that the lower length limit is 13 and that the limit of 10 in “10-20” has no effect in that embodiment.
Further, where an oligonucleotide is described by an overall length range and by regions having specified lengths, and where the sum of specified lengths of the regions is less than the upper limit of the overall length range, the oligonucleotide may have additional nucleosides, beyond those of the specified regions, provided that the total number of nucleosides does not exceed the upper limit of the overall length range. For example, an oligonucleotide consisting of 20-25 linked nucleosides comprising a 5′-wing consisting of 5 linked nucleosides; a 3′-wing consisting of 5 linked nucleosides and a central gap consisting of 10 linked nucleosides (5+5+10=20) may have up to 5 nucleosides that are not part of the 5′-wing, the 3′-wing, or the gap (before reaching the overall length limitation of 25). Such additional nucleosides may be 5′ of the 5′-wing and/or 3′ of the 3′ wing.
Certain Oligonucleotides
In certain embodiments, oligonucleotides of the present invention are characterized by their sugar motif, internucleoside linkage motif, nucleobase modification motif and overall length. In certain embodiments, such parameters are each independent of one another. Thus, each internucleoside linkage of an oligonucleotide having a gapmer sugar motif may be modified or unmodified and may or may not follow the gapmer modification pattern of the sugar modifications. Thus, the internucleoside linkages within the wing regions of a sugar-gapmer may be the same or different from one another and may be the same or different from the internucleoside linkages of the gap region. Likewise, such sugar-gapmer oligonucleotides may comprise one or more modified nucleobase independent of the gapmer pattern of the sugar modifications. Herein if a description of an oligonucleotide or oligomeric compound is silent with respect to one or more parameter, such parameter is not limited. Thus, an oligomeric compound described only as having a gapmer sugar motif without further description may have any length, internucleoside linkage motif, and nucleobase modification motif. Unless otherwise indicated, all chemical modifications are independent of nucleobase sequence.
Certain Conjugate Groups
In certain embodiments, oligomeric compounds are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligomeric compound including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligomeric compound, such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (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), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a 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-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
In certain embodiments, a conjugate group comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
In certain embodiments, conjugate groups are directly attached to oligonucleotides in oligomeric compounds. In certain embodiments, conjugate groups are attached to oligonucleotides by a conjugate linking group. In certain such embodiments, conjugate linking groups, including, but not limited to, bifunctional linking moieties such as those known in the art are amenable to the compounds provided herein. Conjugate linking groups are useful for attachment of conjugate groups, such as chemical stabilizing groups, functional groups, reporter groups and other groups to selective sites in a parent compound such as for example an oligomeric compound. In general a bifunctional linking moiety comprises a hydrocarbyl moiety having two functional groups. One of the functional groups is selected to bind to a parent molecule or compound of interest and the other is selected to bind essentially any selected group such as chemical functional group or a conjugate group. In some embodiments, the conjugate linker comprises a chain structure or an oligomer of repeating units such as ethylene glycol or amino acid units. Examples of functional groups that are routinely used in a bifunctional linking moiety include, but are not limited to, electrophiles for reacting with nucleophilic groups and nucleophiles for reacting with electrophilic groups. In some embodiments, bifunctional linking moieties include amino, hydroxyl, carboxylic acid, thiol, unsaturations (e.g., double or triple bonds), and the like.
Some nonlimiting examples of conjugate linking moieties include pyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and 6-aminohexanoic acid (AHEX or AHA). Other linking groups include, but are not limited to, substituted C1-C10 alkyl, substituted or unsubstituted C2-C10 alkenyl or substituted or unsubstituted C2-C10 alkynyl, wherein a nonlimiting list of preferred substituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and alkynyl.
Conjugate groups may be attached to either or both ends of an oligonucleotide (terminal conjugate groups) and/or at any internal position.
In certain embodiments, conjugate groups are at the 3′-end of an oligonucleotide of an oligomeric compound. In certain embodiments, conjugate groups are near the 3′-end. In certain embodiments, conjugates are attached at the 3′ end of an oligomeric compound, but before one or more terminal group nucleosides. In certain embodiments, conjugate groups are placed within a terminal group.
In certain embodiments, the present invention provides oligomeric compounds. In certain embodiments, oligomeric compounds comprise an oligonucleotide. In certain embodiments, an oligomeric compound comprises an oligonucleotide and one or more conjugate and/or terminal groups. Such conjugate and/or terminal groups may be added to oligonucleotides having any of the chemical motifs discussed above. Thus, for example, an oligomeric compound comprising an oligonucleotide having region of alternating nucleosides may comprise a terminal group.
Antisense Compounds
In certain embodiments, oligomeric compounds of the present invention are antisense compounds. Such antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity. In certain embodiments, antisense compounds specifically hybridize to one or more target nucleic acid. In certain embodiments, a specifically hybridizing antisense compound has a nucleobase sequence comprising a region having sufficient complementarity to a target nucleic acid to allow hybridization and result in antisense activity and insufficient complementarity to any non-target so as to avoid non-specific hybridization to any non-target nucleic acid sequences under conditions in which specific hybridization is desired (e.g., under physiological conditions for in vivo or therapeutic uses, and under conditions in which assays are performed in the case of in vitro assays).
In certain embodiments, the present invention provides antisense compounds comprising oligonucleotides that are fully complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain embodiments, oligonucleotides are 99% complementary to the target nucleic acid. In certain embodiments, oligonucleotides are 95% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 90% complementary to the target nucleic acid.
In certain embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In certain embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In certain embodiments, an antisense compound comprises a region that is fully complementary to a target nucleic acid and is at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide. In certain such embodiments, the region of full complementarity is from 6 to 14 nucleobases in length.
In certain embodiments antisense compounds and antisense oligonucleotides comprise single-strand compounds. In certain embodiments antisense compounds and antisense oligonucleotides comprise double-strand compounds.
