NZ624471B2 - Modified rnai agents - Google Patents

Modified rnai agents Download PDF

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Publication number
NZ624471B2
NZ624471B2 NZ624471A NZ62447112A NZ624471B2 NZ 624471 B2 NZ624471 B2 NZ 624471B2 NZ 624471 A NZ624471 A NZ 624471A NZ 62447112 A NZ62447112 A NZ 62447112A NZ 624471 B2 NZ624471 B2 NZ 624471B2
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kjm
annotation
nucleotides
strand
double
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NZ624471A
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NZ624471A (en
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Klaus Charisse
Satyanarayana Kuchimanchi
Martin Maier
Muthiah Manoharan
Kallanthottathil G Rajeev
Tracy Zimmermann
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Alnylam Pharmaceuticals Inc
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Priority claimed from PCT/US2012/065601 external-priority patent/WO2013074974A2/en
Publication of NZ624471A publication Critical patent/NZ624471A/en
Publication of NZ624471B2 publication Critical patent/NZ624471B2/en

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Abstract

Disclosed is a double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene. The dsRNA duplex comprises one or more motifs of three identical modifications on three consecutive nucleotides in one or both strand, particularly at or near the cleavage site of the strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents and the use of such dsRNA agents in the manufacture of a medicament for delivering a polynucleotide to a specific target in a subject. nd. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents and the use of such dsRNA agents in the manufacture of a medicament for delivering a polynucleotide to a specific target in a subject.

Description

[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Modified RNAi Agents RELATED APPLICATION This application claims priority to U.S. Provisional Application No. 61/561,710, filed on November 18, 201 1, the entire contents of which are hereby incorporated herein by reference.
FIELD OF THE INVENTION The ion relates to RNAi duplex agents having particular motifs that are advantageous for tion of target gene expression, as well as RNAi compositions suitable for therapeutic use. Additionally, the invention provides methods of ting the expression of a target gene by administering these RNAi duplex agents, e.g., for the treatment of various diseases.
BACKGROUND RNA interference or "RNAi" is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNAi (dsRNA) can block gene expression (Fire et al. (1998) Nature 391, 806-81 1; Elbashir et al. (2001) Genes Dev. 15, 188-200).
Short dsRNA s gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi is mediated by duced silencing complex (RISC), a sequence-specific, multicomponent se that destroys messenger RNAs homologous to the silencing r.
RISC is known to contain short RNAs (approximately 22 nucleotides) derived from the -stranded RNA r, but the protein components of this activity remained unknown.
Double-stranded RNA (dsRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA induced silencing x (RISC), which requires degradation of the sense strand of the dsRNA duplex. Sense strand was known to act as the first RISC substrate that is cleaved by Argonaute 2 in the middle of the duplex region Immediately after the d 5'-end and 3'-end fragments of the sense strand are removed from the endonuclease Ago2, the RISC becomes ted by the nse strand (Rand et al. (2005) Cell 123, 621).
It was believed that when the cleavage of the sense strand is inhibited, the endonucleo lytic cleavage of target mRNA is impaired (Leuschner et al. (2006) EMBO Rep., 7, 314; Rand et al. (2005) Cell 123, 621; Schwarz et al. (2004) Curr. Biol. 14, 787).
Leuschner et al. showed that incorporation of a 2'Me ribose to the Ago2 cleavage site in the sense strand inhibits RNAi in HeLa cells (Leuschner et al. (2006) EMBO Rep., 7, 314). A similar effect was observed with phosphorothioate modifications, showing that cleavage of the sense strand was required for efficient RNAi also in mammals.
Morrissey et al. used a siRNA duplex containing 2'-F modified residues, among other sites and modifications, also at the Ago2 cleavage site, and obtained compatible silencing compared to the unmodified siRNAs (Morrissey et al. (2005) Hepatology 41, 1349). However, Morrissey's modification is not motif specific, e.g., one modification includes 2'-F modifications on all pyrimidines on both sense and antisense strands as long as pyrimidine residue is present, without any selectivity; and hence it is uncertain, based on these teachings, if specific motif cation at the cleavage site of sense strand can have any actual effect on gene ing acitivity.
Muhonen et al. used a siRNA duplex containing two 2'-F modified residues at the Ago2 cleavage site on the sense or antisense strand and found it was ted (Muhonen et al. (2007) Chemistry & Biodiversity 4, 858-873). r, Muhonen's modification is also sequence specific, e.g., for each particular strand, Muhonen only modifies either all pyrimidines or all purines, without any selectivity.
Choung et al. used a siRNA duplex containing ative modifications by 2'- OMe or various combinations of 2'-F, 2'-OMe and phosphorothioate modifications to stabilize siRNA in serum to Surl0058 (Choung et al. (2006) Biochemical and Biophysical Research Communications 342, 7). Choung suggested that the residues at the ge site of the antisense strand should not be modified with 2'-OMe in order to increase the stability of the siRNA.
There is thus an g need for iRNA duplex agents to improve the gene ing efficacy of siRNA gene therapeutics. This invention is directed to that need.
SUMMARY In one aspect, the invention provides a -stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the duplex is represented by formula (III): sense: 5' np -Na -(X X X)i-Nb -Y Y Y -Nb -(Z Z Z)j -Na - nq 3' antisense: 3' np’-Na’-(X’X′X′)k-Nb’-Y′Y′Y′-Nb’-(Z′Z′Z′)l-Na’- nq’ 5' (III) wherein: i, j, k, and l are each ndently 0 or 1; p and q are each independently 0-6; each Na and Na’independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each ce comprising at least two differently modified nucleotides, each Nb and Nb’independently ents an oligonucleotide ce comprising 1-10 modified nucleotides; each np, np’, nq and nq’ independently represents an overhang tide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; wherein the modification on Nb is different than the modification on Y and the cation on Nb’is different than the modification on Y’; and n the Y’Y’Y’ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5’- end.
In another aspect, the present invention provides a double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, one of said motifs ing at the cleavage site in the strand and at least one of said motifs occurring at another portion of the strand that is separated from the motif at the cleavage site by at least one ed nucleotide, wherein the modification on the at least one modified nucleotide is different than the modification on the three consecutive modified nucleotides; and wherein the antisense strand contains at least a first motif of three identical modifications on three consecutive nucleotides, one of said motifs occurring at or near the cleavage site in the strand and a second motif occurring at another portion of the strand that is separated from the first motif by at least one modified nucleotide, wherein the modification on the at least one modified nucleotide is different than the modification on the three consecutive modified nucleotides; wherein the modification in the motif occurring at the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand; and wherein the first or the second motif in the antisense strand occurs at the 11, 12 and 13 positions of the antisense strand from the 5’-end.
In another aspect, the present invention provides a double-stranded RNAi agent capable of inhibiting the sion of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three utive nucleotides, one of said motifs occurring at or near the cleavage site in the strand, and wherein a polynucleotide adjacent to the three consecutive nucleotides is a modified nucleotide having a modification other than a 2’-F modification; and wherein the nse strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides, one of said motifs occurring at or near the cleavage site, and wherein a cleotide adjacent to the three consecutive nucleotides is a modified nucleotide having a modification other than a 2’-O-methyl modification; and wherein one of the at least one motif in the antisense strand occurs at the 11, 12 and 13 positions of the nse strand from the ’-end.
In another , the present invention provides a pharmaceutical composition sing the -stranded RNAi agent according to the invention alone or in combination with a pharmaceutically acceptable carrier or excipient.
In another aspect, the present invention provides the use of the double-stranded RNAi agent ing to the invention in the manufacture of a medicament for inhibiting the expression of a target gene.
In r aspect, the present invention provides the use of a dsRNA agent represented by formula (III): sense: 5' np -Na -(X X X) i-Nb -Y Y Y -Nb -(Z Z Z)j -Na - nq 3' antisense: 3' np’-Na’-(X’X′X′)k-Nb’-Y′Y′Y′-Nb’-(Z′Z′Z′)l-Na’- nq’ 5' (III) i, j, k, and l are each independently 0 or 1; p and q are each independently 0-6; each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified tides; each Nb and Nb′ ndently represents an oligonucleotide sequence comprising 1-10 modified nucleotides; each np, np’, nq and nq’ independently represents an overhang tide sequence comprising 0-6 nucleotide sequence; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; and n the modifications on Nb is different than the modification on Y and the modification on Nb’ is different than the modification on Y’; and wherein the Y’Y’Y’ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5’-end in the manufacture of a medicament for delivering a cleotide to a ic target in a subject.
In r aspect, the present invention provides the use of the dsRNAi agent of the invention in the manufacture of a medicament for delivering a cleotide to a specific target of a subject, wherein the ment is provided for subcutaneous administration.
This invention provides effective nucleotide or chemical motifs for dsRNA agents optionally conjugated to at least one ligand, which are advantageous for inhibition of target gene sion, as well as RNAi compositions suitable for therapeutic use.
The inventors surprisingly discovered that introducing one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of a dsRNA agent that is comprised of ed sense and antisense strands enhances the gene silencing activity of the dsRNA agent.
In one aspect, the invention relates to a double-stranded RNAi (dsRNA) agent capable of inhibiting the sion of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The dsRNA duplex is represented by formula (III): sense: 5' np -Na -(X X X) i-Nb -Y Y Y -Nb -(Z Z Z)j -Na - nq 3' antisense: 3' np’-Na’-(X’X′X′)k-Nb’-Y′Y′Y′-Nb’-(Z′Z′Z′)l-Na’- nq’ 5' (III), In a (III), i, j, k, and l are each independently 0 or 1; p and q are each independently 0-6; n represents a nucleotide; each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides; each Nb and Nb′ independently represents an ucleotide sequence comprising 0-10 nucleotides which are either modified or unmodified or combinations thereof; each np and nq independently represents an overhang nucleotide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; wherein the modifications on Nb is ent than the modification on Y and the modifications on Nb’ is different than the modification on Y’. At least one of the Y nucleotides forms a base pair with its complementary Y’ nucleotides, and wherein the modification on the Y tide is ent than the modification on the Y’ nucleotide.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Each np and nq independently represents an overhang nucleotide sequence comprising 0-6 nucleotides; each n and n ' represents an overhang nucleotide; and p and q are each independently 0-6.
In another aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent ses a sense strand and an nse strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one nucleotide. The antisense strand contains at least one motif of three cal modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site within the strand and at least one of the motifs occurs at another portion of the strand that is ted from the motif at or near cleavage site by at least one nucleotide. The cation in the motif occurring at or near the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand.
In another , the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand ns at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand. The antisense strand contains at least one motif of three 2'methyl cations on three consecutive nucleotides at or near the cleavage site.
In r aspect, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense , each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9,10,1 1 from the 5'end. The antisense strand contains at least one motif of three 2'methyl modifications on three consecutive tides at positions 11,12,13 from the 5'end.
[Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In another , the invention further provides a method for delivering dsRNA to a ic target in a t by subcutaneous or intravenenuous administration.
DETAILED DESCRIPTION A superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand and/or antisense strand of a dsRNA agent, particularly at or near the ge site. The sense strand and antisense strand of the dsRNA agent may otherwise be completely modified.
The introduction of these motifs interrupts the modification pattern, if present, of the sense and/or antisense strand. The dsRNA agent optionally conjugates with a GalNAc derivative ligand, for instance on the sense strand. The resulting dsRNA agents present superior gene ing activity.
The inventors surprisingly discovered that having one or more motifs of three identical modifications on three consecutive nucleotides at or near the ge site of at least one strand of a dsRNA agent superiorly ed the gene silencing acitivity of the dsRNA agent.
Accordingly, the invention provides a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand. Each strand of the dsRNA agent can range from 12- nucleotides in length. For example, each strand can be between 14-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in , 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
The sense strand and antisense strand typically form a duplex dsRNA. The duplex region of a dsRNA agent may be 12-30 nucleotide pairs in length. For example, the duplex region can be between 14-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-30 nucleotides in , 27-30 nucleotide pairs in , 17 - 23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 tide pairs in length, 19-25 nucleotide pairs in , 19-23 nucleotide pairs in length, 19- 2 1 nucleotide pairs in [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another e, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27.
In one embodiment, the dsRNA agent of the invention comprises may contain one or more overhang regions and/or capping groups of dsRNA agent at the 3'-end, or 5'-end or both ends of a . The ng can be 1-6 nucleotides in length, for ce 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in , 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mR A or it can be mentary to the gene ces being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
In one embodiment, the nucleotides in the overhang region of the dsRNA agent of the invention can each independently be a modified or unmodified nucleotide including, but no limited to 2'-sugar modified, such as, 2-F 2'-Omethyl, thymidine (T), 2' methoxyethylmethyluridine (Teo), 2'methoxyethyladenosine (Aeo), 2' methoxyethylmethylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being ed or can be other ce.
