HK1178566B

HK1178566B - Oligonucleotide compounds comprising non-nucleotide overhangs

Info

Publication number: HK1178566B
Application number: HK13105356.2A
Authority: HK
Inventors: 莎伦．阿夫金-纳祖姆; 埃琳娜．费因斯坦
Original assignee: 夸克制药公司
Priority date: 2010-01-07
Filing date: 2011-01-06
Publication date: 2017-05-19

Abstract

The invention relates to siRNA compounds comprising one non-nucleotide moiety covalently attached to at least one of the sense or antisense strands to down-regulate the expression of human genes. The invention also relates to pharmaceutical compositions comprising such compounds and a pharmaceutically acceptable carrier and to methods of treating and/or preventing the incidence or severity of various diseases or conditions associated with the target genes and/or symptoms associated with such diseases or conditions.

Description

Oligonucleotide compounds comprising non-nucleotide overhangs

Throughout this application, various patent and scientific publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

RELATED APPLICATIONS

The present application claims priority from U.S. provisional patent application No.61/292878 filed on 7/1/2010, PCT patent application No. PCT/US2010/049047 filed on 16/9/2010, and PCT patent application No. PCT/US2010/059578 filed on 8/12/2010, which are hereby incorporated by reference in their entirety.

Technical Field

Disclosed herein are modified double-stranded nucleic acid molecules, pharmaceutical compositions comprising the nucleic acid molecules, and methods of their use for inhibiting mammalian and non-mammalian target genes. These compounds and compositions are therefore useful for treating subjects suffering from diseases or disorders in which gene expression has adverse consequences and/or symptoms associated with such diseases or disorders. In particular embodiments, the invention provides compositions comprising the nucleic acid molecules and methods of use thereof.

Background

RNA interference (RNAi) in mammals is mediated by small interfering RNA (siRNA) (Fire et al, Nature1998, 391: 806) or microRNA (miRNA) (Ambros, Nature2004, 431 (7006): 350-. The corresponding process in plants is commonly referred to as specific post-transcriptional gene silencing (PTGS) or RNA silencing, and in fungi is also referred to as suppression (quelling).

siRNA is a double-stranded RNA or modified RNA molecule that down-regulates or silences (prevents) the expression of the gene/mRNA of its endogenous (cellular) counterpart (counterpart). The mechanism of RNA interference is described in detail below.

PCT publication No. wo2008/050329 and U.S. No.11/978,089, assigned to the assignee of the present invention, are directed to inhibitors of pro-apoptotic genes and are incorporated by reference in their entirety. PCT patent publication nos. WO2008/104978 and WO2009/044392, assigned to the assignee of the present invention, are directed to chemically modified siRNA structures, which are incorporated by reference in their entirety.

Summary of The Invention

The present invention provides chemically and/or structurally modified siRNA compounds for use in inhibiting gene expression in general, and mammalian and prokaryotic gene expression in particular. Provided herein are novel structural motifs useful for preparing siRNA oligonucleotides comprising one or more non-nucleotide moieties as 3 ' (or 2 ') overhangs at the 3 ' -end, as well as compositions comprising the oligonucleotides and methods of use thereof. The applicants have determined that the addition of one, and preferably two or three, non-nucleotide moieties to the 3' end of the siRNA provides the siRNA with advantageous properties in terms of activity and/or stability and/or delivery. Thus, existing sirnas can be advantageously modified, and future sirnas can be designed and prepared to take advantage of this finding. Without wishing to be bound by theory, the nucleic acid molecules disclosed herein and having a 3 'non-nucleotide overhang (Z or Z', i.e., C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb) are recognized by the PAZ domain of Argonaute and are capable of performing RNAi while exhibiting good stability and activity.

It should be understood that although the term "3 'terminus" is used throughout this application to refer to the end of the siRNA strand to which the non-nucleotide moiety is attached, unless specifically stated otherwise, the non-nucleotide moiety or "overhang" may be attached at the 3' position of the (deoxy) ribose moiety at the 3 '-terminus of the oligonucleotide strand or at the 2' position of the (deoxy) ribose moiety at the 3 '-terminus of the oligonucleotide strand, e.g., at the 3' -terminus of the oligonucleotide strand(attached to the 3' -terminal (deoxy) ribose moiety

The 3' position of the molecule; in the figure, B is a nucleotide base and R is H or OH) or(attached at the 2 '-position of the 3' -terminal (deoxy) ribose moiety).

Provided herein are novel structures of double stranded nucleic acid molecules that have advantageous properties and are applicable to sirnas against any target sequence, comprising non-nucleotide overhangs at one or both 3' ends of the duplex. The chemically modified siRNA modifications disclosed herein can be used to prepare stable and active siRNA compounds useful for RNA interference (RNAi).

The present application also provides pharmaceutical compositions comprising one or more such oligonucleotides and methods of treating or preventing the incidence or severity of a disease or disorder in a subject in need thereof, wherein the disease or disorder and/or a symptom associated therewith is associated with expression of a target gene. In some embodiments, the disease or disorder is selected from the group consisting of: hearing loss, Acute Renal Failure (ARF), glaucoma, Acute Respiratory Distress Syndrome (ARDS) and other acute lung and respiratory tract injuries, ischemia-reperfusion injury following lung transplantation, ocular ischemic conditions, injury in organ transplantation (including lung, liver, heart, pancreas and kidney transplantation) and including delayed graft function recovery (DGF), nephrotoxicity and neurotoxicity, spinal cord injury, bedsores, age-related macular degeneration (AMD), dry eye syndrome, oral mucositis, Ischemic Ocular Neuropathy (ION) and Chronic Obstructive Pulmonary Disease (COPD). Such methods involve administering to a mammal in need of such treatment a prophylactically or therapeutically effective amount of one or more such compounds that inhibits or reduces the expression or activity of at least one such gene. Such compounds may be administered with or in place of other therapies.

The oligonucleotides are selected to target any mammalian or non-mammalian gene. In various embodiments, the modified compound comprises a peptide as set forth in seq id no: 97-68654 (disclosed in U.S. patent No.11/978,089 and PCT patent application No. PCT/IL2007/001278, which are hereby incorporated by reference in their entirety).

In one aspect, double stranded siRNA compounds comprising at least one non-nucleotide 3' end overhang are provided. The present application provides a synthetic double stranded siRNA compound comprising a sense strand and an antisense strand, wherein at least one of the sense or antisense strand comprises 1, 2, 3, 4, or 5, preferably 2 or 3, non-nucleotide moieties covalently attached at the 3' terminus, wherein the non-nucleotide moieties are selected from the group consisting of inverted abasic moieties, alkyl (hydrocarbon) moieties or derivatives thereof, and phosphate-based moieties. In some embodiments, the non-nucleotide moiety is selected from an inverted abasic moiety, an alkyl (hydrocarbon) moiety or derivative thereof, and a phosphate-based moiety. In some embodiments, the non-nucleotide moiety comprises an alkyl (hydrocarbon) moiety or a derivative thereof. Provided herein are double stranded nucleic acid molecules comprising a sense strand and an antisense strand, wherein at least one strand comprises a non-nucleotide moiety covalently attached at the 3 ' or 2' position of the sugar residue at the 3 ' terminal nucleotide of the strand in which it is present; wherein the non-nucleotide moiety is selected from the group consisting of: propanol, a C3 alkyl moiety linked to a phosphodiester, a C3 alkyl moiety linked to a phosphorothioate, a deoxyribose abasic moiety, a ribose abasic moiety, and combinations thereof.

In some embodiments, the non-nucleotide moiety is linked to the sugar residue by a phosphate-based linkage (preferably a phosphodiester or phosphorothioate linkage). In some embodiments, the non-nucleotide moiety comprises a C3 alkyl moiety covalently attached at the 3 ' or 2' position of the sugar residue at the 3 ' terminus of the antisense strand. In various embodiments, the C3 alkyl moiety is selected from C3Pi and C3 OH.

In some embodiments, the molecule comprises two or three C3 alkyl moieties covalently linked by phosphodiester or phosphorothioate linkages or one C3 alkyl moiety covalently linked by phosphodiester or phosphorothioate linkages to abasic moieties. In a preferred embodiment, the molecule comprises two C3 alkyl moieties covalently linked by a phosphodiester or phosphorothioate linkage.

In some embodiments, the C3 alkyl moiety is selected from: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Pi-C3Pi-C3OH, C3Ps-C3Ps-C3OH, C3Pi-C3Ps-C3OH, C3Ps-C3Pi-C3OH, C3Pi-C3Pi-C3Pi, C3Ps-C3Ps-C3Ps, C3Pi-C3Ps-C3Ps, C3Ps-C3Pi-C3Ps, C3Ps-C3Ps-C3Pi, C3Pi-C3Pi-C3Ps, C3Ps-C3Pi-C3Pi or C3Pi-C3Ps-C3 Pi.