Certain Pathways and Mechanisms Associated With Fibrosis
TGFβ1 and its associated pathways contribute to many processes associated with wound healing and tissue repair. After an injury, TGFβ1 contributes to the healing and restoration of normal tissue by, among other things, stimulating the production of certain extracellular matrix proteins and inhibiting the degradation of certain matrix proteins. In certain embodiments, TGFβ1 stimulates the production of fibronectin. In certain embodiments, TGFβ1 stimulates the production of both the EDA+ and EDA− isoforms of fibronectin. In certain embodiments, excessive amounts of the EDA+ fibronectin isoform causes tissue fibrosis. In certain embodiments, excessive tissue fibrosis induced by TGFβ1/EDA+ impairs normal organ function, impairs cellular function, and/or causes cells to change or lose their phenotype. In certain embodiments, the release and/or activation of TGFβ1 causes the formation of fibrosis and consequent changes in cell phenotype. In certain embodiments, changes in cell phenotype due to fibrosis include, but are not limited to, modulation of cadherin expression, induction of α Smooth Muscle Actin (αSMA), alteration of cortical f-actin localization, induction of connexin 43 (Cx 43) expression, alteration of vimentin, alteration of tight junction protein ZO-1, and/or increased secretion of MMP2 & MMP9. In certain embodiments, the release and/or activation of TGFβ1 causes a loss of cell phenotype. In certain embodiments, the loss of cell phenotype due to fibrosis impairs the structure or function of a cell. In certain embodiments, the loss of cell phenotype due to fibrosis destroys the function of a cell.
In certain embodiments, it is therefore desirable to reduce fibrosis without affecting the healing and/or restoration process. In certain embodiments, it is therefore desirable to reduce the formation of fibrosis in a cell without reducing or altering the amount and/or activity of TGFβ1 in the cell. In certain embodiments, it is therefore desirable to reduce the amount of EDA+ fibronectin in a cell without reducing or altering the amount of EDA− fibronectin in the cell. In certain embodiments, the reduction of the amount of EDA+ fibronectin in a cell in response to TGFβ1 will result in wound healing and tissue repair without incurring excessive fibrosis. In certain embodiments, the selective reduction of the amount of EDA+ fibronectin in a cell, relative to the amount of EDA− fibronectin in the cell in the response to TGFβ1 will stimulate wound healing and tissue repair without incurring changes in cell phenotype associated with fibrosis. In certain embodiments, the reduction of the amount of EDA+ fibronectin in a cell, relative to the amount of EDA− fibronectin in the cell in response to TGFβ1 will stimulate wound healing and tissue repair without incurring the loss of cell phenotype due to fibrosis.
In certain embodiments, it is desirable to reverse the formation of fibrosis in a cell without reducing or altering the wound healing function of TGFβ1 in the cell. In certain embodiments, it is therefore desirable to reverse the changes caused by fibrosis in the phenotype of a cell without reducing or altering the wound healing function of TGFβ1 in the cell. In certain embodiments, it is therefore desirable to reverse the loss of phenotype in a cell caused by fibrosis without reducing or altering the wound healing function of TGFβ1 in the cell.
Certain Target Nucleic Acids and Mechanisms
In certain embodiments, antisense compounds comprise or consist of an oligonucleotide comprising a region that is complementary to a target nucleic acid. In certain embodiments, the target nucleic acid is an endogenous RNA molecule. In certain embodiments, the target nucleic acid is a pre-mRNA. In certain embodiments, the target nucleic acid is a fibronectin transcript. In certain embodiments, the target RNA is a fibronectin pre-mRNA.
In certain embodiments, an antisense compound is complementary to a region of fibronectin pre-mRNA. In certain embodiments, an antisense compound is complementary within a region of fibronectin pre-mRNA comprising an exon encoding EDA. In certain embodiments, an antisense compound is complementary to a region of fibronectin pre-mRNA comprising an intron-exon splice junction. In certain embodiments, an antisense compound is complementary to a region of fibronectin pre-mRNA comprising the intron-exon splice junction adjacent to the EDA exon. In certain embodiments, an antisense compound is complementary within a region of fibronectin pre-mRNA consisting of an exon encoding EDA. In certain embodiments, an antisense compound is complementary within a region of fibronectin pre-mRNA comprising an exonic splicing silencer within an exon encoding EDA. In certain embodiments, an antisense compound is complementary within a region of fibronectin pre-mRNA comprising an exonic splicing enhancer within an exon encoding EDA.
In certain embodiments, an antisense compound comprises a modified oligonucleotide consisting of 8 to 30 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a fibronectin transcript. In certain embodiments, the target region is within nucleobase 55469 and nucleobase 55790 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 55469 and nucleobase 55511 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 55511 and nucleobase 55732 of SEQ ID NO.: 1. In certain embodiments, the target region is within nucleobase 55732 and nucleobase 55790 of SEQ ID NO.: 1.
In certain embodiments, an antisense oligonucleotide modulates splicing of a pre-mRNA. In certain embodiments, an antisense oligonucleotide modulates splicing a fibronectin pre-mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of fibronectin mRNA. In certain embodiments, an antisense oligonucleotide increases the amount of EDA− fibronectin mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of EDA+ fibronectin mRNA. In certain embodiments, an antisense oligonucleotide decreases the amount of EDA+ fibronectin mRNA in the presence of TGFβ1. In certain embodiments, an antisense oligonucleotide decreases the amount of EDA+ fibronectin mRNA in a cell without substantially affecting the healing and/or restoration functions of the cell.
In certain embodiments, an antisense oligonucleotide alters the ratio of EDA+/EDA− fibronectin. In certain embodiments, an antisense oligonucleotide increases the ratio of EDA+/EDA− fibronectin. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin to create a fibrosis model. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin to create a fibrosis phenotype. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis model. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis phenotype. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin to create a fibrosis mouse model. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin to create a fibrosis mouse phenotype. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis mouse model. In certain embodiments, it is desirable to increase the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis mouse phenotype.