The 5'- or 3'- overhangs at the sense , antisense strand or both strands of the dsRNA agent of the invention may be phosphorylated. In some embodiments, the overhang region contains two tides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3'-end of the sense strand, antisense strand or both strands.
In one embodiment, this 3'-overhang is present in the antisense strand. In one embodiment, this 3'-overhang is present in the sense strand.
The dsRNA agent of the invention comprises only single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall ity.
For example, the -stranded overhang is located at the 3'-terminal end ofthe sense [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm strand or, alternatively, at the 3'-terminal end of the antisense strand. The dsRNA may also have a blunt end, d at the 5'-end of the antisense strand (or the 3'-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3'-end, and the 5'-endis blunt. While not bound by theory, the asymmetric blunt end at the 5'-end of the antisense strand and 3'-end overhang of the antisense strand favor the guide strand loading into RISC process.
In one embodiment, the dsRNA agent of the invention may also have two blunt ends, at both ends of the dsRNA duplex.
In one embodiment, the dsRNA agent of the ion is a double ended er of 19 nt in length, wherein the sense strand contains at least one motif of three 2'-F cations on three consecutive nucleotides at ons 7,8,9 from the 5'end. The antisense strand ns at least one motif of three 2'methyl modifications on three consecutive tides at positions 11,12,13 from the 5'end.
In one embodiment, the dsRNA agent of the invention is a double ended bluntmer of 20 nt in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 8,9,10 from the 5'end. The antisense strand contains at least one motif of three 2'methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5'end.
In one embodiment, the dsRNA agent of the ion is a double ended bluntmer of 2 1 nt in length, wherein the sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides at positions 9,10,1 1 from the 5'end. The antisense strand contains at least one motif of three 2'methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 5'end.
In one embodiment, the dsRNA agent of the invention comprises a 2 1 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the sense strand contains at least one motif of three 2'-F cations on three consecutive nucleotides at positions 9,10,1 1 from the 5'end; the antisense strand contains at least one motif of three 2' methyl modifications on three consecutive nucleotides at positions 11,12,13 from the 'end, wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang. Preferably, the 2 nt overhang is at the 3'-end of the antisense. Optionally, the dsRNA further comprises a ligand (preferably GalNAc ) .
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In one embodiment, the dsR A agent of the ion comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5'terminal nucleotide (position 1) positions 1 to 23 of said first strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3'terminal nucleotide, comprises at least 8 cleotides in the positions paired with positions 1- 23 of sense strand to form a duplex; wherein at least the 3 ' terminal nucleotide of antisense strand is unpaired with sense , and up to 6 consecutive 3'terminal nucleotides are ed with sense strand, thereby forming a 3' single stranded overhang of 1-6 nucleotides; wherein the inus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5'overhang; wherein at least the sense strand 5'terminal and 3'terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum mentarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently mentary to a target R A along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is uced into a ian cell; and wherein the sense strand contains at least one motif of three 2'-F cations on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2'methyl modifications on three consecutive nucleotides at or near the cleavage site.
In one embodiment, the dsRNA agent of the invention sing a sense and antisense s, wherein said dsRNA agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2'methyl modifications on three consecutive nucleotides at position 11,12,13 from the 5' end; wherein said 3' end of said first strand and said 5' end of said second strand form a blunt end and said second strand is 1-4 nucleotides longer at its 3' end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and said second strand is sufficiently complemenatary to a target mR A along at least 19 nt of said second strand length to reduce target gene expression when said dsRNA agent is ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm introduced into a mammalian cell, and wherein dicer cleavage of said dsR A preferentially s in an siR A comprising said 3' end of said second strand, thereby reducing expression of the target gene in the mammal. ally, the dsRNA agent further comprises a ligand.
In one embodiment, the sense strand of the dsRNA agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.
In one embodiment, the antisense strand of the dsRNA agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the ge site in the antisense strand.
For dsRNA agent having a duplex region of 17-23 nt in length, the cleavage site of the nse strand is typically around the 10, 11 and 12 positions from the 5'-end.
Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1st nucleotide from the 5'-end of the nse strand, or, the count starting from the 1st paired nucleotide within the duplex region from the 5'- end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNA from the 5'- end.
The sense strand of the dsRNA agent comprises at least one motif of three cal modifications on three consecutive nucleotides at the cleavage site of the ; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the ge site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the nse strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand.
Alternatively, at least two nucleotides of the motifs from both strands may overlap, or all three nucleotides may overlap.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In one embodiment, the sense strand of the dsR A agent comprises more than one motif of three identical modifications on three consecutive tides. The first motif should occur at or near the cleavage site of the strand and the other motifs may be a wing modifications. The term "wing modification" herein refers to a motif occurring at r portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adajacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide the chemistries of the motifs can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, the wing modifications may both occur at one end of the duplex region relative to the first motif which is at or near the cleavage site or each of the wing cations may occur on either side of the first motif.
Like the sense strand, the antisense strand of the dsRNA agent comprises at least two motifs of three cal modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that is present on the sense .
In one embodiment, the wing modification on the sense strand, antisense strand, or both strands of the dsRNA agent typically does not include the first one or two terminal nucleotides at the 3'-end, 5'-end or bothends of the .
In r embodiment, the wing modification on the sense strand, nse strand, or both strands of the dsRNA agent typically does not include the first one or two paired nucleotides within the duplex region at the 3'-end, 5'-end or bothends of the strand.
When the sense strand and the antisense strand of the dsRNA agent each n at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.
When the sense strand and the antisense strand of the dsRNA agent each contain at least two wing modifications, the sense strand and the antisense strand can be aligned so that two wing modifications each from one strand fall on one end of the duplex region, ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex , having an overlap of one, two or three nucleotides.
In one ment, every nucleotide in the sense strand and antisense strand of the dsRNA agent, including the tides that are part of the motifs, may be modified.
Each nucleotide may be modified with the same or different modification which can include one or more tion of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2' hydroxyl onthe ribose sugar; wholesale replacement of the phosphate moiety with "dephospho" linkers; modification or ement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the t positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, e.g., at a position on a terminal tide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a R A or may only occur in a single strand region of a RNA. E.g., a phosphorothioate modification at a nking O position may only occur at one or both termini, may only occur in a terminal , e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at i. The 5' end or ends can be phosphorylated.
It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3' or ' overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of cations at the 2' position of the ribose sugar with [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy- 2'-fluoro (2'-F) or 2'methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., orothioate modifications. Overhangs need not be homologous with the target sequence.
In one ment, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'- O-methyl, 2' allyl, 2'-C- allyl, 2'-deoxy, or 2'-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2'- O-methyl or 2'-fluoro.
At least two different modifications are lly present on the sense strand and nse strand. Those two modifications may be the 2'- O-methyl or 2'-fluoro modifications, or others.
In one embodiment, the sense strand and antisense strand each contains two differently modified nucleotides selected from 2'methyl or 2'-fluoro.
In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2'methyl nucleotide, xyfluoro tide, 2 -O-N- methylacetamido (2'NMA) nucleotide, a 2'dimethylaminoethoxyethyl (2' DMAEOE) nucleotide, 2'aminopropyl (2'AP) nucleotide, or 2'-ara-Fnucleotide.
In one embodiment, the Na and/or b comprise modifications of an alternating pattern. The term "alternating motif or "alternative pattern" as used herein refers to a motif having one or more modifications, each modification occurring on ating nucleotides of one strand. The ating nucleotide may refer to one per every other tide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB ...," "AABAABAABAAB "AAABAAABAAAB ...," "AAABBBAAABBB ...," or "ABCABCABC ABC ...," etc.
In one embodiment, the Na' and/or Nb' se modifications of an alternating pattern. The term "alternating motif or "alternative pattern" as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other tide or one per every three tides, or a similar pattern. For example, if A, B [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm and C each represent one type of cation to the nucleotide, the alternating motif can be "ABABABABABAB ...," "AABBAABB AABB ...," BAABAAB "AAABAAABAAAB ...," "AAABBBAAABBB ...," or "ABCABCABC ABC ...." etc.
The type of cations contained in the alternating motif may be the same or ent. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the ating motif such as "ABABAB. ..", "ACACAC..." "BDBDBD ..." or "CDCDCD ...," etc.
In one embodiment, the dsR A agent of the invnetion comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the nse strand is shifted.The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with "ABABAB" from 5'-3' of the strand and the alternating motif in the antisense strand may start with "BABABA" from 3'-5 f the strand within the duplex region. As r example, the alternating motif in the sense strand may start with "AABBAABB" from 5'-3' of the strand and the alternating motif in the antisenese strand may start with "BBAABBAA" from 3'-5 f the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the nse strand.
In one embodiment, the dsRNA agent of the invention comprises the pattern of the alternating motif of 2'methyl modification and 2'-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2'methyl modification and 2'-F modification on the antisense strand initially, i.e., the 2'methyl modified nucleotide on the sense strand base pairs with a 2'-F ed tide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2'-F modification, and the 1 position of the antisense strand may start with the 2'- O-methyl modification.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm The introduction of one or more motifs of three identical modifications on three utive nucleotides to the sense strand and/or antisense strand interrupts the initial modification n present in the sense strand and/or nse . This interruption of the modification pattern of the sense and/or antisense strand by introducing one or more motifs of three cal modifications on three consecutive nucleotides to the sense and/or antisense strand singly enhances the gene silencing acitivty to the target gene.
In one embodiment, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif.
For example, the portion of the sequence containing the motif is " . ..NaYYYNb. ..," where "Y" represents the modification of the motif of three identical modifications on three consecutive nucleotide, and "Na" and "Nb" represent a modification to the nucleotide next to the motif "YYY" that is different than the modification of Y, and where Na and b can be the same or different modifications. Altnernatively, Na and/or b may be present or absent when there is a wing modification present.
The dsRNA agent of the invention may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or nse strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or nse strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense ; or the sense strand or nse strand comprises both internucleotide linkage modifications in an alternating pattern.
The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating n of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the nse strand.
In one ment, the dsRNA comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm phosphonate intemucleotide linkage between the two nucleotides. Intemucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate intemucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate intemucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate intemucleotide es between the terminal three nucleotides, in which two of the three nucleotides are overhang tides, and the third is a paried nucleotide next to the overhang nucleotide. ably, these terminal three nucleotides may be at the 3'-end of the antisense strand.
In one embodiment the sense strand of the dsR A comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate intemucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate cleotide es, wherein one of the phosphorothioate or methylphosphonate intemucleotide es is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and ate intemucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or ate linkage.
In one embodiment the antisense strand of the dsRNA comprises two blocks of two phosphorothioate or methylphosphonate cleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate intemucleotide linkages, wherein one of the phosphorothioate or methylphosphonate intemucleotide linkages is placed at any position in the oligonucleotide sequence and the said nse strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one embodiment the antisense strand of the dsRNA comprises two blocks of three phosphorothioate or phosphonate intemucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate intemucleotide linkages, wherein one of the phosphorothioate or methylphosphonate intemucleotide linkages is placed at [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one embodiment the nse strand of the dsR A comprises two blocks of four phosphorothioate or methylphosphonate internucleotide es separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an nse strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one embodiment the antisense strand of the dsRNA comprises two blocks of five phosphorothioate or methylphosphonate ucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said nse strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an nse strand comprising either phosphorothioate or methylphophonate or phosphate e.
In one embodiment the antisense strand of the dsRNA comprises two blocks of six orothioate or methylphosphonate internucleotide es separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate ucleotide es or an antisense strand comprising either phosphorothioate or phophonate or phosphate linkage.
In one embodiment the antisense strand of the dsRNA comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any ation of orothioate, methylphosphonate and phosphate internucleotide es or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one embodiment the antisense strand of the dsR A comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5 or 6 phosphate internucleotide es, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one embodiment the antisense strand of the dsRNA ses two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3 or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any on in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an nse strand comprising either phosphorothioate or methylphophonate or phosphate linkage.
In one ment, the dsRNA of the invention further comprises one or more orothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense and/or antisense strand. For e, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense and/or antisense strand.
In one embodiment, the dsRNA of the invention r comprises one or more orothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense and/or antisense strand. For e, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm phosphorothioate methylphosphonate cleotide linkage at position 8-16 of the duplex region counting from the 5'-end of the sense strand; the dsR A can optionally further comprise one or more phosphorothioate or methylphosphonate intemucleotide linkage modification within 1-10 of the termini position(s).