In other embodiments, the molecule comprises a C3 alkyl moiety covalently linked to an abasic moiety through a phosphodiester or phosphorothioate linkage, wherein the abasic moiety is selected from a deoxyribose abasic moiety or a ribose abasic moiety. In some embodiments, the C3 alkyl moiety covalently attached to the abasic moiety by a phosphodiester or phosphorothioate linkage is selected from C3Pi-rAb, C3Pi-dAb, rAb-C3OH, rAb-C3Pi, dAb-C3OH, or dAb-C3 Pi.

In some embodiments, a double stranded nucleic acid molecule having the structure (a1) is provided:

(A1)5 '(N) x-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

Wherein each of N and N' is an unmodified or modified nucleotide, or an unconventional moiety;

wherein each of (N) x and (N ') y is an oligonucleotide in which each successive N or N ' is joined to the next N or N ' by a covalent bond;

wherein at least one of Z or Z 'is present and comprises a non-nucleotide moiety covalently attached at the 3' end of the strand in which it is present;

wherein z "may be present or absent, but if present is a capping moiety covalently attached to the 5 'terminus of (N') y;

wherein each of x and y is independently an integer between 18 and 40;

wherein the sequence of (N') y is complementary to the sequence of (N) x; and wherein the sequence of (N) x has complementarity to a contiguous sequence in the target RNA.

In some embodiments, the covalent bond joining each successive N or N' is a phosphodiester bond.

In some embodiments, x-y-19 to 27, such as 19, 20, 21, 22, 23, 24, 25, 26, 27. In some embodiments, x ═ y, and each of x and y is 19, 20, 21, 22, or 23. In various embodiments, x-y-19.

In some embodiments, x ═ y ═ 19 and one of Z or Z' is present and consists of two non-nucleotide moieties.

In some embodiments, x ═ y ═ 19 and Z' is present and consists of two non-nucleotide moieties.

In a preferred embodiment, x ═ y ═ 19 and Z is present and consists of two non-nucleotide moieties.

In a preferred embodiment, x ═ y ═ 19 and Z is present and consists of two non-nucleotide moieties; and Z' is present and consists of a non-nucleotide moiety.

In further embodiments, x ═ y ═ 19 and Z' are present, and each independently comprises two non-nucleotide moieties.

In some embodiments, the double stranded nucleic acid molecule comprises a DNA portion or mismatch to a target at position 1 (5' end) of the antisense strand. This structure will be described herein. According to one embodiment, a double stranded nucleic acid molecule having the structure (a2) shown below is provided:

(A2)5 'N1- (N) x-Z3' (antisense strand)

3 ' Z ' -N2- (N ') y-Z ' 5 ' (sense strand)

Wherein each of N2, N and N' is an unmodified or modified ribonucleotide, or an unconventional moiety;

wherein each of (N) x and (N ') y is an oligonucleotide in which each successive N or N ' is joined to an adjacent N or N ' by a covalent bond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N') y is complementary to the sequence of (N) x, and (N) x is complementary to a contiguous sequence in the target RNA;

wherein N1 is covalently bound to (N) x and is mismatched with or is a portion of DNA complementary to the target RNA;

wherein N1 is a moiety selected from the group consisting of: natural or modified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or deoxyadenosine;

wherein z "may be present or absent, but if present is a capping moiety covalently attached at the 5 'end of N2- (N') y; and is

Wherein at least one of Z or Z 'is present and comprises a non-nucleotide moiety covalently attached at the 3' end of the strand in which it is present.

In some embodiments, x ═ y ═ 18 and one of Z or Z' is present and consists of two non-nucleotide moieties.

In some embodiments, x ═ y ═ 18 and Z' is present and consists of two non-nucleotide moieties.

In a preferred embodiment, x ═ y ═ 18 and Z is present and consists of two non-nucleotide moieties.

In a preferred embodiment, x ═ y ═ 18 and Z is present and consists of two non-nucleotide moieties; and Z' is present and consists of a non-nucleotide moiety.

In further embodiments, x-y-18 and Z' are present, each independently comprising two non-nucleotide moieties.

In some embodiments, the sequence of (N') y is fully complementary to the sequence of (N) x. In various embodiments, the sequence of N2- (N') y is complementary to the sequence of N1- (N) x. In some embodiments, (N) x comprises an antisense sequence that is fully complementary to about 17 to about 39 consecutive nucleotides in the target RNA. In other embodiments, (N) x comprises an antisense sequence substantially complementary to about 17 to about 39 contiguous nucleotides in the target RNA.

In some embodiments, N1 and N2 form a Watson-Crick base pair. In some embodiments, N1 and N2 form non-Watson-Crick base pairs. In some embodiments, a base pair is formed between a ribonucleotide and a deoxyribonucleotide.

In some embodiments, x-y-18, x-y-19, or x-y-20. In a preferred embodiment, x-y-18. When x is 18 in N1- (N) x, N1 refers to position 1, and positions 2 to 19 are included in (N) 18. When y is 18 in N2- (N ') y, N2 refers to the 19 th bit, and the 1 st to 18 th bits are included in (N') 18.

In some embodiments, N1 is covalently bound to (N) x and is mismatched to the target RNA. In various embodiments, N1 is covalently bound to (N) x and is the portion of DNA complementary to the target RNA.

In some embodiments, N1 and N2 form a base pair (rU-rA, rU-dA, dU-rA, dU-dA) between uridine or deoxyuridine and adenosine or deoxyadenosine. In other embodiments, N1 and N2 form a base pair between deoxyuridine and adenosine.

In some embodiments, the double stranded nucleic acid molecule is a siRNA, siNA, or miRNA. Double-stranded nucleic acid molecules as provided herein are also referred to as "duplexes".

In certain preferred embodiments, x-y-18. In some embodiments, N1 and N2 form a Watson-Crick base pair. In other embodiments, N1 and N2 form non-Watson-Crick base pairs. In certain embodiments, N1 is selected from the group consisting of: riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine. In other embodiments, N1 is selected from the group consisting of: ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine.

In some embodiments, each of N and N' is an unmodified nucleotide. In some embodiments, at least one of N or N' comprises a chemically modified nucleotide or an unconventional moiety. In some embodiments, the non-canonical moiety is selected from the group consisting of a mirror nucleotide, an abasic ribose moiety, and an abasic deoxyribose moiety. In some embodiments, the non-canonical portion is a mirror nucleotide, preferably an L-DNA portion. In some embodiments, at least one of N or N 'comprises a 2' OMe sugar modified ribonucleotide.

In some embodiments, the sequence of (N') y is fully complementary to the sequence of (N) x. In other embodiments, the sequence of (N') y is substantially complementary to the sequence of (N) x.

In some embodiments, (N) x comprises an antisense sequence that is fully complementary to about 17 to about 39 consecutive nucleotides in the target RNA. In other embodiments, (N) x comprises an antisense sequence substantially complementary to about 17 to about 39 contiguous nucleotides in the target RNA.

In some embodiments, the nucleic acid molecule disclosed herein is an siRNA, siNA, or miRNA.

In some embodiments of structures a1 and a2, Z is present and Z' is absent. In other embodiments, Z' is present and Z is absent. In other embodiments, both Z and Z' are present. In some embodiments, Z and Z' are present and the same. In other embodiments, Z and Z' are present and different. In some embodiments, Z and Z' are independently 2, 3, 4, or 5 non-nucleotide moieties, or a combination of 2, 3, 4, or 5 non-nucleotide moieties and nucleotides. In some embodiments, each of Z and/or Z 'is comprised of two (2) non-nucleotide moieties covalently linked to the 3' end of the siRNA strand by phosphodiester bonds.

The non-nucleotide moiety is selected from the group consisting of: an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, and an inorganic phosphate. In some embodiments, the non-nucleotide moiety is an alkyl moiety or a derivative thereof. In some embodiments, the alkyl moiety comprises a terminal functional group selected from the group consisting of: alcohol, terminal amine, terminal phosphate, and terminal phosphorothioate moieties.

In some embodiments, Z is present and comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety or derivative thereof, and an inorganic phosphate. In some embodiments, Z is present and consists of two alkyl moieties or derivatives thereof.

In further embodiments, Z' is present and comprises one or more non-nucleotide moieties selected from the group consisting of: abasic moieties, reverse abasic moieties, hydrocarbon moieties, and inorganic phosphate esters. In some embodiments, Z' is present and comprises one or more alkyl moieties or derivatives thereof.

In some embodiments, Z is present and consists of two alkyl moieties or derivatives thereof, and Z' is present and consists of a single alkyl moiety or derivative thereof.

In some embodiments, each of Z and Z' comprises an abasic moiety, such as a deoxyriboabasic moiety (referred to herein as a "dAb") or a riboabasic moiety (referred to herein as a "rAb"). In some embodiments, each of Z and/or Z ' comprises two covalently linked abasic moieties, and is, for example, a 5 ' > 3 ' dAb-dAb or rAb-rAb or dAb-rAb or rAb-dAb. Each moiety is covalently conjugated to an adjacent moiety through a covalent bond, preferably a phosphorus-based bond. In some embodiments, the phosphorus-based linkage is a phosphorothioate, phosphoacetate, or phosphodiester linkage.