In certain embodiments, an antisense oligonucleotide alters the ratio of EDA+/EDA− fibronectin. In certain embodiments, an antisense oligonucleotide decreases the ratio of EDA+/EDA− fibronectin. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin to create a fibrosis model. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin to create a fibrosis phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis model. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin to create a fibrosis mouse model. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin to create a fibrosis mouse phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis mouse model. In certain embodiments, it is desirable to decrease the ratio of EDA+/EDA− fibronectin in the presence of TGFβ1 to create a fibrosis mouse phenotype.
In certain embodiments, an antisense oligonucleotide alters the ratio of EDA−/EDA+ fibronectin. In certain embodiments, an antisense oligonucleotide increases the ratio of EDA−/EDA+ fibronectin. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin to create a fibrosis model. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin to create a fibrosis phenotype. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis model. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis phenotype. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse model. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse phenotype. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis mouse model. In certain embodiments, it is desirable to increase the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis mouse phenotype.
In certain embodiments, an antisense oligonucleotide alters the ratio of EDA−/EDA+ fibronectin. In certain embodiments, an antisense oligonucleotide decreases the ratio of EDA−/EDA+ fibronectin. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin to create a fibrosis model. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin to create a fibrosis phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis model. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse model. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin to create a fibrosis mouse phenotype. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis mouse model. In certain embodiments, it is desirable to decrease the ratio of EDA−/EDA+ fibronectin in the presence of TGFβ1 to create a fibrosis mouse phenotype.
Certain Pharmaceutical Compositions
In certain embodiments, the present invention provides pharmaceutical compositions comprising one or more antisense compound. In certain embodiments, such pharmaceutical composition comprises a suitable pharmaceutically acceptable diluent or carrier. In certain embodiments, a pharmaceutical composition comprises a sterile saline solution and one or more antisense compound. In certain embodiments, such pharmaceutical composition consists of a sterile saline solution and one or more antisense compound. In certain embodiments, the sterile saline is pharmaceutical grade saline. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and sterile water. In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile water. In certain embodiments, the sterile saline is pharmaceutical grade water. In certain embodiments, a pharmaceutical composition comprises one or more antisense compound and phosphate-buffered saline (PBS). In certain embodiments, a pharmaceutical composition consists of one or more antisense compound and sterile phosphate-buffered saline (PBS). In certain embodiments, the sterile saline is pharmaceutical grade PBS.
In certain embodiments, antisense compounds may be admixed with pharmaceutically acceptable active and/or inert substances for the preparation of pharmaceutical compositions or formulations. Compositions and methods for the formulation of pharmaceutical compositions depend on a number of criteria, including, but not limited to, route of administration, extent of disease, or dose to be administered.
Pharmaceutical compositions comprising antisense compounds encompass any pharmaceutically acceptable salts, esters, or salts of such esters. In certain embodiments, pharmaceutical compositions comprising antisense compounds comprise one or more oligonucleotide which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense compounds, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.
A prodrug can include the incorporation of additional nucleosides at one or both ends of an oligomeric compound which are cleaved by endogenous nucleases within the body, to form the active antisense oligomeric compound.
Lipid moieties have been used in nucleic acid therapies in a variety of methods. In certain such methods, the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids. In certain methods, DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
In certain embodiments, pharmaceutical compositions provided herein comprise one or more modified oligonucleotides and one or more excipients. In certain such embodiments, excipients are selected from water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose and polyvinylpyrrolidone.
In certain embodiments, a pharmaceutical composition provided herein comprises a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those comprising hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
In certain embodiments, a pharmaceutical composition provided herein comprises one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
In certain embodiments, a pharmaceutical composition provided herein comprises a co-solvent system. Certain of such co-solvent systems comprise, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase. In certain embodiments, such co-solvent systems are used for hydrophobic compounds. A non-limiting example of such a co-solvent system is the VPD co-solvent system, which is a solution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol 300. The proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics. Furthermore, the identity of co-solvent components may be varied: for example, other surfactants may be used instead of Polysorbate 80™; the fraction size of polyethylene glycol may be varied; other biocompatible polymers may replace polyethylene glycol, e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides may substitute for dextrose.
In certain embodiments, a pharmaceutical composition provided herein is prepared for oral administration. In certain embodiments, pharmaceutical compositions are prepared for buccal administration.
In certain embodiments, a pharmaceutical composition is prepared for administration by injection (e.g., intravenous, subcutaneous, intramuscular, etc.). In certain of such embodiments, a pharmaceutical composition comprises a carrier and is formulated in aqueous solution, such as water or physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. In certain embodiments, other ingredients are included (e.g., ingredients that aid in solubility or serve as preservatives). In certain embodiments, injectable suspensions are prepared using appropriate liquid carriers, suspending agents and the like. Certain pharmaceutical compositions for injection are presented in unit dosage form, e.g., in ampoules or in multi-dose containers. Certain pharmaceutical compositions for injection are suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, such suspensions may also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions.
In certain embodiments, a pharmaceutical composition is prepared for transmucosal administration. In certain of such embodiments penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
In certain embodiments, a pharmaceutical composition provided herein comprises an oligonucleotide in a therapeutically effective amount. In certain embodiments, the therapeutically effective amount is sufficient to prevent, alleviate or ameliorate symptoms of a disease or to prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.