In one embodiment, the dsRNA of the invention further comprises one to five phosphorothioate or methylphosphonate intemucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or phosphonate intemucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5'- end), and one to five phosphorothioate or methylphosphonate intemucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand ing from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises one orothioate intemucleotide linkage modification within on 1-5 and one phosphorothioate or methylphosphonate intemucleotide linkage modification within position 18-23 of the sense strand (counting from the ), and one phosphorothioate intemucleotide e modification at positions 1 and 2 and two phosphorothioate or methylphosphonate intemucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises two orothioate intemucleotide linkage modifications within position 1-5 and one phosphorothioate intemucleotide linkage modification within position 18-23 of the sense strand (counting from the 5'-end), and one phosphorothioate intemucleotide linkage modification at positions 1 and 2 and two phosphorothioate intemucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the ).
In one embodiment, the dsRNA of the invention further comprises two orothioate intemucleotide linkage modifications within on 1-5 and two phosphorothioate intemucleotide linkage modifications within position 18-23 of the sense strand ing from the 5'-end), and one phosphorothioate intemucleotide linkage modification at positions 1 and 2 and two phosphorothioate intemucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end).
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In one embodiment, the dsR A of the invention further comprises two phosphorothioate intemucleotide e modifications within position 1-5 and two phosphorothioate intemucleotide linkage cations within on 18-23 of the sense strand (counting from the 5'-end), and one phosphorothioate intemucleotide linkage modification at positions 1 and 2 and one phosphorothioate intemucleotide linkage modification within ons 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises one phosphorothioate intemucleotide linkage modification within position 1-5 and one phosphorothioate cleotide linkage modification within position 18-23 of the sense strand (counting from the 5'-end), and two orothioate intemucleotide linkage modifications at positions 1 and 2 and two phosphorothioate intemucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the ion further comprises one orothioate intemucleotide e modification within position 1-5 and one within position 18-23 of the sense strand ing from the 5'-end), and two phosphorothioate intemucleotide linkage modification at positions 1 and 2 and one phosphorothioate intemucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the ion further comprises one phosphorothioate intemucleotide linkage modification within position 1-5 (counting from the 5'-end), and two phosphorothioateintemucleotide linkage modifications at positions 1 and 2 and one phosphorothioate intemucleotide linkage modification within positions 18- 23 of the antisense strand (counting from the 5'-end).
In one ment, the dsRNA of the ion further comprises two phosphorothioate intemucleotide linkage modifications within position 1-5 (counting from the 5'-end), and one phosphorothioateintemucleotide linkage modification at positions 1 and 2 and two phosphorothioate intemucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the ion further ses two phosphorothioate intemucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5'-end), and two [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm phosphorothioate intemucleotide linkage modifications at positions 1 and 2 and one phosphorothioate intemucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsR A of the invention further comprises two phosphorothioate intemucleotide linkage cations within position 1-5 and one phosphorothioate intemucleotide linkage modification within position 18-23 of the sense strand (counting from the 5'-end), and two orothioate intemucleotide linkage modifications at positions 1 and 2 and two phosphorothioate intemucleotide e modifications within positions 18-23 of the nse strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises two phosphorothioate intemucleotide e modifications within position 1-5 and one phosphorothioate intemucleotide linkage modification within position 18-23 of the sense strand (counting from the 5'-end), and one orothioate intemucleotide linkage cation at positions 1 and 2 and two phosphorothioate intemucleotide e modifications within positions 18-23 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises two orothioate intemucleotide linkage modifications at position 1 and 2, and two phosphorothioate intemucleotide linkage modifications at position 20 and 2 1 of the sense strand (counting from the 5'-end), and one phosphorothioate intemucleotide linkage modification at positions 1 and one at position 2 1 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the ion further comprises one phosphorothioate intemucleotide linkage modification at on 1, and one phosphorothioate intemucleotide linkage modification at position 2 1 of the sense strand (counting from the 5'-end), and two phosphorothioate intemucleotide linkage modifications at ons 1 and 2 and two phosphorothioate intemucleotide linkage modifications at ons 20 and 2 1 the antisense strand (counting from the 5'-end).
In one ment, the dsRNA of the invention further comprises two phosphorothioate cleotide linkage modifications at position 1 and 2, and two phosphorothioate intemucleotide linkage modifications at position 2 1 and 22 of the sense strand (counting from the 5'-end), and one phosphorothioate intemucleotide linkage [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm modification at positions 1 and one phosphorothioate internucleotide e modification at position 2 1 of the antisense strand (counting from the 5'-end).
In one ment, the dsR A of the invention further comprises one orothioate internucleotide linkage cation at on 1, and one phosphorothioate internucleotide linkage modification at position 1 of the sense strand (counting from the 5'-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 2 1 and 22 the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide e modifications at position 22 and 23 of the sense strand ing from the 5'-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 2 1 of the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA of the invention further comprises one phosphorothioate ucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 2 1 of the sense strand (counting from the 5'-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5'-end).
In one embodiment, the dsRNA agent of the invention ses mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote iation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to e the pairs on an individual pair basis, though next neighbor or r analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) gs; and pairings which e a sal base are preferred over canonical pairings.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In one embodiment, the dsR A agent of the ion comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5'- end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than cal pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5'-end of the duplex.
In one embodiment, the nucleotide at the 1 position within the duplex region from the 5'-end in the antisense strand is selected from the group consisting ofA, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5'- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5'- end of the antisense strand is an AU base pair.
In one embodiment, the sense strand ce may be represented by formula (I): 'np-Na-(X X X )i-Nb-Y Y Y -Nb-(Z Z Z )j- a- q 3' (I) wherein: i and j are each independently 0 or 1; p and q are each independently 0-6; each Na independently represents an oligonucleotide sequence sing 0-25 modified tides, each sequence comprising at least two ently modified nucleotides; each b independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each np and nq independently represent an overhang nucleotide; wherein N b and Y do not have the same modification; and XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2'-F modified nucleotides.
In one embodiment, the Na and/or b comprise cations of ating pattern.
In one embodiment, the YYY motif occurs at or near the ge site of the sense strand. For example, when the dsRNA agent has a duplex region of 17-23 nucleotide pairs in length, the YYY motif can occur at or the vicinity of the cleavage site ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11,12 or 11, 12, 13) of - the sense strand, the count starting from the 1st nucleotide, from the 5'-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5'- end.
In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following as: np-Na -YYY-Nb-ZZZ-Na-nq 3 (la); np-Na -XXX-Nb-YYY-Na-nq 3 (lb); or np-Na -XXX-Nb-YYY-Nb-ZZZ-Na-nq 3 (Ic).
When the sense strand is represented by formula (la), b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.
Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the sense strand is represented as formula (lb), b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 ed nucleotides.
When the sense strand is represented as formula (Ic), each N b ndently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5 or 6 Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified tides.
Each of X, Y and Z may be the same or different from each other.
In one embodiment, the antisense strand sequence of the dsRNA may be ented by formula (II): n '-Na '-(Z'Z'ZVNb '-Y'Y'Y'-Nb'-(X'X'X')i-N'a-np' 3 (II) wherein: k and 1are each ndently 0 or 1; p and q are each independently 0-6; each Na' independently represents an oligonucleotide sequence comprising 0-25 modified tides, each sequence comprising at least two differently modified nucleotides; [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm each b' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; each np'and nq' independently represent an ng nucleotide comprising 0-6 nucleotides; wherein Nb' and Y ' do not have the same modification; C 'C 'C ', Y'Y'Y' and Z'Z'Z' each ndently represent one motif of three identical modifications on three consecutive nucleotides.
In one embodiment, the Na' and/or Nb' comprise modifications of alternating The UΎ Ύ ' motif occurs at or near the ge site of the antisense strand. For example, when the dsRNA agent has a duplex region of 17-23 nt in length, the U Ύ Ύ ' motif can occur at positions 9, 10, 11;10, 11, 12; 11, 12, 13; 12, 13, 14 ; or 13, 14, 15 of the antisense strand, with the count starting from the 1st nucleotide, from the 5'-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5'- end. Preferably, the U Ύ Ύ ' motif occurs at ons 11, 12, 13.
In one embodiment, UΎ Ύ ' motif is all 2'-OMe modified nucleotides.
In one embodiment, k is 1 and 1is 0, or k is 0 and 1is 1, or both k and 1are 1.
The antisense strand can therefore be represented by the following formulas: nq'-Na'-Z'Z'Z'-Nb'-Y'Y'Y'-Na'-np' 3 (Ila); nq'-Na'-YYY'-N 'X'-np' 3 (lib); or *nq'-Na'- Z'Z'Z'-Nb'-Y'Y'Y'-Nb'- X'X'X'-Na'-np' 3* (lie).
When the antisense strand is represented by formula (Ila), N b represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na' ndently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the antisense strand is represented as formula (lib), Nb' represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na' independently represents an ucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm When the antisense strand is represented as formula (He), each Nb' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na' independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N b is 0, 1, 2, 3, 4, 5 or 6 .
Each of X', Y'and Z' may be the same or different from each other.
Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, ethyl, 2'allyl, 2'-C-allyl, or 2'-fluoro. For example, each nucleotide of the sense strand and nse strand is independently modified with 2'methyl or 2'-fluoro. EachX, Y, Z, X', Y'and Z', in particular, may represent a 2'methyl modification or a 2'-fluoro cation.
In one embodiment, the sense strand of the dsRNA agent comprisesYYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 2 1 nt, the count starting from the 1st nucleotide from the 5'-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5'- end; and Y represents 2'-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2'-OMe modification or 2'-F modification.
In one ment the antisense strand may contain U Ύ Ύ ' motif occurring at positions 11, 12, 13 of the strand, the count ng from the 1st nucleotide from the '-end, or ally, the count starting at the 1st paired nucleotide within the duplex , from the 5'- end; and Y' represents 2'methyl modification. The antisense strand may additionally contain X'X'X' motif or Z'Z'Z' motifsas wing cations at the opposite end of the duplex region; and X'X'X'and Z'Z'Z'each independently ents a 2'-OMe modification or 2'-F modification.
The sense strand represented by any one of the above formulas (la), (lb) and (Ic) forms a duplex with a antisense strand being represented by any one of formulas (Ha), (lib) and (He), respectively.
Accordingly, the dsRNA agent may comprise a sense strand and an nse strand, each strand having 14 to 30 tides, the dsRNA duplex represented by formula (III): sense: 5 ' np -Na- (X X X ) -N - Y Y Y -N -(Z Z Z) -Na-n 3 ' ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm antisense: 3 ' n p ' - N a ' - ( C ' X ' X ' ) k - N ' - Y Ύ Ύ ' - N ' - ( Z ' Z ' Z ' ) i-N a ' - n ' 5 ' (III) wherein: i, j , k, and 1are each independently 0 or 1; p and q are each independently 0-6; each Na and Na' ndently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each b and b' independently represents an oligonucleotide ce comprising 0-10 modified nucleotides; wherein each np' , np, nq' , and nq independently ents an overhang nucleotide sequence; and XXX, YYY, ZZZ ,C 'C 'C ',U Ύ Ύ ', and Z'Z'Z' each independently represent one motif of three cal modifications on three consecutive tides.
In one embodiment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are l.In another embodiment, k is 1 and 1is 0; k is 0 and 1is 1; or both k and 1are 1.
In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex ented by formula (V): sense: 5 ' Na - ( X x X ) - N - Y Y Y - N - ( Z z Z ) - N a - n 3 ' antisense: 3 ' n p ' - N ' - ( X ' X ' X ' ) k - N ' - Y Ύ Ύ ' - N ' - ( Z ' Z ' Z ' ) i-N ' 5 ' wherein: i, j , k, and lare each independently 0 or 1; p and q are each independently 2; each Na and Na' independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides; each N b and N b' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm wherein each np' , and nq independently represents an overhang tide sequence; and XXX, YYY, ZZZ, C 'C 'C',UΎ 'U ', and Z'Z'Z'each independently represent one motif of three identical modifications on three consecutive nucleotides.
In one ment, i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1. In another embodiment, k is 1 and 1is 0; k is 0 and 1is 1; or both k and 1are 1.
In one embodiment, the dsRNA agent of the invention comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the dsRNA duplex represented by formula (Va): sense: 5 ' N - ( X x x ) - N - Y Y Y - N - ( Z z Z ) j - N 3 ' a a antisense: 3 ' n ' - N ' - ( X ' X ' X ' ) - N ' - Y ' Y ' Y ' - N ' - ( ' Z ' Z ' ) i-N ' 5 ' p a k a (Va) wherein: i, j , k, and lare each independently 0 or 1; p and q are each ndently 2; each Na and Na' independently represents an oligonucleotide sequence sing 0-25 modified nucleotides, each sequence comprising at least two ently modified nucleotides; each b and N b' independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides; wherein np' represents an overhang nucleotide sequence; and XXX, YYY, ZZZ, C 'C 'C',UΎ 'U ', and Z'Z'Z'each independently represent one motif of three identical modifications on three consecutive nucleotides.