In some embodiments, each of Z and/or Z' independently comprises a C2, C3, C4, C5, or C6 alkyl moiety, optionally C3[ propane, - (CH2)₃-]Moieties or derivatives thereof, such as propanol (C3-OH), propylene glycol or the phosphodiester derivative of propylene glycol ("C3 Pi"). In preferred embodiments, each of Z and/or Z' comprises two hydrocarbon moieties, and in some examples is C3-C3. Each C3 is covalently conjugated to the adjacent C3 by a covalent bond, preferably a phosphorus-based bond. In some embodiments, the phosphorus-based linkage is a phosphorothioate, phosphoacetate, or phosphodiester linkage.

In some embodiments of structure a1 and structure a2, at least one of Z or Z' is present and comprises at least two non-nucleotide moieties covalently linked to the chain in which it is present. In some embodiments, each of Z and Z' independently comprises a C3 alkyl, C3 alcohol, or C3 ester moiety. In some embodiments, Z' is absent and Z is present and comprises a non-nucleotide C3 moiety. In some embodiments, Z is absent and Z' is present and comprises a non-nucleotide C3 moiety.

In some embodiments of structures a1 and a2, each of N and N' is an unmodified nucleotide. In some embodiments, at least one of N or N' comprises a chemically modified nucleotide or an unconventional moiety. In some embodiments, the non-canonical moiety is selected from the group consisting of a mirror nucleotide, an abasic ribose moiety, and an abasic deoxyribose moiety. In some embodiments, the non-canonical portion is a mirror nucleotide, preferably an L-DNA portion. In some embodiments, at least one of N or N 'comprises a 2' OMe sugar modified ribonucleotide.

In other embodiments, the compound of structure a1 or structure a2 comprises at least one ribonucleotide modified in a sugar residue. In some embodiments, the compound comprises a modification at the 2' position of the sugar residue. In some embodiments, the modification at the 2' position includes the presence of an amino, fluoro, alkoxy, or alkyl moiety. In certain embodiments, the 2' modification comprises an alkoxy moiety. In a preferred embodiment, the alkoxy moiety is a methoxy moiety (also known as 2' -O-methyl, 2' OMe, 2' -OCH 3). In some embodiments, the nucleic acid compound comprises a 2' OMe sugar modified alternating ribonucleotide in one or both of the antisense strand and the sense strand. In other embodiments, the compound comprises a 2' OMe sugar modified ribonucleotide in the antisense strand (N) x or N1- (N) x only. In certain embodiments, the intermediate ribonucleotides of the antisense strand, such as the 10 th ribonucleotide in the 19-mer strand, are unmodified. In various embodiments, the nucleic acid compound comprises at least 5 alternating 2' OMe sugar modified and unmodified ribonucleotides. In further embodiments, the compound of structure a1 or structure a2 comprises modified ribonucleotides in alternating positions, wherein each ribonucleotide at the 5 ' and 3 ' ends of (N) x or N1- (N) x is modified in its sugar residue, and each ribonucleotide at the 5 ' and 3 ' ends of (N ') y or N2- (N) y is unmodified in its sugar residue.

In some embodiments, the double-stranded molecule comprises one or more of the following modifications

a) N in at least one of positions 5,6, 7, 8 or 9 from the 5 ' end of the antisense strand is selected from a 2' 5 ' nucleotide or a mirror nucleotide;

b) n 'in at least one of positions 9 or 10 from the 5' terminus of the sense strand is selected from a2 '5' nucleotide and pseudouridine; and

c) n ' in 4, 5 or 6 consecutive positions of the terminal position of (N ') y3 ' comprises a 2' 5 ' nucleotide.

In some embodiments, the double-stranded molecule comprises a combination of the following modifications

a) The antisense strand comprises a 2' 5 ' nucleotide or a mirror nucleotide in at least one of positions 5,6, 7, 8 or 9 from the 5 ' terminus; and

b) the sense strand comprises at least one of a 2' 5 ' nucleotide and a pseudouridine in positions 9 or 10 from the 5 ' terminus.

c) the sense strand comprises 4, 5, or 6 consecutive 2 '5' nucleotides at the 3 'penultimate or 3' terminal position.

In some embodiments, the sense strand [ (N) x or N1- (N) x ] comprises 1, 2, 3, 4, 5,6, 7, 8, or 9 2' OMe sugar modified ribonucleotides. In some embodiments, the antisense strand comprises 2' OMe modified ribonucleotides at positions 2, 4,6, 8, 11, 13, 15, 17 and 19. In other embodiments, the antisense strand comprises 2' OMe modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In other embodiments, the antisense strand comprises 2' OMe modified ribonucleotides at positions 3, 5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments, the antisense strand comprises one or more 2' OMe sugar-modified pyrimidines. In some embodiments, all pyrimidine nucleotides in the antisense strand are 2' OMe sugar modified. In some embodiments, the sense strand comprises a 2' OMe sugar modified pyrimidine.

In some embodiments of structure a1 and structure a2, the sense strand and the antisense strand are independently phosphorylated or non-phosphorylated at the 3 'terminus and the 5' terminus. In some embodiments of structure a1 and structure a2, the sense strand and the antisense strand are not phosphorylated at the 3 'and 5' ends. In other embodiments, the sense strand and the antisense strand are phosphorylated at the 3' terminus.

In some embodiments of structure a1 and structure a2, (N) y comprises at least one non-canonical moiety selected from the group consisting of a mirror nucleotide, a2 '5' nucleotide, and TNA. In some embodiments, the non-canonical portion is a mirror nucleotide. In various embodiments, the mirror nucleotide is selected from the group consisting of an L-ribonucleotide (L-RNA) and an L-deoxyribonucleotide (L-DNA). In a preferred embodiment, the mirror nucleotide is L-DNA. In certain embodiments, the sense strand comprises a non-canonical moiety at position 9 or 10 (from the 5' terminus). In a preferred embodiment, the sense strand comprises a non-canonical moiety at position 9 (from the 5' end). In some embodiments, the sense strand is 19 nucleotides in length and comprises 4, 5, or 6 consecutive non-regular moieties at position 15 (from the 5' end). In some embodiments, the sense strand comprises 4 consecutive 2 '5' ribonucleotides at positions 15, 16, 17 and 18. In some embodiments, the sense strand comprises 5 consecutive 2 '5' ribonucleotides at positions 15, 16, 17, 18 and 19. In various embodiments, the sense strand further comprises Z'. In some embodiments, Z' comprises a C3OH moiety or a C3Pi moiety.

In some embodiments of structure A1, (N') y comprises at least one L-DNA moiety. In some embodiments, x ═ y ═ 19 and (N ') y consists of unmodified ribonucleotides at positions 1-17 and 19 and one L-DNA at the 3' penultimate position (position 18). In other embodiments, x ═ y ═ 19 and (N ') y consists of unmodified ribonucleotides at positions 1 to 16 and 19 and two consecutive L-DNAs at the 3' penultimate positions (positions 17 and 18). In various embodiments, the non-canonical moiety is a nucleotide that is joined to an adjacent nucleotide by a2 '-5' internucleotide phosphate linkage. According to various embodiments, (N ') y comprises 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3' end linked by 2 '-5' internucleotide linkages. In one embodiment, four consecutive nucleotides at the 3 'terminus of (N') y are joined by three 2 '-5' phosphodiester linkages, wherein one or more of the 2 '-5' nucleotides forming the 2 '-5' phosphodiester linkage further comprises a 3 '-O-methyl (3' OMe) sugar modification. Preferably, the 3 ' terminal nucleotide of (N ') y comprises a 2' OMe sugar modification. In certain embodiments, x ═ y ═ 19 and (N ') y comprise two or more contiguous nucleotides at positions 15, 16, 17, 18, and 19, joined to an adjacent nucleotide (2 ' -5 ' nucleotide) by a 2' -5 ' internucleotide linkage. In various embodiments, the nucleotides forming the 2' -5 ' internucleotide linkage include 3 ' deoxyribonucleotides or 3 ' methoxynucleotides (3 ' H or 3 ' OMe instead of 3 ' OH). In some embodiments, x ═ y ═ 19 and (N ') y comprise 2' -5 ' nucleotides at positions 15, 16, and 17, such that adjacent nucleotides are linked by 2' -5 ' internucleotide linkages between positions 15-16, 16-17, and 17-18; or comprises 2 '-5' nucleotides at positions 15, 16, 17, 18 and 19 such that adjacent nucleotides are linked by 2 '-5' internucleotide linkages between positions 15-16, 16-17, 17-18 and 18-19 and a 3 'OH group is available at the 3' terminal nucleotide or positions 16, 17 and 18 such that adjacent nucleotides are linked by 2 '-5' internucleotide linkages between positions 16-17, 17-18 and 18-19. In some embodiments, x ═ y ═ 19 and (N ') y comprises 2' -5 ' nucleotides at positions 16 and 17 or positions 17 and 18 or positions 15 and 17, such that adjacent nucleotides are linked by a 2' -5 ' internucleotide linkage between positions 16-17 and 17-18 or positions 17-18 and 18-19 or positions 15-16 and 17-18, respectively. In other embodiments, the pyrimidine ribonucleotides (rU, rC) in (N ') y are substituted with nucleotides that are joined to adjacent nucleotides by 2' -5 ' internucleotide linkages. In some embodiments, x ═ y ═ 19 and (N ') y comprise at the 3' end five consecutive nucleotides joined by four 2 '-5' linkages, in particular linkages between nucleotides 15-16, 16-17, 17-18 and 18-19.