In certain embodiments, one or more modified oligonucleotide provided herein is formulated as a prodrug. In certain embodiments, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically more active form of an oligonucleotide. In certain embodiments, prodrugs are useful because they are easier to administer than the corresponding active form. For example, in certain instances, a prodrug may be more bioavailable (e.g., through oral administration) than is the corresponding active form. In certain instances, a prodrug may have improved solubility compared to the corresponding active form. In certain embodiments, prodrugs are less water soluble than the corresponding active form. In certain instances, such prodrugs possess superior transmittal across cell membranes, where water solubility is detrimental to mobility. In certain embodiments, a prodrug is an ester. In certain such embodiments, the ester is metabolically hydrolyzed to carboxylic acid upon administration. In certain instances the carboxylic acid containing compound is the corresponding active form. In certain embodiments, a prodrug comprises a short peptide (polyaminoacid) bound to an acid group. In certain of such embodiments, the peptide is cleaved upon administration to form the corresponding active form.
In certain embodiments, the present invention provides compositions and methods for reducing the amount or activity of a target nucleic acid in a cell. In certain embodiments, the cell is in an animal. In certain embodiments, the animal is a mammal. In certain embodiments, the animal is a rodent. In certain embodiments, the animal is a primate. In certain embodiments, the animal is a non-human primate. In certain embodiments, the animal is a human.
In certain embodiments, the present invention provides methods of administering a pharmaceutical composition comprising an oligomeric compound of the present invention to an animal. Suitable administration routes include, but are not limited to, oral, rectal, transmucosal, intestinal, enteral, topical, suppository, through inhalation, intrathecal, intracerebroventricular, intraperitoneal, intranasal, intraocular, intratumoral, and parenteral (e.g., intravenous, intramuscular, intramedullary, and subcutaneous). In certain embodiments, pharmaceutical intrathecals are administered to achieve local rather than systemic exposures. For example, pharmaceutical compositions may be injected directly in the area of desired effect (e.g., into the eyes, ears).
In certain embodiments, a pharmaceutical composition is administered to an animal having at least one symptom associated with fibrosis. In certain embodiments, such administration results in amelioration of at least one symptom. In certain embodiments, administration of a pharmaceutical composition to an animal results in a decrease of EDA+ fibronectin mRNA in a cell of the animal. In certain embodiments, such administration results in an increase in EDA− fibronectin mRNA. In certain embodiments, such administration results in a decrease in EDA+ fibronectin protein and an increase EDA− fibronectin protein. In certain embodiments, a fibronectin protein lacking EDA amino acids is preferred over a fibronectin protein having EDA amino acids. In certain embodiments, the administration of certain antisense oligonucleotides delays the onset of fibrosis. In certain embodiments, the administration of certain antisense oligonucleotides slows the progression of fibrosis. In certain embodiments, the administration of certain antisense oligonucleotides prevents the formation of fibrosis. In certain embodiments, the administration of certain antisense oligonucleotides reverses fibrosis. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular phenotype. In certain embodiments, the administration of certain antisense oligonucleotides rescues cellular morphology.
Nonlimiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references, GenBank accession numbers, and the like recited in the present application is incorporated herein by reference in its entirety.
Although the sequence listing accompanying this filing identifies each sequence as either “RNA” or “DNA” as required, in reality, those sequences may be modified with any combination of chemical modifications. One of skill in the art will readily appreciate that such designation as “RNA” or “DNA” to describe modified oligonucleotides is, in certain instances, arbitrary. For example, an oligonucleotide comprising a nucleoside comprising a 2′-OH sugar moiety and a thymine base could be described as a DNA having a modified sugar (2′-OH for the natural 2′-H of DNA) or as an RNA having a modified base (thymine (methylated uracil) for natural uracil of RNA).
Accordingly, nucleic acid sequences provided herein, including, but not limited to those in the sequence listing, are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to such nucleic acids having modified nucleobases. By way of further example and without limitation, an oligomeric compound having the nucleobase sequence “ATCGATCG” encompasses any oligomeric compounds having such nucleobase sequence, whether modified or unmodified, including, but not limited to, such compounds comprising RNA bases, such as those having sequence “AUCGAUCG” and those having some DNA bases and some RNA bases such as “AUCGATCG” and oligomeric compounds having other modified or naturally occurring bases, such as “ATmeCGAUCG,” wherein meC indicates a cytosine base comprising a methyl group at the 5-position.
EXAMPLESThe following examples illustrate certain embodiments of the present invention and are not limiting. Moreover, where specific embodiments are provided, the inventors have contemplated generic application of those specific embodiments. For example, disclosure of an oligonucleotide having a particular motif provides reasonable support for additional oligonucleotides having the same or similar motif. And, for example, where a particular high-affinity modification appears at a particular position, other high-affinity modifications at the same position are considered suitable, unless otherwise indicated.
Example 1: In Vitro Screening of Human Fibronectin Splicing with Antisense Oligonucleotides in HKC-8 CellsAntisense oligonucleotides were designed targeting a fibronectin nucleic acid and were tested for their effects on the alternative splicing of the fibronectin gene sequence in vitro. The newly designed antisense oligonucleotides in Table 1 were designed as uniform MOE oligonucleotides. Each nucleoside in the oligonucleotide has a 2′-MOE modification. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Stop site” indicates the 3′-most nucleoside to which the oligonucleotide is targeted human gene sequence. Each oligonucleotide listed in Table 1 is targeted to SEQ ID NO: 1 (the complement of GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708). ISIS 141923 (CCTTCCCTGAAGGTTCCTCC (SEQ ID NO: 25), no known human target) was used as a negative control.