Exemplary combinations of the sense strand and antisense strand forming a dsRNA duplex include the formulas below: ' r p - N - Y Y Y - N - Z Z Z - N - n 3 ' 3 ' n ' - N ' - Y ' Y ' Y ' - N ' - Z ' Z ' Z ' - N ' n ' 5 ' (Ilia) ' n - N - X X X - N - Y Y Y - N - n 3 ' 3 ' n ' - N ' - X ' X ' X ' - N ' - Y ' Y ' Y ' - N ' - n ' 5 ' [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm (nib) ' r p - N - X X X - N - Y Y Y - N - Z Z Z - N a - n 3 ' 3 ' n p ' - Na ' - X ' X ' X ' - N ' - Y Ύ Ύ ' - N ' - Z ' Z ' Z ' - N a - n ' 5 ' (IIIc) When the dsR A agent is represented by formula (Ilia), each N b and b' independently represents an oligonucleotide sequence sing 1-10, 1-7, 1-5 or 1-4 modified tides. Each Na and Na'independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the dsRNA agent is represented as formula (Illb), each N b and Nb' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0- , 0-4, 0-2 or Omodified nucleotides. Each Na and Na' independently represents an ucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.
When the dsRNA agent is represented as formula (IIIc), each N b and Nb' independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0- , 0-4, 0-2 or 0 ed tides. Each Na and Na' independently represents an oligonucleotide ce comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na' ,N b and N b independently comprises modifications of alternating pattern.
Each of X, Y and Z in formulas (III), (Ilia), (Illb) and (IIIc) may be the same or different from each other.
When the dsRNA agent is represented by formula (III), (Ilia), (Illb) or (IIIc), at least one of the Y tides may form a base pair with one of the Y' nucleotides.
Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y ' nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y' nucleotides.
It is understood that Na nucleotides from base pair with Na' ,N b nucleotides from base pair with Nb', X nucleotides from base pair with X', Y nucleotides from base pair with Y', and Z nucleotides from base pair with Z'.
When the dsRNA agent is represented by a (Ilia) or (IIIc), at least one of the Z nucleotides may form a base pair with one of the Z' nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the ponding Z' nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z' nucleotides.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm When the dsRNA agent is represented as formula (Illb) or (IIIc), at least one of the X nucleotides may form a base pair with one of the X' nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X' nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X' nucleotides.
In one embodiment, the modification on the Y tide is different than the modification on the Y ' nucleotide, the modification on the Z nucleotide is different than the cation on the Z' nucleotide, and/or the modification on the X nucleotide is different than the modification on the X ' tide.
In one embodiment, the dsRNA agent is a multimer containing at least two duplexes represented by formula (III), , (Illb) or (IIIc), wherein said duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, said multimer further comprise a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.
In one embodiment, the dsRNA agent is a multimer containing three, four, five, six or more duplexes represented by a (III), (Ilia), (Illb) or (IIIc), wherein said es are connected by a linker. The linker can be cleavable or non-cleavable.
Optionally, said multimer further comprises a . Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.
In one embodiment, two dsRNA agent represented by formula (III), , (Illb) or (IIIc) are linked to each other at the 5' end, and one or both of the 3' ends of the are optionally conjugated to to a ligand. Each of the dsRNA can target the same gene or two different genes; or each of the dsRNA can target same gene at two different target sites.
Various publications described multimeric siRNA and can all be used with the dsRNA of the invention. Such publications include WO2007/091269, US Patent No. 7858769, WO2010/14151 1, WO2007/1 17686, WO2009/014887 and WO201 1/031520 which are hereby orated by their entirely.
The dsRNA agent that contains ations of one or more carbohydrate moieties to a dsRNA agent can optimize one or more properties of the dsRNA agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the dsRNA agent. E.g., the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit . A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., en, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.
The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one "backbone ment " preferably two "backbone attachment points" and (ii) at least one "tethering attachment point." A "backbone attachment point" as used herein refers to a functional group, e.g. a hydroxy1group, or generally, a bond available for, and that is suitable for oration of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A "tethering attachment point" (TAP) in some embodiments refers to a constituent ring atom of the cyclic r, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment , that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, haride, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide.
Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier.
Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of r chemical entity, e.g., a ligand to the constituent ring.
In embodimentn the dsR A of the invention is conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; ably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [l,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, azolidinyl, quinoxalinyl, pyridazinonyl, ydrofuryl and and decalin; preferably, the acyclic group is selected from serinol ne or diethanolamine backbone. ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm The double-stranded R A (dsRNA) agent of the invention may optionally be conjugated to one or more ligands. The ligand can be attached to the sense strand, antisense strand or both strands, at the 3'-end, 5'-end or bothends. For instance, the ligand may be conjugated to the sense , in particular, the 3'-end of the sense strand.
Ligands A wide variety of entities can be coupled to the ucleotides of the present invention. Preferred moieties are ligands, which are coupled, preferably covalently, either directly or ctly via an intervening tether.
In preferred embodiments, a ligand alters the bution, targeting or lifetime of the molecule into which it is incorporated. In red embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, receptor e.g., a cellular or organ tment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Ligands providing enhanced affinity for a ed target are also termed targeting ligands.
Some ligands can have endosomolytic properties. The endosomolytic ligands promote the lysis of the me and/or transport of the composition of the invention, or its components, from the endosome to the asm of the cell. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In one embodiment, the endosomolytic ligand assumes its active conformation at endosomal pH. The "active" conformation is that conformation in which the molytic ligand promotes lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include the GALA peptide (Subbarao et al, Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al, J .
Am. Chem. Soc, 1996, 118: 1581-1586), and their derivatives (Turk et al, Biochem.
Biophys. Acta, 2002, 1559: 56-68). In one embodiment, the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH. The molytic component may be linear or branched.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Ligands can improve transport, hybridization, and specificity ties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule sing any combination of monomers described herein and/or natural or modified ribonucleotides.
Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and se-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, ns, es, polyamines, and peptide mimics.
Ligands can include a naturally occurring substance, such as a protein (e.g., human serum n (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); a carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, -lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride mer, N-(2-hydroxypropyl)methacrylamide copolymer , polyethylene glycol (PEG), polyvinyl alcohol (PVA), ethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide. s can also include targeting , e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, rotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, alent fucose, glycosylated polyaminoacids, multivalent ose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic or an aptamer.
Table 2 shows some examples of targeting ligands and their associated receptors.
Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or a chelator (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis- 0(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3- propanediol, heptadecyl group, palmitic acid, myristic acid,03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), ting , ate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. ), ort/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, ole clusters, acridine-imidazole conjugates, Eu3+ xes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.
Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, s, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent e, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an tor of NF-KB.
The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate nts. The drug can be, for example, taxon, stine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or vin.
The ligand can increase the uptake of the oligonucleotide into the cell by activating an inflammatory se, for example. Exemplary ligands that would have [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm such an effect include tumor necrosis factor alpha pha), eukin-1 beta, or gamma interferon.
In one aspect, the ligand is a lipid or based molecule. Such a lipid or lipid- based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a nonkidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.
A lipid based ligand can be used to modulate, e.g., control the g of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the ate to the kidney.
In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the ate will be preferably distributed to a non-kidney tissue. However, it is preferred that the ty not be so strong that the gand binding cannot be reversed.
In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.
In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K . Other exemplary vitamins include B vitamins, e.g., folic acid, B12, avin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also ed are HAS, low density lipoprotein (LDL) and high-density lipoprotein (HDL).
[Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In r aspect, the ligand is a ermeation agent, preferably a helical cell- permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D- amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The e or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation e, cationic peptide, amphipathic e, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another ative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An ary hobic MTS-containing peptide is RFGF having the amino acid ce AAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, ucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK) have been found to be e of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A e moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.An RGD peptide moiety can be [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, , spleen, or liver (Aoki et al, Cancer Gene Therapy 8:783-787, 2001). Preferably, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a ylated RGD peptide can deliver an iRNA agent to a tumor cell expressing 3 (Haubner et al, Jour. Nucl. Med., 42:326-336, 2001). Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing es and peptidomimetics can target cancer cells, in particular cells that exhibit an integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other es that target the integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type lignads that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.
A "cell tion peptide" is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating e can be, for example, an a-helical linear e (e.g., LL-37 or Ceropin PI), a disulfide bond-containing peptide (e.g., a -defensin, b-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also e a r zation signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV- 1 gp41 and the NLS of SV40 large T antigen (Simeoni et al, Nucl. Acids Res. 31:2717-2724, 2003).
In one embodiment, a targeting peptide can be an amphipathic a-helical e.
Exemplary amphipathic a-helical peptides e, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S . clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, ins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm to maintain the integrity of helix stability. For example, a m number of helix stabilization es will be utilized (e.g., leu, ala, or lys), and a m number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix.
Formation of salt bridges between residues with opposite charges, separated by i ± 3, or i ± 4 positions can provide stability. For e, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt s with the anionic residues glutamate or aspartate.
Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, b, or g peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.
The targeting ligand can be any ligand that is capable of targeting a specific receptor. Examples are: folate, GalNAc, galactose, mannose, mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster, galactose cluster, or an er. A r is a combination of two or more sugar units. The targeting ligands also include integrin receptor ligands, Chemokine receptor ligands, transferrin, biotin, nin receptor ligands, PSMA, elin, GCPII, somatostatin, LDL and HDL s. The ligands can also be based on nucleic acid, e.g., an aptamer. The aptamer can be unmodified or have any combination of modifications disclosed herein.
Endosomal release agents include imidazoles, poly or oligoimidazoles, PEIs, peptides, fusogenic peptides, polycaboxylates, polyacations, masked oligo or poly cations or , acetals, polyacetals, ketals/polyketyals, orthoesters, polymers with masked or unmasked ic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges.
PK tor stands for pharmacokinetic modulator. PK modulator include iles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Examplary PK tor include, but are not limited to, terol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Oligonucleotides that se a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g. oligonucleotides of about 5 bases, bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbaone are also amenable to the present invention as ligands (e.g. as PK modulating ligands).
In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands.
Other ligand ates amenable to the invention are described in U.S. Patent Applications USSN: ,185, filed August 10, 2004; USSN: 10/946,873, filed ber 21, 2004; USSN: 10/833,934, filed August 3, 2007; USSN: 11/1 15,989 filed April 27, 2005 and USSN: 11/944,227 filed November 21, 2007, which are incorporated by reference in their entireties for all purposes.
When two or more s are present, the ligands can all have same properties, all have different ties or some ligands have the same ties while others have different properties. For example, a ligand can have targeting properties, have molytic activity or have PK modulating properties. In a preferred ment, all the ligands have different properties.
Ligands can be coupled to the oligonucleotides at s places, for example, 3'- end, 5'-end, and/or at an internal position. In preferred embodiments, the ligand is attached to the ucleotides via an intervening tether, e.g. a carrier described herein.
The ligand or tethered ligand may be present on a monomer when said monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated via coupling to a "precursor" monomer after said "precursor" monomer has been incorporated into the growing strand. For example, a monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH2) N H 2 may be incorporated into a g oligonucelotide strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor monomer by coupling the ophilic group of the ligand with the al nucleophilic group of the precursor monomer's tether.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm In another example, a r having a chemical group suitable for taking part in Click Chemistry reaction may be incorporated e.g., an azide or alkyne terminated tether/linker. In a subsequent operation, i.e., after incorporation of the precursor monomer into the , a ligand having mentary chemical group, e.g. an alkyne or azide can be attached to the precursor monomer by coupling the alkyne and the azide together.
For double- ed oligonucleotides, ligands can be attached to one or both strands. In some embodiments, a double-stranded iRNA agent contains a ligand conjugated to the sense . In other embodiments, a double-stranded iRNA agent contains a ligand conjugated to the antisense strand.
In some embodiments, ligand can be conjugated to nucleobases, sugar moieties, or internucleosidic es of nucleic acid molecules. Conjugation to purine nucleobases or derivatives thereof can occur at any position including, endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase are attached to a ate moiety. Conjugation to pyrimidine nucleobases or derivatives thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a dine nucleobase can be substituted with a conjugate moiety. Conjugation to sugar moieties of nucleosides can occur at any carbon atom. Example carbon atoms of a sugar moiety that can be attached to a conjugate moiety include the 2', 3', and 5' carbon atoms. The G position can also be attached to a ate moiety, such as in an abasic e. Internucleosidic linkages can also bear conjugate moieties. For phosphoruscontaining linkages (e.g., phosphodiester, phosphorothioate, orodithiotate, phosphoroamidate, and the like), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleosidic linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.