In some embodiments, x ═ y ═ 19 and (N ') y comprise five consecutive nucleotides at the 3' terminus joined by four 2 '-5' linkages, and optionally further comprise Z 'and Z' independently selected from inverted abasic moieties and C3 alkyl [ C3, 1, 3-propanediol mono (dihydrogen phosphate) ] caps. The C3 alkyl cap is covalently attached to the 3 'or 5' terminal nucleotide. In some embodiments, the 3 'C3 end cap further comprises a 3' phosphate ester. In some embodiments, the 3 'C3 terminal cap further comprises a 3' terminal hydroxyl group.

In some embodiments, x ═ y ═ 19 and (N') y comprises L-DNA at position 18; and (N') y optionally further comprises an abasic moiety independently selected from inverted and C3 alkyl [ C3; 1, 3-propanediol mono (dihydrogen phosphate) ] capped Z 'and Z'.

In some embodiments, (N ') y comprises a 3 ' terminal phosphate (i.e., phosphorylated at the 3 ' terminus). In some embodiments, (N ') y comprises a 3' terminal hydroxyl group.

In some embodiments, x ═ y ═ 19 and (N) x comprises 2' OMe sugar modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or positions 2, 4,6, 8, 11, 13, 15, 17, 19. In some embodiments, x ═ y ═ 19 and (N) x comprises a 2' OMe sugar modified pyrimidine. In some embodiments, all pyrimidines in (N) x comprise a 2' OMe sugar modification.

In some embodiments of structure a2, x-y-18 and N2 is a riboadenosine moiety. In some embodiments, x-y-18 and N2- (N ') y comprises five consecutive nucleotides at the 3' end connected by four 2 '-5' linkages, in particular linkages between nucleotides 15-16, 16-17, 17-18 and 18-19. In some embodiments, the linkage comprises a phosphodiester linkage. In some embodiments, x ═ y ═ 18 and N2- (N ') y comprises five consecutive nucleotides at the 3' terminus joined by four 2 '-5' linkages, and optionally further comprises an abasic moiety independently selected from inverted abasic moieties and C3 alkyl [ C3; 1, 3-propanediol mono (dihydrogen phosphate) ] capped Z 'and Z'. In some embodiments, x ═ y ═ 18 and N2- (N') y comprises L-DNA at position 18; and (N ') y optionally further comprises Z ' and Z ' independently selected from inverted abasic moieties and C3 alkyl [ C3, 1, 3-propanediol mono (dihydrogen phosphate) ] caps. In some embodiments, N2- (N ') y comprises a 3' terminal phosphate. In some embodiments, N2- (N ') y comprises a 3' terminal hydroxyl group. In some embodiments, x ═ y ═ 18 and N1- (N) x comprises a 2' OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or position 1, 3, 5, 9, 11, 13, 15, 17, 19 or position 3, 5, 9, 11, 13, 15, 17 or position 2, 4,6, 8, 11, 13, 15, 17, 19. In some embodiments, x-y-18 and N1- (N) x comprise 2'OMe sugar modified ribonucleotides at positions 11, 13, 15, 17 and 19 (from the 5' terminus). In some embodiments, x-y-18 and N1- (N) x comprises a 2' OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or at position 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments, x-y-18 and N1- (N) x comprise a 2' OMe sugar modified ribonucleotide at positions 2, 4,6, 8, 11, 13, 15, 17, 19.

In some embodiments, x ═ y ═ 18 and N1- (N) x comprise 2' OMe sugar modified pyrimidines. In some embodiments, all pyrimidines in (N) x comprise a 2' OMe sugar modification. In some embodiments, the antisense strand further comprises L-DNA or2 '-5' nucleotides at positions 5,6, or 7 (5 '> 3'). In other embodiments, the antisense strand further comprises ribonucleotides that create a2 '5' internucleotide linkage between the ribonucleotides in positions 5-6 or 6-7 (5 '> 3').

In additional embodiments, N1- (N) x further comprises Z, wherein Z comprises a non-nucleotide overhang. In some embodiments, the non-nucleotide overhang is C3-C3[1, 3-propanediol mono (dihydrogen phosphate) ] 2.

In some embodiments of structure A2, (N) y comprises at least one L-DNA moiety. In some embodiments, x ═ y ═ 18 and (N ') y consists of unmodified ribonucleotides at positions 1-16 and 18 and one L-DNA at the 3' penultimate position (position 17). In other embodiments, x ═ y ═ 18 and (N ') y consists of unmodified ribonucleotides at positions 1 to 15 and 18 and two consecutive L-DNAs at the 3' penultimate positions (positions 16 and 17). In various embodiments, the non-canonical moiety is a nucleotide that is joined to an adjacent nucleotide by a2 '-5' internucleotide phosphate linkage. According to various embodiments, (N ') y comprises 2, 3, 4, 5, or 6 consecutive ribonucleotides at the 3' end linked by 2 '-5' internucleotide linkages. In one embodiment, four consecutive nucleotides at the 3 'terminus of (N') y are joined by three 2 '-5' phosphodiester linkages, wherein one or more of the 2 '-5' nucleotides forming the 2 '-5' phosphodiester linkage further comprises a 3 '-O-methyl (3' OMe) sugar modification. Preferably, the 3 ' terminal nucleotide of (N ') y comprises a 2' OMe sugar modification. In certain embodiments, two or more consecutive nucleotides at positions 14, 15, 16, 17 and 18 in (N ') y and x ═ y ═ 18 comprise nucleotides joined to adjacent nucleotides by 2' -5 ' internucleotide linkages. In various embodiments, the nucleotides forming 2 '-5' internucleotide linkages include 3 'deoxyribonucleotides or 3' methoxynucleotides. In some embodiments, x ═ y ═ 18 and (N ') y comprise nucleotides that are joined to adjacent nucleotides by 2' -5 ' internucleotide linkages between positions 15-16, 16-17 and 17-18 or between positions 16-17 and 17-18. In some embodiments, x ═ y ═ 18 and (N ') y comprises nucleotides that are joined to adjacent nucleotides by 2' -5 ' internucleotide linkages between positions 14-15, 15-16, 16-17, and 17-18 or between positions 16-17 and 17-18 or between positions 15-16 and 17-18. In other embodiments, the pyrimidine ribonucleotides (rU, rC) in (N ') y are substituted with nucleotides that are joined to adjacent nucleotides by 2' -5 ' internucleotide linkages.

The C3 alkyl moiety may be covalently linked to the 3 ' terminus of (N ') y and/or the 3 ' terminus of (N) x via a phosphodiester bond. In some embodiments, the alkyl moiety comprises propanol, propyl phosphate, or propyl thiophosphate. In some embodiments, each of Z and Z' is independently selected from propanol, propyl phosphate, propyl phosphorothioate, a combination thereof or a plurality thereof, especially 2 or 3 covalently linked propanols, propyl phosphate, propyl phosphorothioate or a combination thereof.

In some embodiments, each of Z and Z' is independently selected from propyl phosphate, propyl thiophosphate, propyl phosphorus-propanol; propyl phosphorus-propyl thiophosphate; propyl phosphorus-propyl phosphate; (propyl phosphate) 3, (propyl phosphate) 2-propanol, (propyl phosphate) 2-propyl thiophosphate. Any propane or propanol conjugate moiety may be included in Z or Z'.

In further embodiments, each of Z and/or Z' comprises a combination of an abasic moiety and an unmodified deoxyribonucleotide or ribonucleotide, or a combination of a hydrocarbon moiety and an unmodified deoxyribonucleotide or ribonucleotide, or a combination of an abasic moiety (deoxyribose or ribose) and a hydrocarbon moiety. In such embodiments, each of Z and/or Z' comprises a C3Pi-rAb, a C3Ps-rAb, a C3Ps-dAb, or a C3 Pi-dAb.

According to certain embodiments, the present invention provides siRNA compounds further comprising one or more modified ribonucleotides or unconventional moieties, wherein the modified nucleotides have modifications in the sugar moiety, in the base moiety or in the internucleotide linkage moiety. In some embodiments, one or more of N or N 'comprises a 2' OMe modified ribonucleotide, a2 '5' or an L-nucleotide.