Cultured HKC-8 cells, which are SV40-transformed human proximal tubular cells, were transfected using 3 μL LipofectAMINE2000®/mL OptiMEM with 200 nM antisense oligonucleotide. After a treatment period of approximately 4 hours, the medium was removed and new medium was added, left in culture overnight. RNA was isolated from the cells and the ratio of Extra Domain A positive fibronectin (EDA+FN) to EDA negative fibronectin (EDA−FN) was measured by conventional PCR. Human primers with forward sequence GGAGAGAGTCAGCCTCTGGTTCAG, designated herein as SEQ ID NO: 2; reverse sequence TGTCAACTGGGCGCTCAGGCTTGTG, designated herein as SEQ ID NO: 3) was used to measure mRNA levels. To compare the efficacy of antisense treatments performed in different experiments and allow for inter-assay variability, ratios were indexed on the corresponding negative control. Results are presented in Table 1 and demonstrate that treatment with antisense oligonucleotides targeted to the EDA region of fibronectin resulted in decreased expression of the EDA+FN isoform compared to the negative control. ‘null’ indicates that the EDA+ band was undetectable for that sample.
The antisense oligonucleotides described in Example 1 were also tested for their effects on the alternative splicing of the fibronectin gene sequence in primary human proximal tubular cells (PTEC). Cultured PTEC cells were transfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM antisense oligonucleotide for 4 hours, the medium was removed and new medium added, left in culture overnight, and then treated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 0.1% BSA for 24 hrs. RNA was isolated from the cells and levels were measured by conventional PCR. The ratio of EDA+FN to EDA−FN for the given oligonucleotide-treated cells to the ratio for the negative control-treated cells was calculated. Results are presented in Table 2 and indicate that treatment with antisense oligonucleotides targeted to the EDA region of fibronectin resulted in decreased expression of the EDA+FN isoform compared to the negative control, even after induction with TGFβ1. ‘null’ indicates that the EDA+ band was undetectable for that sample.
Antisense oligonucleotides selected from the studies described above were tested for their effects on the alternative splicing of the fibronectin gene sequence in primary human proximal tubular cells (PTEC) treated with TGFβ1. One set of cultured PTEC cells were transfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM antisense oligonucleotide. These cells were treated for 4 hours with antisense oligonucleotide; the medium was removed and new medium added; left in culture overnight; and then treated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 0.1% BSA for 24 hrs. These cells were designated as pre-TGFβ1. Another set of cells were first treated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 01% BSA for 24 hrs; then transfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM antisense oligonucleotide for 4 h; the medium was removed and new medium added; and then left in culture overnight. These cells were designated as post-TGFβ1. RNA was isolated from the cells and levels were measured by conventional PCR. The ratio of EDA+FN to EDA−FN for the given oligonucleotide-treated cells to the ratio for the negative control-treated cells was calculated. Results are presented in Tables 3 and 4, and indicate that treatment with antisense oligonucleotides targeted to the EDA region of fibronectin resulted in decreased expression of the EDA+FN isoform compared to the negative control, irrespective of whether the treatment with antisense oligonucleotides took place before or after induction with TGFβ1 ‘null’ indicates that the EDA+ band was undetectable for that sample.
The antisense oligonucleotides described above were tested for their effects on the alternative splicing of the fibronectin gene sequence in primary human proximal tubular cells (PTEC) treated with TGFβ1. Cultured PTEC cells were transfected using 2 μL LipofectAMINE2000/mL OptiMEM with 100 nM antisense oligonucleotide. The cells were treated for 4 hours with antisense oligonucleotide; the medium was removed and new medium added; left in culture overnight; and then treated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 01% BSA for 48 hrs. The cells were lysed in lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X, 0.5% sodium deoxycholate, 0.1% SDS; pH 7.2) and protein was extracted as described in Phanish M K et al., Biochem J. 2006 Jan. 15; 393(Pt 2):601-7. The protein samples were run on an SDS-PAGE and analyzed via western analysis using the anti-Fibronectin antibody [IST-9] (Abcam, ab6328) that reacts with an epitope located in the ED-A sequence of cellular fibronectin. Results are presented in Table 5, and indicate that treatment with antisense oligonucleotides targeted to the EDA region of fibronectin resulted in decreased protein expression of the EDA+FN isoform compared to the negative control.
ISIS 511403 was tested for its effect on the alternative splicing of the fibronectin gene sequence in primary human proximal tubular cells (PTEC) treated with TGFβ1. Cultured PTEC cells were treated with 0.1% BSA (vehicle) or with 2.5 ng/mL TGFβ1 in 01% BSA for 24 hrs and then transfected using 2 μL LipofectAMINE2000®/mL OptiMEM with 100 nM ISIS 511403 or 100 nM ISIS 141923 for 24 h. After a recovery period of 24 h in normal growth medium, RNA was isolated and the ratio of EDA+FN to EDA□FN was measured by conventional PCR. In addition, the individual expressions of EDA+FN and EDA−□FN normalized to 18s RNA were also measured (human primer probe set for 18S: forward sequence: GTAACCCGTTGAACCCCATT (SEQ ID NO: 26), reverse sequence: CCATCCAATCGGTAGTAGCG (SEQ ID NO: 27)). In addition, the expression of total fibronectin was also measured by quantitative real-time PCR (probe set Hs01549940_m1, Applied Biosystems). The results are presented in Table 6 and indicate that treatment with ISIS 511403 decreased the ratio of EDA+FN to EDA−FN, decreased expression of EDA+FN, increased expression of EDA−FN, and had no effect on total fibronectin expression compared to that of the negative control cells.
The effect of treatment with ISIS 511403 on lactate dehydrogenase (LDH) release by the cells was also measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (Promega, G1780). The results are presented in Table 7 and indicate the decrease in LDH release in cells treated with ISIS 511403 compared to the cells treated with the negative control. This demonstrates that ISIS 511403 can rescue certain pronounced changes in cell phenotype caused by TGFβ1 induction, e.g. the release of LDH.
The effect of treatment with ISIS 511403 on αSMA mRNA expression by the cells was also measured by quantitative real-time PCR, using primer probe set Hs00909449_m1 (Applied Biosystems). The results are presented in Table 8 and indicate the decrease in αSMA in cells treated with ISIS 511403 compared to the cells treated with the negative control. The numbers in parentheses indicate the range. This demonstrates that ISIS 511403 can rescue certain pronounced changes in cell phenotype caused by TGFβ1 induction, e.g. the induction of αSMA.