Any suitable ligand in the field of RNA interference may be used, although the ligand is typically a carbohydrate e.g. monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, polysaccharide.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Linkers that conjugate the ligand to the nucleic acid include those discussed above. For example, the ligand can be one or more GalNAc (N -acetylglucosamine) derivatives ed through a bivalent or trivalent ed .
In one embodiment, the dsRNA of the invention is ated to a bivalent and trivalent branched linkers include the structures shown in an of formula IV - VII : Formula (IV) Formula (V) Formula (VI) or Formula (VII) q A, q , q A, q , q4A, q4 , q5A, q5 and q5C represent ndently for each occurrence 0-20 and wherein the repeating unit can be the same or different; 2A 2B A B 4A ,4B A B C 2A 3A 4A 4B 4A 5B T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH20 ; Q A, Q2 , Q A, Q , Q4A, Q4 , Q5A, Q5 , Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherin one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), S0 2, N(RN), C(R')=C(R"), CºC or C(O); R A, R , R A, R , R4A, R4 , R5A, R5 , R5C are each independently for each occurrence absent, NH, O, S, CH2, C(0)0, C(0)NH, NHCH(Ra)C(0), -C(0)-CH(R a)- [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm cyclyl; L2A, L2 , L A, L , L4A, L4 , L5A, L5 and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as ), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain.
Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (VII): Formula (VII) wherein L5A, L5 and L5C represent a monosaccharide, such as GalNAc tive.
Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the following compounds: [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm HO HO HO HoO H< Hfik' O\/\O/\/O\/\N/(\/O0%“ HO HO H O 0 HowHO i O\/\O/\/O\/\N o HO HO O\/\O/\/O\/\ N HO HoO H/K Hfim‘- O\/\O/\/O\/\Nip03W HO HO H O HO ‘0 O\/\O/\/O\/\ N 0 HO O\//\O/\\/0 NHAc \ HO NM' HO O\/\O/\/0 HO NHAc [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm O H HO O\/\/\n/N NHAC O HO OH NHACHO OH NHAC O NHAC O H AcHN OMNWNWH AcHN H O ON‘LHWNX AcHN H HO OH HO&&’OM0 i AcHN HO OH AcHN H O=( Ho&g/O\/\)LHO 0 )=0 AcHN IZ 4343 [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm ation] kjm Unmarked set by kjm Definitions As used herein, the terms "dsRNA", "siRNA", and "iRNA agent" are used interchangeably to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.
As used , the phrase "mediates RNAi" refers to the ability to silence, in a sequence specific manner, a target RNA. While not g to be bound by theory, it is believed that silencing uses the RNAi machinery or s and a guide RNA, e.g., an siRNA agent of 2 1 to 23 nucleotides.
As used herein, "specifically hybridizable" and ementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a nd of the invention and a target RNA molecule.
Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric nd to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 tides.
In one embodiment, a dsRNA agent of the invention is "sufficiently complementary" to a target RNA, e.g., a target mRNA, such that the dsRNA agent silences production of protein encoded by the target mRNA. In r ment, the dsRNA agent of the ion is "exactly complementary" to a target RNA, e.g., the target RNA and the dsRNA duplex agent anneal, for example to form a hybrid made exclusively of Watson-Crick base pairs in the region of exact complementarity. A "sufficiently complementary" target RNA can include an internal region {e.g., of at least nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the dsRNA agent of the invention specifically discriminates a singlenucleotide difference. In this case, the dsRNA agent only mediates RNAi if exact complementary is found in the region {e.g., within 7 nucleotides of) the single-nucleotide difference.
[Annotation] kjm None set by kjm ation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm As used , the term "oligonucleotide" refers to a nucleic acid le (RNA or DNA) for example of length less than 100, 200, 300, or 400 nucleotides.
The term "halo" refers to any radical of fluorine, chlorine, bromine or iodine. The term "alkyl" refers to saturated and rated non-aromatic hydrocarbon chains that may be a straight chain or branched chain, containing the indicated number of carbon atoms (these include t limitation propyl, allyl, or propargyl), which may be optionally ed with N , O, or S . For example, Ci-Cio indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. The term "alkoxy" refers to an -O-alkyl radical. The term "alkylene" refers to a divalent alkyl (i.e., -R-). The term enedioxo" refers to a divalent species of the structure -, in which R represents an alkylene. The term "aminoalkyl" refers to an alkyl substituted with an aminoThe term "mercapto" refers to an -SH radical. The term "thioalkoxy" refers to an - S-alkyl radical.
The term "aryl" refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. es of aryl groups include phenyl, naphthyl and the like. The term "arylalkyl" or the term "aralkyl" refers to alkyl substituted with an aryl. The term "arylalkoxy" refers to an alkoxy substituted with aryl.
The term "cycloalkyl" as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, for example, 3 to 8 carbons, and, for example, 3 to 6 carbons, wherein the cycloalkyl group additionally may be optionally substituted. Cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.
The term "heteroaryl" refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 atoms if tricyclic, said heteroatoms selected from O, N , or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N , O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring may be substituted by a substituent. Examples of heteroaryl groups include pyridyl, furyl or l, imidazolyl, benzimidazolyl, dinyl, thiophenyl or thienyl, [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm quinolinyl, indolyl, thiazolyl, and the like. The term "heteroarylalkyl" or the term "heteroaralkyl" refers to an alkyl substituted with a heteroaryl. The term "heteroarylalkoxy" refers to an alkoxy substituted with heteroaryl.
The term "heterocyclyl" refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 ed tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if ic, or 1-9 heteroatoms if tricyclic, said atoms ed from O, N , or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N , O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclyl groups include trizolyl, tetrazolyl, piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.
The term "oxo" refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.
The term "acyl" refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further tuted by substituents.
The term "substituted" refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkyIt hioalkyl, arylthioalkyl, ulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylamino carbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, ino, alkylaminoalkyl, arylaminoalkyl, lkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, yl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent can be further tuted.
Cleavable Linking Groups A ble linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm together. In a preferred embodiment, the cleavable g group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second nce condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).
Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood.
Examples of such degradative agents include: redox agents which are selected for particular ates or which have no substrate specificity, including, e.g., ive or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.
A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired tment of the cell.
A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable g group orated into a linker can depend on the cell to be ed. For e, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.
Linkers that n peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue.
Thus one can determine the relative tibility to cleavage between a first and a second condition, where the first is ed to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other s or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole s. It may be useful to make initial evaluations in cell-free or e conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).
Redox cleavable linking groups One class of cleavable linking groups are redox cleavable linking groups that are cleaved upon reduction or oxidation. An e of reductively cleavable linking group is a disulphide linking group (-S-S-). To determine if a ate cleavable linking group is a le "reductively cleavable linking " or for example is suitable for use with a particular iR A moiety and particular targeting agent one can look to methods bed herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be ted under conditions which are ed to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most % in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be ined using standard enzyme kinetics assays under conditions chosen to [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm ation] kjm Unmarked set by kjm mimic intracellular media and compared to conditions chosen to mimic extracellular media.
Phosphate-based cleavable linking groups Phosphate-based cleavable linking groups are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves ate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are P(0)(ORk), )(ORk), P(S)(SRk), -S-P(0)(ORk), - 0-P(0)(ORk)-S-, -S-P(0)(ORk)-S-, P(S)(ORk)-S-, -S-P(S)(ORk), P(0)(Rk)- 0-, P(S)(Rk), -S-P(0)(Rk), -S-P(S)(Rk), -S-P(0)(Rk)-S-, P(S)( Rk)-S-.
Preferred embodiments are P(0)(OH), P(S)(OH), P(S)(SH), -S- P(0)(OH), P(0)(OH)-S-, -S-P(0)(OH)-S-, )(OH)-S-, -S-P(S)(OH), -O- R(0)( H), P(S)(H), -S-P(0)(H), -S-P(S)(H), -S-P(0)(H)-S-, P(S)(H)- S-. A preferred embodiment is P(0)(OH). These candidates can be evaluated using s analogous to those described above.
Acid cleavable linking groups Acid cleavable linking groups are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic nment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups e but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN- C(0)0, or -OC(O). A red embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, tuted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.
Ester-based linking groups Ester-based cleavable linking groups are cleaved by s such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm linking groups have the general formula -C(0)0-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.
Peptide-based cleaving groups Peptide-based cleavable linking groups are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., ides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (- C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding es and proteins and does not include the entire amide functional group. Peptide-based ble linking groups have the general formula - NHCHRAC(0)NHCHR C(0)-, where RA and R are the R groups of the two adjacent amino acids. These candidates can be evaluated using s analogous to those described above.As used , "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which may be , branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic), with an , nitrogen or sulfur atom bonded to each carbon atom.
Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides ning from about 4-9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and ccharide gums. Specific monosaccharides include C and above (preferably C -C8) sugars; di- and trisaccharides e sugars having two or three monosaccharide units (preferably C -C8) .
Alternative embodiments In another embodiment, the invention s to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the antisense strand. Every nucleotide in the sense strand and nse strand has been ed. The modifications on sense strand and antisense strand each independently comprises at least two different cations.
In another embodiment, the invention relates to a dsR A agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least one motif of three identical modifications on three utive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the antisense strand. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides. The cation pattern of the antisense strand is shifted by one or more nucleotides relative to the modification pattern of the sense strand.
In another embodiment, the ion relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an nse strand, each strand having 14 to 30 nucleotides. The sense strand ns at least two motifs of three identical modifications on three consecutive nucleotides, when at least one of the motifs occurs at the cleavage site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at the cleavage site by at least one tide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site in the strand and at least one of the motifs occurs at another n of the strand that is separated from the motif at or near cleavage site by at least one nucleotide.
In another embodiment, the invention relates to a dsRNA agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 30 nucleotides. The sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand and at least one of the motifs occurs at r portion of the strand that is separated from the [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm motif at the ge site by at least one tide. The antisense strand contains at least one motif of three identical modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the ge site in the strand and at least one of the motifs occurs at another portion of the strand that is separated from the motif at or near cleavage site by at least one nucleotide. The modification in the motif occurring at the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand. In another embodiment, the invention relates to a dsR A agent e of ting the expression of a target gene. The dsR A agent comprises a sense strand and an antisense strand, each strand having 1 to nucleotides. The sense strand contains at least one motif of three 2'-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at the cleavage site in the strand. The antisense strand contains at least one motif of three 2'methyl modifications on three consecutive tides.
The sense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at r portion of the strand that is separated from the three 2'-F modifications at the cleavage site by at least one nucleotide. The antisense strand may further comprises one or more motifs of three identical modifications on three consecutive nucleotides, where the one or more additional motifs occur at another portion of the strand that is separated from the three ethyl modifications by at least one nucleotide. At least one of the nucleotides having a 2'-F cation may form a base pair with one of the nucleotides having a 2'methyl modification.
In one embodiment, the dsRNA of the ion is administered in buffer.
In one embodiment, siR A compounds described herein can be formulated for administration to a subject. A formulated siRNA ition can assume a variety of states. In some examples, the composition is at least lly crystalline, mly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, e.g., in a solution that includes water.
The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the siRNA [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self- assembly.
A siRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a siRNA, e.g., a protein that complexes with siRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg +), salts, RNAse tors (e.g., a broad icity RNAse inhibitor such as RNAsin) and so forth.
In one embodiment, the siRNA ation includes another siNA compound, e.g., a second siRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species. Such siRNAs can e RNAi with respect to a similar number of different genes.
In one embodiment, the siRNA preparation es at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA). For example, a siRNA composition for the treatment of a viral disease, e.g., HIV, might include a known antiviral agent (e.g., a se inhibitor or reverse transcriptase inhibitor). In another e, a siRNA ition for the treatment of a cancer might further comprise a chemotherapeutic agent.
Exemplary formulations are discussed below.
Liposomes. For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNAs, and such practice is within the invention. An siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a sor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a -stranded siRNA compound, or ssiRNA nd, or precursor thereof) [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm preparation can be ated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an s or. The aqueous portion contains the siR A composition. The lipophilic material isolates the aqueous interior from an s exterior, which typically does not include the siRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological nes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular nes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the siRNA are red into the cell where the siRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the mes are also specifically targeted, e.g., to direct the siRNA to ular cell types.
A liposome containing a siRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an athic ic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be ic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The siRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the siRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of siRNA.
If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid {e.g., ne or spermidine). pH can also adjusted to favor sation.
Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a cleotide/cationic lipid x as structural [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in r, P. L . et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M . Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim.
Biophys. Acta 728:339, 1983; and Fukunaga, et al. inol. 115:757, 1984.
Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging siRNA preparations into liposomes.
Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. heless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to r DNA encoding the thymidine kinase gene to cell monolayers in e. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).
One major type of liposomal composition es phospholipids other than naturally-derived atidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl atidylcholine (DPPC). Anionic liposome itions generally are formed from dimyristoyl phosphatidylglycerol, while c fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or atidylcholine and/or cholesterol.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Examples of other methods to uce liposomes into cells in vitro and include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 50, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; n, m. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.
In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to r siRNAs to macrophages.
Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation f, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker , 1988, volume 1, p . 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the s volume of the liposomes.
A positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]- N ,N ,N -trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell nes of tissue culture cells, resulting in ry of siRNA (see, e.g., Feigner, P. L . et al., Proc. Natl.
Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).
A DOTMA analogue, l,2-bis(oleoyloxy)(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise vely d DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged mes are used, the net charge on the resulting complexes is also positive.
Positively charged complexes prepared in this way spontaneously attach to negatively [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. r commercially ble cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, a) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.
Other reported cationic lipid compounds include those that have been conjugated to a variety o f moieties including, for example, carboxyspermine which has been conjugated to one o f two types of lipids and includes compounds such as 5- carboxyspermylglycine dioctaoleoylamide ("DOGS") (Transfectam™, Promega, Madison, Wisconsin) and itoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).
Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Choi") which has been formulated into liposomes in combination with DOPE (See, Gao, X . and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991).
Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to b e effective for transfection in the presence o f serum (Zhou, X . et al., Biochim. Biophys.
Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated ic lipids, are said to exhibit lower toxicity and provide more ent transfection than the DOTMA-containing compositions. Other cially available cationic lipid products include DMRIE and DMRIE-HP (Vical, L a Jo 11a, California) and Lipofectamine (DOSPA) (Life Technology, Inc., rsburg, nd). Other cationic lipids suitable for the delivery of oligonucleotides are described in W O 98/39359 and W O 96/37194. mal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA, into the skin. In some implementations, liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal s, e.g., into skin. For example, the mes can b e applied topically.
Topical delivery o f drugs formulated as liposomes to the skin has been nted (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm Antiviral Research, 18, 1992, 259-265; Mannino, R . J . and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C . et al.
Meth. Enz. 149:157-176, 1987; Straubinger, R . M . and Papahadjopoulos, D . Meth. Enz. 101:512-527, 1983; Wang, C . Y . and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851- 7855, 1987).
Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems sing non-ionic tant and cholesterol. Non-ionic liposomal ations comprising me I (glyceryl dilaurate/cholesterol/polyoxyethylenestearyl ether) and Novasome II (glyceryl distearate/ cholesterol/polyoxyethylenestearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with siRNA are useful for ng a dermatological disorder.
Liposomes that include siRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding e edge activators, y surfactants, to a standard liposomal composition. Transfersomes that include siRNA can be delivered, for e, subcutaneously by infection in order to deliver siRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self- loading.
Other ations amenable to the present ion are described in United States provisional application serial nos. 61/018,616, filed January 2, 2008; 61/018,61 1, filed January 2, 2008; 61/039,748, filed March 26, 2008; 61/047,087, filed April 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no , filed October 3, 2007 also describes formulations that are amenable to the t invention.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm tants. For ease of exposition the formulations, itions and methods in this section are discussed largely with regard to fied siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNA nds, and such ce is within the scope of the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). siRNA (or a precursor, e.g., a larger dsiRNA which can be processed into a siRNA, or a DNA which encodes a siRNA or precursor) compositions can include a surfactant. In one embodiment, the siRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and g the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in "Pharmaceutical Dosage Forms," Marcel Dekker, Inc., New York, NY, 1988, p . 285).
If the surfactant le is not ionized, it is classified as a ic surfactant.
Nonionic tants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants e nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxy ethylene surfactants are the most popular members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and lated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl es and sulfosuccinates, and phosphates.
The most important members of the anionic tant class are the alkyl sulfates and the soaps.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.
If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as eric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.
The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in "Pharmaceutical Dosage " Marcel Dekker, Inc., New York, NY, 1988, p . 285).
Micelles and other Membranous Formulations. For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified siRNA compounds. It may be understood, however, that these micelles and other ations, compositions and methods can be practiced with other siRNA compounds, e.g., modified siRNA compounds, and such practice is within the invention. The siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, {e.g., a precursor, e.g., a larger siRNA nd which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof)) composition can be provided as a micellar formulation. "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed , leaving the hydrophilic portions in contact with the surrounding aqueous phase.
The se arrangement exists if the nment is hobic.
A mixed micellar formulation suitable for delivery h transdermal membranes may be ed by mixing an aqueous solution of the siRNA composition, an alkali metal C to C alkyl sulphate, and a micelle forming compounds. ary e forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile t, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, urates, borage oil, evening of primrose oil, l, trihydroxy oxo cholanyl [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm glycine and pharmaceutically acceptable salts thereof, in, polyglycerin, lysine, polylysine, triolein, polyoxy ethylene ethers and ues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.
The e forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.
In one method a first micellar composition is prepared which contains the siR A ition and at least the alkali metal alkyl te. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by on of the remaining micelle forming compounds, with vigorous mixing.
Phenol and/or ol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial . Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as in may also be added after formation of the mixed micellar composition.
For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The lant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and lant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.
Propellants may include hydrogen-containing chlorofluorocarbons, hydrogencontaining fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.
The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption h the oral cavities, it is often desirable to se, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract. ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Particles. For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds.
It may be understood, however, that these particles, formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the invention. In another embodiment, an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, {e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be ed by spray-drying, but may also be produced by other methods including lyophilization, ation, fluid bed drying, vacuum drying, or a combination of these techniques.
Pharmaceutical itions The iRNA agents of the ion may be formulated for pharmaceutical use.
Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the the dsRNA agents in any of the preceding ments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and/or diluents.
The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches us or non-aqueous ons or suspensions), tablets, e.g., those targeted for , sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or ned-release formulation; (3) l application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or ectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly ageous.
The phrase "therapeutically-effective amount" as used herein means that amount of a compound, material, or ition comprising a compound of the invention which [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm is effective for producing some d therapeutic effect in at least a pulation of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without ive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, ed in ng or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being ible with the other ingredients of the ation and not injurious to the t.
Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, seed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; ( 1 1) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl e; (13) agar; (14) buffering agents, such as magnesium hydroxide and um hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) ic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum ent, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in ceutical formulations.
The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of stration.
The amount of active ingredient which can be combined with a r material to produce a single dosage form will lly be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0 .1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, e forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally ilable a compound of the present invention. iR A agent preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg +), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.
Methods of preparing these formulations or compositions include the step of bringing into ation a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and tely bringing into association a compound of the t invention with liquid carriers, or finely divided solid carriers, or both, and then, if ary, shaping the product.
In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or uscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of ution which, in turn, may depend upon crystal size and crystalline form.
[Annotation] kjm None set by kjm ation] kjm ionNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm Alternatively, delayed absorption of a parenterally-administered drug form is lished by dissolving or suspending the drug in an oil vehicle.
The compounds according to the invention may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals .
The term "treatment" is ed to encompass also prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.
Double-stranded RNAi agents are produced in a cell in vivo, e.g., from exogenous DNA templates that are delivered into the cell. For example, the DNA templates can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous ion, local administration (U.S.
Pat. No. 470), or by stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 9 1:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene ry vehicle is imbedded. The DNA templates, for example, can include two transcription units, one that produces a transcript that es the top strand of a dsRNA agent and one that produces a transcript that includes the bottom strand of a dsRNA agent. When the templates are transcribed, the dsRNA agent is produced, and processed into siRNA agent fragments that mediate gene silencing.
Routes of Delivery A composition that es an iRNA can be red to a subject by a variety of routes. ary routes include: enous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.
The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more s of iRNA and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and [Annotation] kjm None set by kjm [Annotation] kjm ionNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm absorption delaying , and the like, compatible with pharmaceutical administration.
The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any tional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. mentary active compounds can also be incorporated into the compositions.
The compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.
The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iR A in aerosol form. The vascular endothelial cells could be targeted by coating a balloon er with the iRNA and ically introducing the DNA.
Dosage In one aspect, the invention features a method of administering a dsRNA agent, e.g., a siRNA agent, to a subject {e.g., a human subject). The method includes administering a unit dose of the dsRNA agent, e.g., a siRNA agent, e.g., double stranded siRNA agent that (a) the double-stranded part is 14-30 nucleotides (nt) long, for example, 21-23 nt, (b) is complementary to a target RNA {e.g., an endogenous or pathogen target RNA), and, ally, (c) includes at least one 3'overhang 1-5 nucleotide long. In one ment, the unit dose is less than 10 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent {e.g., about 4.4 x 10 16 copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, , 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.
The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection {e.g., intravenous, subcutaneous or [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm uscular), an inhaled dose, or a topical application. In some ebmodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg/kg of body weight.
In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency {e.g., not a regular frequency). For example, the unit dose may be stered a single time.
In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than a dsRNA agent, e.g., other than a siR A agent.
In another embodiment, the t has atherosclerosis and the effective dose of a dsRNA agent, e.g., a siRNA agent, is stered in combination with, e.g., after surgical intervention, e.g., angioplasty.
In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a dsRNA agent, e.g., a siRNA agent, {e.g., a precursor, e.g., a larger dsRNA agent which can be processed into a siRNA agent, or a DNA which encodes a dsRNA agent, e.g., a siRNA agent, or precursor thereof). The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 mg to 15 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 1 mg per kg of bodyweight per day. The nance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment n may last for a period of time which will vary depending upon the nature of the particular e, its severity and the overall condition of the t. In certain embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days.
Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the e state is observed, if the disease state has been ablated, or if undesired ffects are observed.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm The effective dose can be administered in a single dose or in two or more doses, as desired or ered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.
In one embodiment, the composition includes a plurality of dsRNA agent s.
In r embodiment, the dsRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence.
In another embodiment, the plurality of dsRNA agent species is specific for different naturally occurring target genes. In another embodiment, the dsRNA agent is allele specific.
The dsRNA agents of the invention described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.
In one ment, the stration of the dsRNA agent, e.g., a siRNA agent, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, aneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a ser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.
The invention provides methods, compositions, and kits, for rectal administration or delivery of dsRNA agents described herein s of inhibiting expression of the target gene ments of the invention also relate to methods for inhibiting the expression of a target gene. The method comprises the step of administering the dsRNA agents in any of the preceding embodiments, in an amount sufficient to inhibit expression of the target gene.
Another aspect the invention relates to a method of ting the expression of a target gene in a cell, comprising ing to said cell a dsRNA agent of this invention.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm ed set by kjm In one embodiment, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CPvK gene, GRB2 gene, RAS gene, MEK gene, JNK gene, RAF gene, Erkl/2 gene, PCNA (p21 ) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, en, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAFl/CIPl) gene, mutations in the p27(KIPl) gene, mutations in the PPM ID gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor ssor genes, and mutations in the p53 tumor suppressor gene.
The invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly orated by reference.
EXAMPLES Example 1. In vitro screening of siRNA duplexes Cell culture and trans fections: Human Hep3B cells or rat H.II.4.E cells (ATCC, Manassas, VA) were grown to near confluence at 37 °C in an atmosphere of 5% C0 2 in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 mΐ of Opti-MEM plus 0.2 mΐ of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778- 150) to 5 mΐ of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 mΐ of complete growth media t antibiotic containing ~2 xlO4 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA cation. Single dose experiments were med at IOhM and O.lnM final duplex concentration and dose response [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm experiments were done using 8, 4 fold serial dilutions with a maximum dose of lOnM final duplex concentration.
Total RNA isolation using DYNABEADS mRNA Isolation Kit flnvitrogen. part # : 610- Cells were harvested and lysed in 150 mΐ of Lysis/Binding Buffer then mixed for minute at 850rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 mΐ Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were ed using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, ic beads were washed 2 times with 150 mΐ Wash Buffer A and mixed for 1 minute. Beads were capture again and supernatant removed. Beads were then washed with 150 mΐ Wash Buffer B, captured and supernatant was removed. Beads were next washed with 150 mΐ n Buffer, captured and supernatant removed. Beads were allowed to dry for 2 minutes. After drying, 50 mΐ of Elution Buffer was added and mixed for 5 s at 70°C. Beads were captured on magnet for 5 minutes. 40 mΐ of atant was d and added to another 96 well plate. cDNA sis using ABI High capacity cDNA reverse transcription kit (Applied Biosvstems, Foster City, CA, Cat #4368813): A master mix of 1 mΐ 10X Buffer, 0.4m125X dNTPs, I mΐ Random primers, 0.5 mΐ Reverse Transcriptase, 0.5 mΐ RNase inhibitor and 1.6m1of H20 per reaction were added into 5 mΐ total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 thermal cycler (Hercules, CA) through the ing steps: 25 °C 10 min, 37 °C 120 min, 85 °C 5 sec, 4 °C hold.