In some embodiments, (N) x comprises modified and unmodified ribonucleotides, each modified ribonucleotide having a 2' -O-methyl group (2 ' OMe modified or 2' OMe sugar modified) on its sugar, wherein the N at the 3 ' terminus of (N) x is a modified ribonucleotide, (N) x comprises at least five alternating modified ribonucleotides from the 3 ' terminus and at least nine modified ribonucleotides in total, and each of the remaining N is an unmodified ribonucleotide.

In some embodiments, (N) x and (N')_yComprises at least one mirror nucleotide. In some embodiments, in (N ') y, there is at least one unconventional moiety selected from the group consisting of an abasic ribose moiety, an abasic deoxyribose moiety, a modified or unmodified deoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide phosphate linkage. In some embodiments, at least one of N' is a mirror nucleotide.

In some embodiments, the non-canonical portion is an L-DNA mirror nucleotide. In further embodiments, x ═ y ═ 19 and at least one unconventional moiety is present at one of positions 15, 16, 17, or 18 in (N') y. In some embodiments, the non-canonical portion is a mirror nucleotide, preferably an L-DNA portion. In some embodiments, an L-DNA moiety is present at position 17, 18 or at positions 17 and 18.

In some embodiments, the non-canonical moiety is a nucleotide that is joined to an adjacent nucleotide by a2 '-5' internucleotide phosphate linkage. In a further embodiment, x ═ y ═ 19 and the nucleotides at positions 15 to 19 or 16 to 19 or 17 to 19 in (N ') y are joined to adjacent nucleotides by 2' -5 ' internucleotide phosphate linkages. In some embodiments, x ═ y ═ 19 and the nucleotides at positions 15-19 or 16-19 or 17-19 or 15-18 or 16-18 in (N ') y are joined to adjacent nucleotides by 2' -5 ' internucleotide phosphate linkages.

In some embodiments, (N) x comprises nine alternating modified ribonucleotides. In other embodiments, (N) x comprises nine alternating modified ribonucleotides, the latter further comprising a 2' OMe modified nucleotide at position 2. In some embodiments, x is 19 and (N) x comprises a 2' OMe modified ribonucleotide in the odd numbered positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19. In other embodiments, (N) x further comprises a 2' OMe modified ribonucleotide at one or both of positions 2 and 18. In other embodiments, (N) x comprises a 2' OMe modified ribonucleotide at positions 2, 4,6, 8, 11, 13, 15, 17, 19. In some embodiments, at least one pyrimidine nucleotide in (N) x comprises a 2' OMe sugar modification. In some embodiments, all of the pyrimidine nucleotides in (N) x comprise a 2' OMe sugar modification. In some embodiments, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pyrimidine nucleotides in n (x) comprise a 2' OMe sugar modification.

In various embodiments, z "is present and is selected from the group consisting of an abasic ribose moiety, an abasic deoxyribose moiety, a deoxyribose moiety, an inverted abasic ribose moiety, a C3 moiety, a C6-amino-Pi, a mirror nucleotide.

In certain embodiments, (N) x is fully complementary to the target sequence. In other embodiments, (N) x is substantially complementary to the target sequence. In some embodiments, (N) x comprises one mismatch to the target sequence. In a preferred embodiment, (N) x comprises a nucleotide mismatch with the target sequence at the 5' end of (N) x (i.e. position 1).

In certain embodiments, (N) x and (N') y are fully complementary. In other embodiments, (N) x and (N') y are substantially complementary.

In some embodiments, (N) x comprises one mismatch with the target sequence at position 1, and (N) x and (N') y are fully complementary. In some embodiments, (N) x comprises one nucleotide mismatch with the target sequence at position 1, and (N') y comprises one nucleotide mismatch with (N) x at position 1.

In some embodiments, x-y-19 and in (N) x, the eighteenth consecutive nucleotides at positions 2-19 are complementary to the eighteenth consecutive nucleotides in the target RNA and the nucleotide at position 1 is mismatched to the target RNA sequence. In various embodiments, the nucleotide at position 1 in (N) x is substituted with a moiety selected from the group consisting of: ribouracil, modified ribouracil, deoxyribouracil, modified deoxyribouracil, pseudouracil, deoxypseudouracil, deoxyribothymine, modified deoxyribothymine, ribocytosine, modified ribocytosine, deoxyribocytosine, modified deoxyribocytosine, an abasic ribose moiety, and an abasic deoxyribose moiety. In some embodiments, the nucleotide at position 1 in (N) x is substituted with a moiety selected from the group consisting of: ribouracil, modified ribouracil, deoxyribouracil, modified deoxyribouracil.

In some embodiments, x-y-19 and 18 consecutive nucleotides at positions 2-19 in (N) x are complementary to 18 consecutive nucleotides in the target RNA, and the nucleotide at position 1 is mismatched to the target RNA sequence, while the nucleotide at position 19 of (N') y is complementary to the nucleotide at position 1 of (N) x. In other embodiments, x ═ y ═ 19 and the nucleotide at position 1 of (N) x is mismatched to the target mRNA sequence, and (N') y is mismatched at position 19 to the nucleotide at position 1 of (N) x.

The advantages of the double stranded nucleic acid molecules disclosed herein are: they exhibit improved stability and/or improved activity and/or reduced off-target effects and/or reduced immune response and/or enhanced cellular uptake when compared to blunt-ended or 3' dTdT-bearing molecules.

Other embodiments are contemplated where x-y-21 or where x-y-23. Structures (a1) and (a2) with known and future pairs of oligonucleotides (sense and antisense strands) can be used for mammalian or non-mammalian (e.g., viral, bacterial, plant) genes. In some embodiments, the mammalian gene is a human gene. In various embodiments, mRNA of a human gene is shown in PCT patent publication No. wo 2009/044392. In further embodiments, the oligonucleotide pairs are shown in PCT patent publication No. wo 2009/044392. In further embodiments, structure (a1) or (a2) further comprises the modifications and motifs shown in PCT patent publication No. wo 2009/044392.

In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of a molecule of the invention to inhibit the expression of a human gene and a pharmaceutically acceptable carrier.

More specifically, the present invention provides methods and compositions useful for treating a subject suffering from: acute Renal Failure (ARF), hearing loss, glaucoma, Acute Respiratory Distress Syndrome (ARDS) and other acute lung and respiratory tract injuries, injuries in organ transplants (including lung, kidney, bone marrow, heart, pancreas, cornea or liver transplants), such as ischemia-reperfusion injury, and including toxic kidney damage in delayed graft function recovery (DGF), spinal cord injury, bedsores, dry eye syndrome, oral mucositis, ischemic ocular neuropathy and Chronic Obstructive Pulmonary Disease (COPD).

The methods of the invention comprise administering to the subject one or more siRNA compounds that inhibit gene expression. The novel structures disclosed herein, when integrated into antisense and corresponding sense nucleic acid sequences directed against any target gene, provide siRNA compounds useful for reducing expression of that target gene. The target gene is a mammalian or non-mammalian gene.

Brief Description of Drawings

FIGS. 1A-1J show the chemical structures of some possible 3 'alkyl/alkyl derivatives highlighted when covalently linked to the 3' terminal nucleotide (DNA or RNA) of the oligonucleotide chain through a phosphodiester linkage. B on the nucleotide moiety is referred to as the nucleotide "base"; r is in each case either H or OH; FIG. 1A shows a 3' terminal nucleotide covalently linked to a propanol moiety by phosphodiester linkage; FIG. 1B shows the 3' terminal nucleotide covalently linked to a C3Pi moiety by phosphodiester linkage; FIG. 1C shows the 3' terminal nucleotide covalently linked to the C3Pi-C3OH moiety through phosphodiester linkage (C3 is covalently linked to C3OH through phosphodiester linkage); FIG. 1D shows the 3' terminal nucleotide covalently linked to the C3Pi-C3Pi moiety by phosphodiester linkage; FIG. 1E shows the 3' terminal nucleotide covalently linked to C3Pi-C3Pi-C3OH by phosphodiester linkage. FIG. 1F shows the 3' terminal nucleotide covalently linked to C3Pi-C3Pi-C3Pi by phosphodiester linkage. FIG. 1G shows the 3' terminal nucleotide covalently linked to C3Pi-rAb or C3Pi-dAb by phosphodiester linkage. Figure 1H shows the 3' terminal nucleotide covalently linked to rAb-C3Pi (R1 ═ OH) or dAb-C3Pi (R1 ═ H) by phosphodiester linkage. Figure 1J shows the 3' terminal nucleotide covalently linked by phosphodiester linkage to rAb-rAb (R1 ═ R2 ═ OH) or dAb-rAb (R1 ═ H, R2 ═ OH) or rAb-dAb (R1 ═ OH, R2 ═ H) or dAb-dAb (R1 ═ R2 ═ H).