The data presented in Tables 7 and 8 indicate that treatment with antisense oligonucleotides inhibiting the splicing and inclusion of the EDA region of fibronectin resulted in decreased fibrosis in primary human PTEC and therefore have therapeutic benefit in the prevention, treatment, or amelioration of fibrosis.
Additionally, by staining it was observed that prevention of EDA inclusion by treatment with ISIS 511403 resulted in a significant reduction in αSMA compared to treatment with a control (ISIS 141923). By western blot analysis, it was also observed that prevention of EDA inclusion by treatment with ISIS 511403 resulted in reduction in secretion of MMP2 & MMP9. The fold-change in the cell motility marker, S 100A4 was measured and is presented in Table 9. Treatment with ISIS 511403 resulted in significant reduction in S100A4 in TGF-β-treated cells.
By staining and by western blot analysis, it was also observed that prevention of EDA inclusion by treatment with ISIS 511403 resulted in near complete inhibition of Connexin 43. By staining, it was also observed that prevention of EDA inclusion by treatment with ISIS 511403 resulted in a moderate increase in f-actin localization.
Example 6: Design of Antisense Oligonucleotides Targeting Human and Murine FibronectinAntisense oligonucleotides were designed targeting a fibronectin nucleic acid. The newly designed chimeric antisense oligonucleotides in Table 10 were designed as uniform MOE oligonucleotides. Each nucleoside in the oligonucleotide has a 2′-MOE modification. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Human Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Murine Start Site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted murine gene sequence. Each oligonucleotide listed in Table 10 is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 (the complement of GENBANK Accession No. NT_005403.14 truncated from nucleotides 66434501 to 66510708) or to murine fibronectin genomic sequence, SEQ ID NO: 29 (the complement of Accession No. NT_039170.2 truncated from nucleotides 20696091 to 20764741), or both. Several of the oligonucleotides are cross-reactive with human and mouse gene sequences. The greater the complementarity between the oligonucleotide and the gene sequence, the more likely the oligonucleotide can target the gene sequence. ‘Mismatches’ indicates the number of nucleotides in the oligonucleotide that are mismatched with the gene sequence. ‘n/a’ indicates that the oligonucleotide contains more than 2 mismatches with the particular gene sequence.
Some of the antisense oligonucleotides presented in Example 6 were tested for potency in cultured primary PTEC cells. ISIS 511403 was also included in the study.
Cultured PTEC cells were transfected using 2 μL LipofectAMINE2000/mL OptiMEM with 100 nM antisense oligonucleotide for 4 hours, the medium was removed and new medium added, left in culture overnight, and then treated with 0.1% BSA (vehicle) or 2.5 ng/mL TGFβ1 in 0.1% BSA for 24 hrs. RNA was isolated from the cells and levels of EDA+FN mRNA were measured by RT-PCR using primer probe sets RTS3963_MGB (forward sequence GCCTTGCACGATGATATGGA, designated herein as SEQ ID NO: 91; reverse sequence TGTGGGTGTGACCTGAGTGAA, designated herein as SEQ ID NO: 92; probe sequence ATTGGAACCCAGTCCAC, designated herein as SEQ ID NO: 93), as well as with primer probe set RTS3964 (forward sequence GAATCCAAGCGGAGAGAGTCA, designated herein as SEQ ID NO: 94; reverse sequence ACATCAGTGAATGCCAGTCCTTT, designated herein as SEQ ID NO: 95; probe sequence TTCAGACTGCAGTAACCAACATTGATCGCC, designated herein as SEQ ID NO: 96), both of which are designed to the EDA+ variant of the FN mRNA transcript (NM_212478.1, designated herein as SEQ ID NO: 97) and which target different regions of the transcript. For data analysis, the levels of EDA+FN mRNA were normalized to the levels of the house-keeping gene, the large ribosomal protein transcript (Human RPLPO, Applied Biosystems, cat#4333761F). For each antisense oligonucleotide, the ratio of EDA+FN to RPLPO in antisense oligonucleotide-treated cells was then normalized to the ratio of EDA+FN mRNA to RPLPO in untreated cells. The results are presented in Table 11. The results indicate that treatment with antisense oligonucleotides reduced expression of the EDA+ transcript compared to untreated cells, both in the presence or absence of TGFβ1.
Antisense oligonucleotides were designed targeting a fibronectin nucleic acid and were tested for their effects on blocking of splicing in vitro. The newly designed chimeric antisense oligonucleotides in Table 12 were designed as uniform MOE oligonucleotides. Each oligonucleotide is 15 nucleosides long and each nucleoside in the oligonucleotide has a 2′-MOE modification. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Human Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Murine Start Site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted murine gene sequence. Each oligonucleotide listed in Table 12 is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 or to murine fibronectin genomic sequence, SEQ ID NO: 22, or both. Several of the oligonucleotides are cross-reactive with human and mouse gene sequences. The greater the complementarity between the oligonucleotide and the gene sequence, the more likely the oligonucleotide can target the gene sequence. ‘Mismatches’ indicates the least number of nucleotides in the oligonucleotide that are mismatched with the gene sequence; the antisense oligonucleotide may target the gene sequence with more mismatches. ‘n/a’ indicates that the antisense oligonucleotide has more than 3 mismatches with the particular gene sequence.