Real time PCR: 2m1of cDNA were added to a master mix containing 0.5m1GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E (human) Cat # 3 (rodent)), 0.5 m1TTR [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm TaqMan probe (Applied Biosystems cat # HS00174914 m l (human) cat # Rn00562124_ml (rat)) and 5m1Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plate (Roche cat # 04887301001). Real time PCR was done in a Roche LC 480 Real Time PCR machine (Roche). Each duplex was tested in at least two ndent transfections and each transfection was assayed in duplicate, unless otherwise noted.
To calculate relative fold change, real time data were analyzed using the DD method and normalized to assays performed with cells transfected with lOnM AD- 1955, or mock transfected cells. IC50S were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD- 1955 or na ve cells over the same dose range, or to its own lowest dose. IC50S were ated for each individual transfection as well as in combination, where a single IC50 was fit to the data from both transfections.
The results of gene silencing of the exemplary siRNA duplex with various motif cations of the invention are shown in the table below.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm Example 2. RNA Synthesis and Duplex Annealing 1. Oligonucleotide Synthesis: All oligonucleotides were synthesized on an AKTAoligopilot synthesizer or an ABI 394 izer. Commercially available lled pore glass solid support (dT- CPG, 500A, Prime Synthesis) and RNA phosphoramidites with standard protecting groups, 5'dimethoxytrityl N6-benzoyl-2'-t-butyldimethylsilyl-adenosine-3 '- - N ,N '- diisopropylcyanoethylphosphoramidite, 5'dimethoxytrityl-N4-acetyl-2 '-t- butyldimethylsilyl-cytidine-3 'N,N'-diisopropylcyanoethylphosphoramidite, 5' dimethoxytrityl-N2~isobutryl-2 '-t-butyldimethylsilyl-guanosine-3 '-O-N,N '-diisopropyl- 2-cyanoethylphosphoramidite, and 5'dimethoxytrityl-2 '-t-butyldimethylsilyl-uridine- 3'N,N'-diisopropylcyanoethylphosphoramidite (Pierce Nucleic Acids Technologies) were used for the oligonucleotide synthesis unless otherwise specified.
The 2'-F phosphoramidites, 5'-O-dimethoxytrityl-N4-acetyl-2'-fluro-cytidine-3 '-O- N,N'-diisopropylcyanoethyl-phosphoramidite and 5'-O-dimethoxytrityl-2 '-flurouridine-3'N ,N'-diisopropylcyanoethyl-phosphoramidite were purchased from (Promega). All phosphoramidites were used at a concentration of 0.2M in acetonitrile (CH3CN) except for guanosine which was used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recycling time of 16 minutes was used. The activator was 5-ethyl thiotetrazole (0.75M, American International Chemicals), for the PO-oxidation Iodine/Water/Pyridine was used and the PS-oxidation PADS (2 %>) in 2,6-lutidine/ACN (1:1 v/v) was used. .
Ligand conjugated s were synthesized using solid support ning the corresponding ligand. For example, the introduction of carbohydrate moiety/ligand (for e.g., ) at the 3'-end of a sequence was achieved by starting the synthesis with the corresponding carbohydrate solid support. Similarly a cholesterol moiety at the 3'-end was uced by ng the synthesis on the cholesterol support. In general, the ligand moiety was tethered to hydroxyprolinol via a tether of choice as described in the previous examples to obtain a ypro lino 1- ligand moiety. The hydroxyprolinolligand moiety was then coupled to a solid support via a succinate linker or was converted to phosphoramidite via standard itylation conditions to obtain the desired ydrate conjugate building blocks. Fluorophore labeled siRNAs were synthesized [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm from the corresponding phosphoramidite or solid support, purchased from Biosearch Technologies. The oleyl lithocholic (GalNAc)3 polymer support made in house at a loading of 38.6 ram. The Mannose (Man)3 polymer support was also made in house at a loading of 42.0 umol/gram.
Conjugation of the ligand of choice at desired on, for example at the 5'-end of the sequence, was achieved by coupling of the corresponding phosphoramidite to the growing chain under standard phosphoramidite ng conditions unless otherwise specified. An extended 15 min coupling of 0.1 M solution of phosphoramidite in anhydrous CH3CN in the ce of 5-(ethylthio)-l H-tetrazole activator to a solid bound oligonucleotide. Oxidation of the internucleotide phosphite to the phosphate was carried out using standard iodine-water as reported (1) or by treatment with tert-butyl hydroperoxide/acetonitrile/water (10: 87: 3) with 10 min oxidation wait time conjugated oligonucleotide. Phosphorothioate was introduced by the oxidation of ite to phosphorothioate by using a sulfur transfer reagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucage reagent The cholesterol phosphoramidite was synthesized in house, and used at a concentration of 0.1 M in dichloromethane. Coupling time for the cholesterol phosphoramidite was 16 s. 2. Deprotection- 1 (Nucleobase Deprotection) After completion of synthesis, the support was transferred to a 100 ml glass bottle (VWR). The ucleotide was cleaved from the support with simultaneous ection of base and phosphate groups with 80 mL of a mixture of ethanolic a [ammonia: ethanol (3:1)] for 6.5h at 55°C. The bottle was cooled briefly on ice and then the ethanolic ammonia mixture was filtered into a new 250 ml bottle. The CPG was washed with 2 x 40 mL portions of l/water (1:1 v/v). The volume of the mixture was then reduced to ~ 30 ml by roto-vap. The mixture was then frozen on dry ice and dried under vacuum on a speed vac. 3. Deprotection-II (Removal of 2' TBDMS group) The dried residue was ended in 26 ml of triethylamine, triethylamine trihydro fluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6) and heated at 60°C for 90 s to remove the t rt-butyldimethylsilyl (TBDMS) groups at the 2' position. The [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm reaction was then quenched with 50 ml of 20mM sodium acetate and pH adjusted to 6.5, and stored in freezer until purification. 4. Analysis The oligoncuelotides were analyzed by high-performance liquid chromatography (HPLC) prior to purification and selection of buffer and column depends on nature of the sequence and or conjugated .
. HPLC Purification The ligand conjugated oligonucleotides were purified reverse phase preparative HPLC. The unconjugated oligonucleotides were ed by anion-exchange HPLC on a TSK gel column packed in house. The buffers were 20 mM sodium phosphate (pH 8.5) in % CH3CN (buffer A) and 20 mM sodium ate (pH 8.5) in 10% CH3CN, 1M NaBr (buffer B). Fractions containing full-length ucleotides were pooled, desalted, and lyophilized. imately 0.15 OD of desalted oligonucleotidess were d in water to 150 mΐ and then pipetted in special vials for CGE and LC/MS analysis.
Compounds were finally analyzed by LC-ESMS and CGE. 6. siRNA preparation For the preparation of siRNA, equimolar amounts of sense and antisense strand were heated in lxPBS at 95°C for 5 min and slowly cooled to room temperature.
Integrity of the duplex was confirmed by HPLC analysis.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm duplex 3 modified Table 2.
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Egg 353000003222053005300330: 80m3202300000H00F00<030<3005m 23530350502323005m Same/0023290350502:2100800% 86m32023506350500<u3230030,... 8093202350635050050208030 80933023290320502523905m EmmefizfiumouEvoke/00329605m Same/62350535050250208030 $30830:008030380302000300 ficfiéiByoSS#0002323833 Same/623530350502323005m a0200230000308§00§<0<EE§0 fic§§<30$o£005230230039... 8093202350538500502000030 0$o£0050053030039... $922:000000022302... 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$08 $08 0003 0008 0003 088 :08 N88 $08 E08 £08 088 808 TE $08 $08 $08 $08 808 B08 0008 0008 0008 808 N008 $08 $08 $08 0008 $08 0008 0008 088 :08 N88 $08 E08 £08 088 0000 000.0 000.0 000.0 00:.0 00:.0 00:.0 “:0 0:.0 00:0 0:.0 0:.0 0:.0 0:.0 0:.0 0:.0 00:0 2:0 2:0 9:0 22:0 :0:.0 00:.0 0:0 00:.0 00:0 00:.0 00.0 2.0 EEEEEEEEEEEEEEEEEEa!EEEEEEEEEaHEEa EE 00.0 :0 00.0 :0 :0 :.0 :.0 00.0 :0 00.0 :.0 :0 :.0 0:.0 00.0 00.0 EggEgg :0 EggEEEEggEggEEEEgg 00°600202:50003005020230052 0000032023000030050200230020 006032023005300502002000020 0000002023005300823220005m 000003202300530050232800320 00000202:030003000302032000.0000m 20230000300523020000300 202:00003005025230.0050 00°60:20m9505300502520505m 00005£02:39003050232000.0000m 0060320230063005000230052 000032023005300502323005m 000003202300003005000200005m 006032023005300502523005m 000000202300003005020200005m 860320230006300823230052 005052023005300820200005m 00°60020230063005003200905m 00°6002023000030082023005m 0060320230063005000220005m 00000020m95053005023220005m 00°6002023006300502:$0905m 0000032023000630050250230020 0050020230063005020200005m 00°600202:500005082023905m 0050:2005:02032000023200.0050 0060020255003005020200005m R0:2 08:2 08:2 000:2 :00:2 000:2 000:2 000:2 000:2 000:2 000:2 000:2 000:2 000:2 :00:2 000:2 000:2 000:2 000:2 000:2 000:2 000:2 000:2 00:2 :0:2 00:2 00:2 300000502303032000230.0302 32303032000230.0302 3200003032030028023032 500000:023020002000£23032 002880502303032000230.0002 500000532300038023032 50300532303032000230.0302 5000005:03030320002230.0302 5000005:03030320000250032 500000532300022822005m 0030053250000002000£23002 50300532303032000230.0302 5000005025500002000023032 50300533030320003230.0302 02000030520300282032 003005323020002000023032 5000005323002532000323000 50805325600602000023032 5000005023020002000£23032 50£003025600602000023032 50£000023030020000250032 50805523000602000023032 500000502303032060230.0302 532303032802002 50805023020002000023032 500000532300008000230.0002 503005302000303200000030032 R0:0 000:0 000:0 000:0 :00:0 00:0 000:0 00:0 000:0 000:0 000:0 00:0 000:0 000:0 00::0 :0:0 00:0 00:0 R0:0 Ea 000:0 :00:0 00:0 000:0 00:0 000:0 000:0 00:0 000:0 000:0 000:0 :00:0 000:0 000:0 00:0 000:0 000:0 00:0 000:0 000:0 00::0 :0:0 00:0 00:0 mod ad :6 ad wad End mod mmupomPEuEEPGEsupbuuFDumsuF<muF0muFDm mmupomPEuEEDMUFOEDUUPDUUEPEUEUFOMBm mmupwmsuguupflabmupogb3933033095m PEuEEPGEEDUSUEPEEOMUFDm mmupwmsuEuEEDmuGEDuu5uup<3<£wmu5m mmupomPEG$053095uu5uup<sup<£oupwu5m mmupwmPEfiEEDmGEDfiDUE:$0595m 80530306033290305$:£<3<<t00t3<t mmupomPEGm3050505EDUUFDUUEPEEOMUFDm mmupomPEuEEDMUFO:u5u050603303m05mu5m 805950393305303059533033033m mmupwm3030603305303059533033095m mmupwmsuEuEEDmuGP5UU5UUF<HU<U£OOUHE mmupwmPEfiflDBmG3059533033033m mmmfl238$30F30505::30605060305030305050503 mmupwmsuEuEEDmuGEDuu5uup<3<£wmu5m mmupwmsuEuEEDmuGEDuu5uup<3<£wmu5m mmupomPEuEEDUPOUGEDUUPDUUEPEEOMBm mmupom:80330530305uu5uup<sup<£oupwu5m uFDuEqu::muFOEDuuFDquPEflmem 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movfio wovfio movfio wovfio movfio Davao HHvHQ NHvHQ mavfio vfivfio mHvHQ ofivfio mfivfio vaHQ mHvHQ Example 3 : In vitro silencing activity with various chemical modifications on TTR siRNA The IC for each modified siRNA is determined in Hep3B cells by standard e transfection using Lipofectamine RNAiMAX. In brief, reverse transfection is carried out by adding 5 m of Opti-MEM to 5 of siRNA duplex per well into a 96- well plate along with 10 of EM plus 0.5 mΐ of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) and incubating at room temperature for -20 minutes. Following tion, 100 mΐ of complete growth media without antibiotic containing 12,000-15,000 Hep3B cells is then added to each well. Cells are incubated for 24 hours at 37°C in an atmosphere of 5% C02 prior to lysis and analysis of ApoB and GAPDH niRNA by bDNA (Quantigene). 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Transfection was carried out by adding 14.8 mΐ of Opti-MEM plus 0.2m1of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad CA. cat # 13778-150) to 5 mΐ of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 s. 80 mΐ of complete growth media without antibiotic containing 2 xlO4 Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA cation. Single dose ments were performed at IOhM and O.lnM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1,0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated. cDNA sis using ABI High capacity cDNA e transcription kit (Applied Biosystems, Foster City, CA, Cat #4368813) A master mix of 2m1 10X Buffer, 0.8 mΐ 25X dNTPs, 2m1Random primers, I mΐ Reverse Transcriptase, 1 mΐ RNase inhibitor and 3.2m1of H20 per reaction were added into IOmI total RNA. cDNA was generated using a Bio-Rad C-1000 or S-1000 l cycler (Hercules, CA) through the following steps: 25°C 10 min, 37°C 120 min, 85°C 5 sec, 4°C hold.