FIGS. 2A-2F are similar to FIGS. 1A-1F except that the 3 ' -terminal non-nucleotide overhang is attached to the 2' -position of the ribose moiety instead of the 3 ' -position.

Figures 3A-3K illustrate some specific examples of nucleotides having an overhanging 3' -terminus, in some cases where the oxygen atom attached to the phosphorus has been replaced with sulfur, according to embodiments of the invention.

Figure 4 provides stability data for two double stranded molecules: s505 (example compound 5 in table 3) which is a blunt-ended 19-mer duplex, and S800(C3Pi-C3OH, example compound 7 in table 3) which is a 19-mer duplex comprising a non-nucleotide C3C 33' overhang. Both compounds were nuclease stable for at least 36 hours in cell extracts, but S800 had an IC50 value of about 0.17nM and S505 had an IC50 value of 1.1 nM. The sequences used to generate these two compounds are shown as seq id no: 1 and 2 (sense strand and antisense strand, respectively).

Detailed Description

The present invention relates to double stranded siRNA compounds comprising at least one non-nucleotide moiety covalently attached to the 3' terminus of one or both of the sense and antisense strands. The non-nucleotide moiety is selected from the group consisting of an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, and an inorganic phosphate.

Structural design

In one aspect, provided herein are double-stranded nucleic acid molecules comprising a sense strand and an antisense strand, wherein at least one strand comprises 1, 2, 3, 4, or 5 non-nucleotide moieties covalently linked at the 3' terminus; wherein the non-nucleotide moiety is selected from the group consisting of an alkyl (hydrocarbon) moiety or a derivative thereof and a phosphate-based moiety. In certain preferred embodiments, the non-nucleotide moiety comprises an alkyl moiety or an alkyl derivative moiety. In some embodiments, the at least one strand is an antisense strand. In a preferred embodiment, the antisense strand comprises two non-nucleotide moieties covalently linked at the 3' terminus, including C3-C3, C3-C3-Pi, C3-C3-Ps, idAb-idAb.

(A1)5 '(N) x-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

Wherein each of N and N' is a nucleotide, which may be unmodified or modified, or an unconventional moiety;

wherein each of x and y is independently an integer between 18 and 40;

(A2)5 'N1- (N) x-Z3' (antisense strand)

3 ' Z ' -N2- (N ') y-Z ' 5 ' (sense strand)

wherein each of x and y is independently an integer between 17 and 39;

In some embodiments, x-y-18, x-y-19, or x-y-20. In a preferred embodiment, x-y-18. When x is 18 in N1- (N) x, N1 refers to position 1, and positions 2 to 19 are included in (N) 1818. When y is 18 in N2- (N ') y, N2 refers to the 19 th bit, and the 1 st to 18 th bits are included in (N') 18.

In some embodiments, the uridine at position 1 of the antisense strand is substituted with N1 selected from adenosine, deoxyadenosine, deoxyuridine (dU), ribothymidine, or deoxythymidine. In various embodiments, N1 is selected from adenosine, deoxyadenosine, or deoxyuridine.

In some embodiments, guanosine at position 1 of the antisense strand is substituted with N1 selected from adenosine, deoxyadenosine, uridine, deoxyuridine, ribothymidine, or deoxythymidine. In various embodiments, N1 is selected from adenosine, deoxyadenosine, uridine, or deoxyuridine.

In some embodiments, the cytidine at position 1 of the antisense strand is substituted with N1 selected from adenosine, deoxyadenosine, uridine, deoxyuridine, ribothymidine, or deoxythymidine. In various embodiments, N1 is selected from adenosine, deoxyadenosine, uridine, or deoxyuridine.

In some embodiments, the adenosine at position 1 of the antisense strand is substituted with N1 selected from deoxyadenosine, deoxyuridine, ribothymidine or deoxythymidine. In various embodiments, N1 is selected from deoxyadenosine or deoxyuridine.

In some embodiments, N1 and N2 form a base pair between uridine or deoxyuridine and adenosine or deoxyadenosine. In other embodiments, N1 and N2 form a base pair between deoxyuridine and adenosine.

In certain embodiments, position 1 (5' end) of the antisense strand comprises deoxyribouridine (dU) or adenosine. In some embodiments, N1 is selected from the group consisting of: riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine, and N2 is selected from the group consisting of: ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine. In certain embodiments, N1 is selected from the group consisting of: riboadenosine and modified riboadenosine, and N2 is selected from the group consisting of: ribouridine and modified ribouridine.

In certain embodiments, N1 is selected from the group consisting of: ribouridine, deoxyribouridine, modified ribouridine and modified deoxyribouridine, and N2 is selected from the group consisting of: riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine. In certain embodiments, N1 is selected from the group consisting of: ribouridine and deoxyribouridine, and N2 is selected from the group consisting of: riboadenosine and modified riboadenosine. In certain embodiments, N1 is ribouridine and N2 is riboadenosine. In certain embodiments, N1 is deoxyribouridine and N2 is riboadenosine.

In some embodiments of structure (a2), N1 comprises a 2'OMe sugar-modified ribouracil or a 2' OMe sugar-modified riboadenosine. In certain embodiments of structure (a2), N2 comprises a 2' OMe sugar-modified ribonucleotide or deoxyribonucleotide.

In some embodiments of structure (a2), N1 comprises a 2'OMe sugar modified ribouracil or a 2' OMe sugar modified ribocytosine. In certain embodiments of structure (a2), N2 comprises a 2' OMe sugar modified ribonucleotide.

c) n ' in 4, 5 or 6 consecutive positions of the 3 ' terminal position of (N ') y comprises a 2' 5 ' nucleotide.

In some embodiments, x ═ y ═ 19 and (N') y comprises L-DNA at position 18; and (N ') y optionally further comprises Z ' and Z ' independently selected from inverted abasic moieties and C3 alkyl [ C3, 1, 3-propanediol mono (dihydrogen phosphate) ] caps.

In some embodiments of structure a2, x-y-18 and N2 is a riboadenosine moiety. In some embodiments, x-y-18 and N2- (N ') y comprises five nucleotides joined by four 2' -5 'linkages at the 3' end, in particular linkages between nucleotides 15-16, 16-17, 17-18 and 18-19. In some embodiments, the linkage comprises a phosphodiester linkage. In some embodiments, x ═ y ═ 18 and N2- (N ') y comprises five consecutive nucleotides joined by four 2' -5 'linkages at the 3' terminus, and optionally further comprises Z 'and Z' independently selected from inverted abasic moieties and C3 alkyl [ C3, 1, 3-propanediol mono (dihydrogen phosphate) ] caps. In some embodiments, x ═ y ═ 18 and N2- (N') y comprises L-DNA at position 18; and (N ') y optionally further comprises Z ' and Z ' independently selected from inverted abasic moieties and C3 alkyl [ C3, 1, 3-propanediol mono (dihydrogen phosphate) ] caps. In some embodiments, N2- (N ') y comprises a 3' terminal phosphate. In some embodiments, N2- (N ') y comprises a 3' terminal hydroxyl group. In some embodiments, x ═ y ═ 18 and N1- (N) x comprises a 2' OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or position 1, 3, 5, 9, 11, 13, 15, 17, 19 or position 3, 5, 9, 11, 13, 15, 17 or position 2, 4,6, 8, 11, 13, 15, 17, 19. In some embodiments, x-y-18 and N1- (N) x comprise 2'OMe sugar modified ribonucleotides at positions 11, 13, 15, 17 and 19 (from the 5' terminus). In some embodiments, x-y-18 and N1- (N) x comprises a 2' OMe sugar modified ribonucleotide at position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 or at position 3, 5, 7, 9, 11, 13, 15, 17, 19. In some embodiments, x-y-18 and N1- (N) x comprises a 2' OMe, sugar-modified ribonucleotide at positions 2, 4,6, 8, 11, 13, 15, 17, 19.

In some embodiments of structure a1 and structure a2, each N consists of an unmodified ribonucleotide. In some embodiments of structure a1 and structure a2, each N' consists of unmodified nucleotides. In a preferred embodiment, at least one of N and N' is a modified ribonucleotide or an unconventional moiety.

In other embodiments, the molecule of structure a1 or structure a2 comprises at least one ribonucleotide modified in a sugar residue. In some embodiments, the compound comprises a modification at the 2' position of the sugar residue. In some embodiments, the modification at the 2' position includes the presence of an amino, fluoro, alkoxy, or alkyl moiety. In certain embodiments, the 2' modification comprises an alkoxy moiety. In a preferred embodiment, the alkoxy moiety is a methoxy moiety (also known as 2' -O-methyl, 2' OMe, 2' -OCH 3). In some embodiments, the nucleic acid compound comprises a 2' OMe sugar modified alternating ribonucleotide in one or both of the antisense strand and the sense strand. In other embodiments, the compound comprises a 2' OMe sugar modified ribonucleotide in the antisense strand (N) x or N1- (N) x only. In certain embodiments, the intermediate ribonucleotides of the antisense strand, such as the 10 th ribonucleotide in the 19-mer strand, are unmodified. In various embodiments, the nucleic acid compound comprises at least 5 alternating 2' OMe sugar modified and unmodified ribonucleotides.