Cultured MHT cells, a mouse hepatocellular carcinoma cell line (Koller, E. et al., Nucleic Acids Research, 2011, 1-13), were transfected using 5 μl LipofectAMINE2000®/mL OptiMEM with 50 nM antisense oligonucleotide. After a treatment period of approximately 4 hours, the medium was removed and new medium was added, and the cells were left in culture overnight. RNA was isolated from the cells and measured by RT-PCR. EDA+FN mRNA expression was measured with mouse primer probe set LTS01050 (forward sequence AAACTGCAGTGACCAACATTGATC, designated herein as SEQ ID NO: 98; reverse sequence CTTGCCCCTGTGGGCTTT, designated herein as SEQ ID NO: 99; probe sequence CTGATGTGGATGTCGATT, designated herein as SEQ ID NO: 100), as well as with LTS01052 (forward sequence GCCAGCCCCTGATTGGA, designated herein as SEQ ID NO: 101; reverse sequence CCGGTAGCCAGTGAGCTGAA, designated herein as SEQ ID NO: 102; probe sequence CACCAATCTGAAGTTC, designated herein as SEQ ID NO: 103). The primer probe sets target different regions of the mouse sequence.
Results are presented in Table 12 and are the average of the values measured in three separate experiments. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value of untreated cells was taken as 1.00. ‘n.d.’ indicates that the mRNA expression level values were not considered because the oligonucleotide targeted an amplicon region of the specific primer probe set.
Additional antisense oligonucleotides were designed based on the ISIS oligonucleotides that demonstrated significant effect on fibronectin splicing in the studies described above. These oligonucleotides were designed by creating oligonucleotides shifted slightly upstream and downstream (i.e. “microwalk”) of ISIS 511417, ISIS 594685, ISIS 594686, ISIS 594686, ISIS 594687, ISIS 594688, ISIS 594689, ISIS 594690, ISIS 594691, and ISIS 598145. The newly designed antisense oligonucleotides in Tables 13 and 14 were designed as uniform MOE oligonucleotides. Each oligonucleotide is 18 nucleosides long and each nucleoside in the oligonucleotide has a 2′-MOE modification. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. The oligonucleotides are presented in the tables below. “Human Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Murine Start Site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted murine gene sequence. Each oligonucleotide listed in the tables is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 or to murine fibronectin genomic sequence, SEQ ID NO: 29, or both. Several of the oligonucleotides are cross-reactive with human and mouse gene sequences. The greater the complementarity between the oligonucleotide and the gene sequence, the more likely the oligonucleotide can target the gene sequence. ‘Mismatches’ indicates the least number of nucleotides in the oligonucleotide that are mismatched with the gene sequence; the antisense oligonucleotide may target the gene sequence with more mismatches. ‘n/a’ indicates that the antisense oligonucleotide has more than 3 mismatches with the particular gene sequence. Cultured MHT cells were transfected using 5 μl LipofectAMINE2000®/mL OptiMEM with 10 nM antisense oligonucleotide. After a treatment period of approximately 4 hours, the medium was removed and new medium was added, left in culture overnight. RNA was isolated from the cells and measured by RT-PCR. EDA+FN mRNA expression was measured with mouse primer probe set LTS01050, as well as with LTS01052.
Results are presented in Tables 13 and 14, and are the average of the values measured in three separate experiments. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value of untreated cells was taken as 1.00. ‘n.d.’ indicates that the mRNA expression level values were not considered because the oligonucleotide targeted an amplicon region of the specific primer probe set.
Antisense oligonucleotides were designed targeting a fibronectin nucleic acid and were tested for their effects on blocking of fibronectin splicing in vitro. ISIS 606793 was also included in the study. The newly designed antisense oligonucleotides in Tables 15-22 were designed as deoxy and (S)-cEt oligonucleotides. Each nucleoside in the oligonucleotide has a 2′-MOE, deoxy, or (S)-cEt modification, as presented in the Chemistry column of the tables. ‘e’ indicates MOE; ‘k’ indicates (S)-cEt; ‘d’ indicates deoxy modifications. The internucleoside linkages throughout each oligonucleotide are phosphorothioate (P═S) linkages. All cytosine residues throughout each oligonucleotide are 5-methylcytosines. “Human Start site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted in the human gene sequence. “Murine Start Site” indicates the 5′-most nucleoside to which the oligonucleotide is targeted murine gene sequence. Each oligonucleotide listed in Tables 15-22 is targeted to the human fibronectin genomic sequence, SEQ ID NO: 1 or to murine fibronectin genomic sequence, SEQ ID NO: 29, or both. Several of the oligonucleotides are cross-reactive with human and mouse gene sequences. The greater the complementarity between the oligonucleotide and the gene sequence, the more likely the oligonucleotide can target the gene sequence. ‘Mismatches’ indicates the least number of nucleotides in the oligonucleotide that are mismatched with the gene sequence; the antisense oligonucleotide may target the gene sequence with more mismatches. ‘n/a’ indicates that the antisense oligonucleotide has more than 3 mismatches with the particular gene sequence.
Cultured b.END cells were transfected using 2 μl Cytofectin/mL with 3 nM antisense oligonucleotide. After a treatment period of approximately 4 hours, the medium was removed and new medium was added, left in culture overnight. RNA was isolated from the cells and measured by RT-PCR. EDA+FN mRNA expression was measured with mouse primer probe set LTS01050, as well as with LTS01052.
Results are presented in Tables 15-22, and are the average of the values measured in three separate experiments. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value of untreated cells was taken as 1.00. ‘n.d.’ indicates that the mRNA expression level values were not considered because the oligonucleotide targeted an amplicon region of the specific primer probe set.