Real time PCR 2 mΐ of cDNA was added to a master mix ning 0.5 m1GAPDH TaqMan Probe (Applied Biosystems Cat #43263 17E), 0.5m1ANGPTL TaqMan probe (Applied Biosystems cat # Hs00205581_ml) and 5m1Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat # 04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the Q) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.
To calculate relative fold change, real time data was analyzed using the AACt method and normalized to assays performed with cells transfected with lOnM AD- 1955, or mock ected cells. IC50S were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD- 1955 or na ve cells over the same dose range, or to its own lowest dose. AD- 1955 sequence, used as a ve control, targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT; antisense : uACUcAGCGuAAGdTsdT .
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, foreign patents, foreign patent applications and non-patent publications referred to in this specification are incorporated herein by reference, in their ty. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, ations and publications to e yet r embodiments.
These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
[Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm ation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm

Claims (45)

1. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the duplex is represented by formula (III): sense: 5' np -Na -(X X X)i-Nb -Y Y Y -Nb -(Z Z Z)j -Na - nq 3' antisense: 3' np’-Na’-(X’X′X′)k-Nb’-Y′Y′Y′-Nb’-(Z′Z′Z′)l-Na’- nq’ 5' (III) wherein: i, j, k, and l are each independently 0 or 1; p and q are each independently 0-6; each Na and Na’independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides, each Nb and Nb’independently represents an oligonucleotide sequence comprising 1-10 modified nucleotides; each np, np’, nq and nq’ independently ents an overhang nucleotide sequence comprising 0-6 nucleotides; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; wherein the modification on Nb is ent than the cation on Y and the modification on Nb’is different than the modification on Y’; and n the Y’Y’Y’ motif occurs at the 11, 12 and 13 positions of the antisense strand from the .
2. The double-stranded RNAi agent of claim 1, wherein i is 1; j is 1; or both i and j are 1.
3. The double-stranded RNAi agent of claim 1 or claim 2, wherein k is 1; l is 1; or both k and l are 1.
4. The double-stranded RNAi agent of any preceding claim, wherein the YYY motif occurs at or near the cleavage site of the sense strand.
5. The -stranded RNAi agent of any preceding claim, n the Y’ is 2’-OMe.
6. The double- stranded RNAi agent of claim 1, wherein formula (III) is represented as formula (IIIa): [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm 5' np -Na -Y Y Y -Nb -Z Z Z -Na-nq 3' 3' np’-Na’-Y′Y′Y′-Nb’-Z′Z′Z′-Na’nq’ 5' (IIIa) wherein each Nb and Nb’ independently represents an oligonucleotide sequence comprising 1-5 ed nucleotides.
7. The double-stranded RNAi agent of claim 1, n formula (III) is ented as formula (IIIb): 5' np-Na-X X X -Nb-Y Y Y -Na-nq 3' 3' np-Na-X′X′X′-Nb-Y′Y′Y′-Na-nq 5' (IIIb) wherein each Nb and Nb’ ndently ents an oligonucleotide sequence comprising 1-5 modified nucleotides.
8. The double-stranded RNAi agent of claim 1, wherein formula (III) is represented as formula (IIIc): 5' np-Na-X X X -Nb-Y Y Y -Nb-Z Z Z -Na-nq 3' 3' np-Na-X′X′X′-Nb-Y′Y′Y′-Nb-Z′Z′Z′-Na-nq 5' (IIIc) wherein each Nb and Nb’ independently represents an oligonucleotide sequence sing 1-5 modified nucleotides and each Na and Na’ independently represents an ucleotide sequence comprising 2-10 modified nucleotides.
9. The double-stranded RNAi agent of any one of claims 1 to 8, wherein the duplex region is 17-30 nucleotide pairs in length.
10. The double-stranded RNAi agent of any one of claims 1 to 8, wherein the duplex region is 17-19 nucleotide pairs in length.
11. The double-stranded RNAi agent of any one of claims 1 to 8, wherein the duplex region is 27-30 nucleotide pairs in length.
12. The double-stranded RNAi agent of any preceding claim, wherein each strand has 17-30 nucleotides. [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm
13. The double-stranded RNAi agent of any preceding claim, wherein the modifications on the nucleotides are ed from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O- alkyl, 2′-O-allyl, 2′-C- allyl, 2′-fluoro, 2’-deoxy, and combinations thereof.
14. The double-stranded RNAi agent of claim 13, wherein the nucleotides are ed with either 2’-OCH3 or 2’-F.
15. The double-stranded RNAi agent of any preceding claim, further comprising at least one ligand.
16. The double-stranded RNAi agent of claim 15, wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
17. The double-stranded RNAi agent of any one of claims 1 to 12, wherein the modifications on the nucleotides are selected from the group ting of 2'-O-methyl nucleotide, xyfluoro nucleotide, 2'-O-N-methylacetamido (2'-O-NMA) nucleotide, a 2'-O-dimethylaminoethoxyethyl (2'- O-DMAEOE) nucleotide, 2'-O-aminopropyl (2'-O-AP) nucleotide, 2'-ara-F, and combinations thereof.
18. The double-stranded RNAi agent of claim 15 or claim 16, wherein the ligand is attached to the 3’ end of the sense strand.
19. The -stranded RNAi agent of any preceding claim, further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.
20. The -stranded RNAi agent of any preceding claim, wherein the nucleotide at the 1 position of the 5’-end of the duplex in the antisense strand is ed from the group consisting of A, dA, dU, U, and dT.
21. The double-stranded RNAi agent of any ing claim, wherein the base pair at the 1 position of the 5’-end of the duplex is an AU base pair.
22. The double-stranded RNAi agent of any preceding claim, wherein the Y tides contain a 2’-fluoro modification.
23. The double-stranded RNAi agent of any preceding claim, wherein the Y’ nucleotides contain a 2’-O-methyl modification. [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm
24. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand contains at least two motifs of three identical modifications on three consecutive nucleotides, one of said motifs ing at the cleavage site in the strand and at least one of said motifs occurring at r portion of the strand that is separated from the motif at the cleavage site by at least one modified nucleotide, wherein the cation on the at least one modified nucleotide is different than the modification on the three consecutive modified nucleotides; and n the antisense strand contains at least a first motif of three identical modifications on three consecutive nucleotides, one of said motifs occurring at or near the ge site in the strand and a second motif occurring at another portion of the strand that is separated from the first motif by at least one modified tide, wherein the modification on the at least one ed nucleotide is different than the modification on the three consecutive modified nucleotides; wherein the modification in the motif occurring at the cleavage site in the sense strand is different than the modification in the motif occurring at or near the cleavage site in the antisense strand; and n the first or the second motif in the antisense strand occurs at the 11, 12 and 13 positions of the antisense strand from the .
25. The double-stranded RNAi agent of claim 24, wherein at least one of the nucleotides occurring at the cleavage site in the sense strand forms a base pair with one of the nucleotides in the motif occurring at or near the cleavage site in the antisense strand.
26. The double-stranded RNAi agent of claim 24 or claim 25, wherein the duplex has 17-30 nucleotides.
27. The double-stranded RNAi agent of claim 24 or claim 25, wherein the duplex has 17-19 nucleotides.
28. The double-stranded RNAi agent of claim 24 or claim 25, wherein each strand has 17-23 nucleotides.
29. The double-stranded RNAi agent of any one of claims 24 to 28, n the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, lkyl, 2′-O-allyl, 2′-C- allyl, 2′-fluoro, 2’-deoxy, and combinations thereof.
30. The double-stranded RNAi agent of claim 29, wherein the modifications on the nucleotide are 2’-OCH3 or 2’-F. [Annotation] kjm None set by kjm ation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm
31. The double-stranded RNAi agent of any one of claims 24 to 30, further comprising a ligand ed to the 3’ end of the sense strand.
32. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand contains at least one motif of three 2’-F modifications on three consecutive nucleotides, one of said motifs occurring at or near the cleavage site in the strand, and wherein a polynucleotide adjacent to the three consecutive nucleotides is a modified nucleotide having a cation other than a 2’-F modification; and wherein the antisense strand contains at least one motif of three 2’-O-methyl modifications on three consecutive nucleotides, one of said motifs occurring at or near the ge site, and wherein a polynucleotide adjacent to the three consecutive tides is a modified tide having a modification other than a 2’-O-methyl modification; and wherein one of the at least one motif in the antisense strand occurs at the 11, 12 and 13 ons of the antisense strand from the 5’-end.
33. The double-stranded RNAi agent of claim 32, wherein the sense strand comprises one or more motifs of three identical modifications on three utive nucleotides, said motifs occurring at another portion of the strand that is separated from the three 2’-F modifications at the cleavage site by at least one nucleotide.
34. The double-stranded RNAi agent of claim 32 or claim 33, wherein the antisense strand comprises one or more motifs of three cal modifications on three consecutive nucleotides, said motifs occurring at another portion of the strand that is separated from the three 2’-O-methyl modifications by at least one nucleotide.
35. The double-stranded RNAi agent of any one of claims 32 to 34, wherein at least one of the nucleotides having a 2’-F cation forms a base pair with one of the nucleotides having a 2’-O- methyl modification.
36. The double-stranded RNAi agent of any one of claims 32 to 34, wherein the duplex is 17-30 nucleotide pairs in length.
37. The double-stranded RNAi agent of any one of claims 32 to 36, wherein the duplex is 17-19 nucleotide pairs in length. ation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm
38. The double-stranded RNAi agent of any one of claims 32 to 37, wherein each strand has 17-23 tides.
39. The double-stranded RNAi agent of any one of claims 32 to 38, further comprising a ligand attached to the 3’ end of the sense strand.
40. A pharmaceutical composition comprising the double-stranded RNAi agent according to any one of the preceding claims alone or in combination with a pharmaceutically acceptable carrier or excipient.
41. Use of the double-stranded RNAi agent according to any one of claims 1 to 39 in the manufacture of a medicament for inhibiting the expression of a target gene.
42. The use of claim 41, wherein the medicament is provided for subcutaneous or intravenous administration.
43. Use of a dsRNA agent ented by formula (III): sense: 5' np -Na -(X X X) i-Nb -Y Y Y -Nb -(Z Z Z)j -Na - nq 3' antisense: 3' np’-Na’-(X’X′X′)k-Nb’-Y′Y′Y′-Nb’-(Z′Z′Z′)l-Na’- nq’ (III) wherein: i, j, k, and l are each independently 0 or 1; p and q are each independently 0-6; each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified tides; each Nb and Nb′ independently represents an oligonucleotide sequence comprising 1-10 ed nucleotides; each np, np’, nq and nq’ ndently represents an overhang nucleotide ce comprising 0-6 nucleotide ce; and XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three utive nucleotides; and wherein the modifications on Nb is different than the modification on Y and the modification on Nb’ is different than the modification on Y’; and wherein the Y’Y’Y’ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5’-end [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm Unmarked set by kjm [Annotation] kjm None set by kjm [Annotation] kjm MigrationNone set by kjm [Annotation] kjm ed set by kjm in the manufacture of a medicament for delivering a polynucleotide to a specific target in a subject.
44. The use of claim 43 , wherein the medicament is provided for uscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous or ospinal administration, or combinations thereof.
45. Use of the dsRNAi agent of any one of claims 1 to 39 in the manufacture of a medicament for delivering a polynucleotide to a specific target of a subject, wherein the medicament is provided for aneous administration.
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US201161561710P 2011-11-18 2011-11-18
US61/561,710 2011-11-18
PCT/US2012/065601 WO2013074974A2 (en) 2011-11-18 2012-11-16 Modified rnai agents

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