In further embodiments, the compound of structure a1 or structure a2 comprises modified ribonucleotides in alternating positions, wherein each ribonucleotide at the 5 ' and 3 ' ends of (N) x or N1- (N) x is modified in its sugar residue, and each ribonucleotide at the 5 ' and 3 ' ends of (N ') y or N2- (N) y is unmodified in its sugar residue.

In some embodiments, (N) x or N1- (N) x comprises 2' OMe modified ribonucleotides at positions 2, 4,6, 8, 11, 13, 15, 17 and 19. In other embodiments, (N) x or N1- (N) x comprises 2' OMe modified ribonucleotides at positions 1, 3, 5, 7, 9, 11, 13, 15, 17 and 19. In some embodiments, (N) x or N1- (N) x comprises a 2' OMe modified pyrimidine. In some embodiments, all pyrimidine nucleotides in (N) x or N1- (N) x are 2' OMe modified. In some embodiments, (N ') y or N2- (N ') y comprises a 2' OMe modified pyrimidine. In further embodiments, the compound of structure a1 or structure a2 comprises modified ribonucleotides in alternating positions, wherein each ribonucleotide at the 5 ' and 3 ' ends of (N) x or N1- (N) x is modified in its sugar residue, and each ribonucleotide at the 5 ' and 3 ' ends of (N ') y or N2- (N) y is unmodified in its sugar residue.

The nucleic acid molecules disclosed herein can have a blunt end at one end, for example when Z and Z "are absent or where Z' is absent. A nucleic acid molecule can be modified with modified nucleotides or unconventional moieties that can be located anywhere along either strand of the sense or antisense strand. A nucleic acid molecule can comprise about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 modified nucleotides. The nucleic acid molecule can comprise about 1, 2, 3, 4, 5,6, 7, or 8 non-canonical moieties. A nucleic acid molecule may comprise a set of about 1, 2, 3, 4, 5,6, 7 or 8, preferably 1, 2, 3 or 4, consecutive modified nucleotides or unconventional moieties. The modified nucleic acid may be present only on the sense strand, only on the antisense strand, or on both the sense and antisense strands. In some embodiments, the modified nucleotides include 2 'sugar modified nucleotides, including 2' O-methyl modified nucleotides, 2 'deoxy fluoro modified nucleotides, 2' -amino modified nucleotides. In some embodiments, the non-canonical moiety includes a mirror nucleotide (i.e., L-DNA or L-RNA) or a nucleotide capable of forming a2 '-5' linkage (a2 '5' nucleotide).

As used herein, the term "duplex region" refers to a region in a double-stranded molecule where two complementary or substantially complementary oligonucleotides form base pairs with each other, typically by Watson-Crick base pairing or by any other means that allows duplex formation. For example, an oligonucleotide having 19 nucleotide units may base pair with a complementary oligonucleotide of 19 nucleotide units, or may base pair with 15, 16, 17, or 18 bases on each strand, such that a "duplex region" consists of 15, 16, 17, or 18 base pairs. The remaining base pairs may exist, for example, as 5 'and 3' overhangs. Furthermore, within the duplex region, 100% complementarity is not required; substantial complementarity is permitted within the duplex region. The overhang region can be comprised of nucleotide or non-nucleotide moieties. As disclosed herein, at least one overhang region is comprised of one or more non-nucleotide moieties.

A general non-limiting nucleic acid molecule pattern is shown below, where N' ═ sense strand nucleotides in the duplex region; z ═ a 5 '-capping moiety covalently attached to the 5' terminus of the sense strand; c3 ═ 3 carbon non-nucleotide moieties; an antisense nucleotide in the duplex region; idB-the inverted abasic deoxyribonucleotide non-nucleotide moiety. Each N, N' is independently a modified or unmodified or unconventional moiety. The length of the sense strand and the antisense strand are each independently 18 to 40 nucleotides. The examples provided below have a 19 nucleotide duplex region; however, the nucleic acid molecules disclosed herein can have a duplex region of any value between 18 and 40 nucleotides, and wherein the length of each strand is independently between 18 and 40 nucleotides. In each duplex, the antisense strand (N) x is shown above. In some embodiments, the double-stranded nucleic acid molecule has the following structure:

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′PiC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-C3pi-C3Pi

3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′HOC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-aB-aB

3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-aB-aB

5′NNNNNNNNNNNNNNNNNNN-idB-idB

3′aB-aB-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-aB-aB

3′aB-aB-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

3′PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Pi

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

3′ROC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

3′ROC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Ps

3′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Ps

3′OHC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3Ps

3′OHC3-PiC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-z″

in some preferred embodiments, the nucleic acid molecules disclosed herein have the following structure:

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

or

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

3′iPC3-N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′N′-Z″

Wherein N and N' are independently ribonucleotides that may be unmodified or modified, or are unconventional moieties;

wherein each N is linked to an adjacent N by a covalent bond;

wherein each N 'is linked to an adjacent N' by a covalent bond; and is

Wherein z "is a capping moiety covalently attached to the 5' terminus of the sense strand. The term "aB" refers to an abasic moiety that can be a ribose abasic moiety or a deoxyribose abasic moiety or an inverted ribose abasic moiety or an inverted deoxyribose abasic moiety.

In some embodiments, a nucleic acid molecule disclosed herein comprises Z. In other embodiments, the nucleic acid molecule disclosed herein comprises Z'. In other embodiments, both Z and Z' are present. In some embodiments, Z and Z' are both present and the same. In other embodiments, Z and Z' are both present and different. In some embodiments, Z and Z' independently comprise 1 or2 non-nucleotide moieties. In some embodiments, Z and Z' independently comprise 2 non-nucleotide moieties.

In some embodiments, Z is present and comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, and an inorganic phosphate moiety.

In further embodiments, Z' is present and comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, or an inorganic phosphate moiety.

In further embodiments, Z and/or Z' are present and independently comprise a combination of one or more nucleotides and one or more non-nucleotide moieties selected from the moieties disclosed herein.

In some embodiments, each of Z and Z' comprises an abasic moiety, optionally a deoxyriboabasic (referred to herein as "dAb") or riboabasic (referred to herein as "rAb") nucleotide. In some embodiments, each of Z and/or Z' is a dAb-dAb or rAb-rAb.

In some embodiments, each of Z and/or Z' independently comprises an alkyl moiety, optionally a phosphodiester derivative of propylene glycol ((CH2)3-Pi, also referred to herein as "C3 Pi") modified moiety. In some embodiments, Z and/or Z' is C3Pi-C3 Pi. In a specific embodiment, x ═ y ═ 19 and Z comprises two propylene glycol derivatives C3-C3 (i.e., -C3-Pi-C3-Pi). In various embodiments, the C3 moiety is covalently linked to the 3' terminus of the sense or antisense strand through a phosphodiester linkage.

In further embodiments, Z and/or Z' comprises a combination of one or more abasic moieties and unmodified nucleotides or a combination of one or more hydrocarbon moieties and unmodified nucleotides or a combination of one or more abasic and hydrocarbon moieties. In such embodiments, Z and/or Z' is optionally C3-rAb or C3-dAb.

In further embodiments directed to structures a1 or a2, the nucleic acid molecule further comprises a 2' O-Me modification on the sugar of the ribonucleotides at positions 2, 4,6, 8, 11, 13, 15, 17 and 19 of the antisense strand. In a further embodiment, the compound further comprises an L-DNA nucleotide at position 18 of the sense strand. In further embodiments, the compound comprises a nucleotide joined to an adjacent nucleotide by a2 '-5' internucleotide phosphate linkage. In a further embodiment, x ═ y ═ 19 and the nucleotides at positions 15 to 19 or 16 to 19 or 17 to 19 in (N ') y are joined to adjacent nucleotides by 2' -5 ' internucleotide phosphate linkages. In some embodiments, x ═ y ═ 19 and the nucleotides at positions 15-19 or 16-19 or 17-19 or 15-18 or 16-18 in (N ') y are joined to adjacent nucleotides through 2' -5 ' internucleotide phosphate linkages.

According to certain embodiments, the present invention provides siRNA compounds further comprising one or more modified nucleotides, wherein the modified nucleotides have a modification in the sugar moiety, in the base moiety, or in the internucleotide linkage moiety.

In some embodiments, (N) x comprises modified and unmodified ribonucleotides, each modified ribonucleotide having a 2' -O-methyl group on its sugar, wherein N at the 3 ' -terminus of (N) x is a modified ribonucleotide, (N) x comprises at least five alternating modified ribonucleotides, starting from the 3 ' -terminus, and at least nine modified ribonucleotides in total, and each of the remaining N is an unmodified ribonucleotide.