Antisense oligonucleotides from the studies described above exhibiting significant in vitro inhibition of EDA+FN mRNA were selected and tested at various doses in b.END cells. Cells were transfected using Cytofectin reagent with 0.19 nM, 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, or 6.25 nM concentrations of antisense oligonucleotide, as specified in Tables 23-27. After a treatment period of approximately 16 hours, RNA was isolated from the cells and EDA+FN mRNA levels were measured by quantitative real-time PCR. Primer probe sets LTS01050 and LTS01052 were used to measure mRNA levels. EDA+FN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value of untreated cells was taken as 1.00. Different primer probe sets were used for different antisense oligonucleotide-treated cells to avoid the amplicon effect. Each table represents a separate experiment.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Tables 23-27. As illustrated in the tables, EDA+FN mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
Antisense oligonucleotides from the studies described above exhibiting significant in vitro inhibition of EDA+FN mRNA were selected and tested at various doses in b.END cells. Cells were transfected using Cytofectin reagent with 0.39 nM, 0.78 nM, 1.56 nM, 3.125 nM, 6.25 nM or 12.5 nM concentrations of antisense oligonucleotide, as specified in Tables 28-31. After a treatment period of approximately 16 hours, RNA was isolated from the cells and EDA+FN mRNA levels were measured by quantitative real-time PCR. Primer probe sets LTS01050 and LTS01052 were used to measure mRNA levels. EDA+FN mRNA levels were adjusted according to total RNA content, as measured by RIBOGREEN®. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value of untreated cells was taken as 1.00. Different primer probe sets were used for different antisense oligonucleotide-treated cells to avoid the amplicon effect. Each table represents a separate experiment.
The half maximal inhibitory concentration (IC50) of each oligonucleotide is also presented in Tables 28-31. As illustrated in the tables, EDA+FN mRNA levels were significantly reduced in a dose-dependent manner in antisense oligonucleotide treated cells.
C57BL/6 mice are a multipurpose mice model, frequently utilized for safety and efficacy testing. The mice were treated with ISIS antisense oligonucleotides selected from studies described above and evaluated for efficacy, as well as changes in the levels of various plasma chemistry markers.
Study with Uniform MOE Oligonucleotides
TreatmentGroups of eight-week old C57BL/6 mice were injected subcutaneously twice a week for 3 weeks with 100 mg/kg of ISIS 594675, ISIS 598145, ISIS 598151, ISIS 598153, ISIS 598163, ISIS 606770, ISIS 606785, ISIS 606787, ISIS 606788, ISIS 606793, ISIS 606804, or ISIS 606812. One group of eight-week old C57BL/6 mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
RNA AnalysisTo evaluate the effect of ISIS oligonucleotides on blocking fibronectin splicing, mRNA levels of EDA+FN were measured by RT-PCR using mouse primer probe set LTS01050 and LTS01052. The results are presented in Table 32, normalized to total fibronectin. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value in untreated mice was taken as 1.00. ‘n.d.’ indicates that the mRNA expression level values were not considered because the oligonucleotide targeted an amplicon region of the specific primer probe set.
To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 33. ISIS oligonucleotides did not cause any changes in the levels of any of the liver or kidney function markers outside the expected range for antisense oligonucleotides.
Liver, spleen and kidney weights were measured at the end of the study, and are presented in Table 34. ISIS oligonucleotides did not cause any changes in organ weights outside the expected range for antisense oligonucleotides.
Study with Deoxy, (S)-cEt and MOE Oligonucleotides
Groups of eight-week old C57BL/6 mice were injected subcutaneously twice a week for 3 weeks with 100 mg/kg of ISIS 607149, ISIS 607186, ISIS 607204, ISIS 607205, ISIS 607207, ISIS 607277, ISIS 607285, ISIS 607286, ISIS 607330, ISIS 607332, ISIS 607341, ISIS 607352, ISIS 607428, or ISIS 607429. One group of eight-week old C57BL/6 mice was injected subcutaneously twice a week for 3 weeks with PBS. Mice were euthanized 48 hours after the last dose, and organs and plasma were harvested for further analysis.
RNA AnalysisTo evaluate the effect of ISIS oligonucleotides on blocking fibronectin splicing, mRNA levels of EDA+FN were measured by RT-PCR using mouse primer probe set LTS01050 and LTS01052. The results are presented in Table 35, normalized to total fibronectin. The results demonstrate blocking of splicing, as represented by EDA+FN expression. The expression value in untreated mice was taken as 1.00. ‘n.d.’ indicates that the mRNA expression level values were not considered because the oligonucleotide targeted an amplicon region of the specific primer probe set.
To evaluate the effect of ISIS oligonucleotides on liver and kidney function, plasma levels of transaminases, bilirubin, albumin, and BUN were measured using an automated clinical chemistry analyzer (Hitachi Olympus AU400e, Melville, N.Y.). The results are presented in Table 36. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.
Liver, spleen and kidney weights were measured at the end of the study, and are presented in Table 37. ISIS oligonucleotides that caused any changes in organ weights outside the expected range for antisense oligonucleotides were excluded from further studies.
Claims
1. A compound comprising a modified oligonucleotide consisting of 18 to 20 linked nucleosides and having a nucleobase sequence comprising a complementary region comprising at least 8 contiguous nucleobases complementary to a target region of equal length of a fibronectin transcript, wherein the target region is within nucleobase 55441 and nucleobase 55790 of SEQ ID NO: 1.
2. The compound of claim 1, wherein the complementary region of the modified oligonucleotide is 100% complementary to the target region.
3-118. (canceled)
119. The compound of claim 1, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.
120. The compound of claim 119, comprising at least one phosphorothioate internucleoside linkage.
121. The compound of claim 1, wherein the modified oligonucleotide comprises at least one 2′-substituted sugar moiety.
122. The compound of claim 121, wherein the 2′-substituted sugar moiety is selected from a group consisting of 2′-MOE, 2′-OMe, and 2′-F.
123. The compound of claim 121, wherein the 2′-substituted sugar moiety is a 2′-MOE.
124. A pharmaceutical composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier or diluent.
Type: Application
Filed: Oct 27, 2017
Publication Date: May 10, 2018
Applicant: Ionis Pharmaceuticals, Inc. (Carlsbad, CA)
Inventors: Susan M. Freier (San Diego, CA), Frank Rigo (Carlsbad, CA)
Application Number: 15/795,663