In some embodiments, at least one of (N) x and (N') y comprises at least one mirror nucleotide. In some embodiments, in (N ') y, there is at least one unconventional moiety, which can be an abasic ribose moiety, an abasic deoxyribose moiety, a modified or unmodified deoxyribonucleotide, a mirror nucleotide, and a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide phosphate linkage or any other unconventional moiety disclosed herein.

In some embodiments, the non-canonical moiety is an L-DNA mirror nucleotide; in further embodiments, there is at least one non-canonical moiety at position 15, 16, 17, or 18 of (N') y. In some embodiments, the non-canonical moiety is selected from the group consisting of a mirror nucleotide, an abasic ribose moiety, and an abasic deoxyribose moiety. In some embodiments, the non-canonical portion is a mirror nucleotide, preferably an L-DNA portion. In some embodiments, an L-DNA moiety is present at position 17, 18 or at positions 17 and 18.

In other embodiments, (N ') y comprises at least five abasic ribose moieties or abasic deoxyribose moieties, and at least one of N' is LNA.

In some embodiments, (N) x comprises nine alternating modified ribonucleotides. In other embodiments, (N) x comprises nine alternating modified ribonucleotides, the latter further comprising a 2' modified nucleotide at position 2. In some embodiments, (N) x comprises a 2' OMe modified ribonucleotide at the odd numbered positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19. In other embodiments, (N) x further comprises a 2' OMe modified ribonucleotide at one or both of positions 2 and 18. In other embodiments, (N) x comprises a 2' OMe modified ribonucleotide at positions 2, 4,6, 8, 11, 13, 15, 17, 19. In some embodiments, at least one pyrimidine nucleotide in (N) x comprises a 2' OMe sugar modification. In some embodiments, all of the pyrimidine nucleotides in (N) x comprise a 2' OMe sugar modification. In some embodiments, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 pyrimidine nucleotides in n (x) comprise a 2' OMe sugar modification.

In various embodiments, z "is present and is selected from the group consisting of an abasic ribose moiety, a deoxyribose moiety, an inverted abasic ribose moiety, a deoxyribose moiety, C6-amino-Pi, a mirror nucleotide.

In one embodiment of the nucleic acid molecule, (N ') y comprises at least two nucleotides joined by a 2' -5 'phosphodiester linkage at either or both of the 5' and 3 'ends of (N') y. In certain embodiments, x ═ y ═ 19; in (N) x, nucleotides alternate between modified ribonucleotides and unmodified ribonucleotides, each modified ribonucleotide being modified so as to have a 2' -O-methyl group on its sugar, while the ribonucleotide located in the middle of (N) x is unmodified; and the three nucleotides at the 3 'end of (N') y are joined by two 2 '-5' phosphodiester bonds. In other embodiments, x ═ y ═ 19; in (N) x, nucleotides alternate between modified ribonucleotides and unmodified ribonucleotides, each modified ribonucleotide being modified so as to have a 2' -O-methyl group on its sugar, while the ribonucleotide located in the middle of (N) x is unmodified; and four consecutive nucleotides at the 5 'end of (N') y are joined by three 2 '-5' phosphodiester bonds. In further embodiments, the additional nucleotide located at the middle position of (N) y may be modified at its sugar with a 2' -O-methyl group. In another embodiment, in (N) x, the nucleotides alternate between 2 '-O-methyl modified ribonucleotides and unmodified ribonucleotides, and in (N') y, four consecutive nucleotides at the 5 'terminus are joined by three 2' -5 'phosphodiester linkages, and the 5' terminal nucleotide or two or three consecutive nucleotides at the 5 'terminus comprise a 3' -O-Me sugar modification.

In certain embodiments of structure (a1), the nucleotides at x ═ y ═ 19 and at least one position in (N ') y include mirror nucleotides, deoxyribonucleotides, and nucleotides joined to adjacent nucleotides by a 2' -5 ' internucleotide linkage;

in certain embodiments of structure (a1), x ═ y ═ 19 and (N') y comprises mirror nucleotides. In various embodiments, the mirror nucleotide is an L-DNA nucleotide. In certain embodiments, the L-DNA is L-deoxyribocytosine. In some embodiments, (N') y comprises L-DNA at position 18. In other embodiments, (N') y comprises L-DNA at positions 17 and 18. In certain embodiments, (N') y comprises L-DNA substitutions at position 2 and at one or both of positions 17 and 18. Other embodiments of structure (a1) are contemplated where x-y-21 or where x-y-23; in these embodiments, the above (N') y modification is not at positions 15, 16, 17, 18, but for the 21mer at positions 17, 18, 19, 20; and at bits 19, 20, 21, 22 for 23 mers; similarly, modifications at one or both of positions 17 and 18 are at one or both of positions 19 or20 for 21 mers and at one or both of positions 21 and 22 for 23 mers. All modifications in the 19mer were similarly adjusted for the 21 and 23 mers.

According to various embodiments of structures a1 or a2, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive ribonucleotides at the 3 ' end of (N ') y or N2- (N ') y are linked by 2' -5 ' internucleotide linkages. In one embodiment, four consecutive nucleotides at the 3 ' terminus of (N ') y are joined by three 2' -5 ' phosphodiester linkages, wherein one or more of the 2' -5 ' nucleotides forming the 2' -5 ' phosphodiester linkage further comprise a 3 ' -O-methyl sugar modification. Preferably, the 3 ' terminal nucleotide of (N ') y comprises a 2' -O-methyl sugar modification. In certain embodiments of structure (a1), x ═ y ═ 19 and in (N ') y the two or more consecutive nucleotides at positions 15, 16, 17, 18, and 19 comprise nucleotides joined to adjacent nucleotides by 2' -5 ' internucleotide linkages. In various embodiments, the nucleotides forming 2 '-5' internucleotide linkages include 3 'deoxyribonucleotides or 3' methoxynucleotides. In some embodiments, the nucleotides at positions 17 and 18 in (N ') y are joined by a 2' -5 ' internucleotide linkage. In other embodiments, the nucleotides at positions 16, 17, 18, 16-17, 17-18, or 16-18 in (N ') y are joined by a 2' -5 ' internucleotide linkage.

In certain embodiments, (N ') y comprises L-DNA at position 2 and 2' -5 ' internucleotide linkages at positions 16, 17, 18, 16-17, 17-18 or 16-18. In certain embodiments, (N ') y comprises 2' -5 'internucleotide linkages at positions 16, 17, 18, 16-17, 17-18, or 16-18, and a 5' end cap nucleotide.

In one embodiment of the nucleic acid molecule, the 3 ' terminal nucleotide of (N ') y or two or three consecutive nucleotides of the 3 ' terminus are L-deoxyribonucleotides.

In other embodiments of the nucleic acid molecule, in (N ') y, the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive ribonucleotides at either terminus or the 2-8 modified nucleotides at each of the 5' and 3 'termini are independently 2' sugar modified nucleotides. In some embodiments, the 2' sugar modification includes the presence of an amino, fluoro, alkoxy, or alkyl moiety. In certain embodiments, the 2 'sugar modification comprises a methoxy moiety (2' -OMe).

In one embodiment, three, four or five consecutive nucleotides of the 5 ' terminus of (N ') y comprise a 2' -OMe modification. In another embodiment, three consecutive nucleotides at the 3 ' terminus of (N ') y comprise a 2' -O-Me sugar modification.

In some embodiments of structures a1 or a2, in (N ') y or N2- (N') y, the 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive ribonucleotides at either end or the 2-8 modified nucleotides at each of the 5 'and 3' ends are independently bicyclic nucleotides. In various embodiments, the bicyclic nucleotide is a Locked Nucleic Acid (LNA). The 2 '-O, 4' -C-ethylene bridged nucleic acid (ENA) is an LNA (see below).

In various embodiments, (N ') y or N2- (N ') y comprises a modified nucleotide at the 5 ' end or at both the 3 ' and 5 ' ends.

In some embodiments of structures a1 or a2, at least two nucleotides at either or both of the 5 ' and 3 ' termini of (N ') y are joined by a P-ethoxy backbone modification. In certain embodiments, x ═ y ═ 19 or x ═ y ═ 23; in (N) x, nucleotides alternate between modified ribonucleotides and unmodified ribonucleotides, each modified ribonucleotide being modified so as to have a 2' -O-methyl group on its sugar, while the ribonucleotide located in the middle position of (N) x is unmodified; and four consecutive nucleotides at the 3 ' end or the 5 ' end of (N ') y are joined by three P-ethoxy backbone modifications. In another embodiment, three consecutive nucleotides at the 3 ' end or the 5 ' end of (N ') y are joined by two P-ethoxy backbone modifications.

In some embodiments of structures a1 or a2, in (N ') y or N2- (N ') y, the 2, 3, 4, 5,6, 7, or 8 consecutive ribonucleotides at each of the 5 ' and 3 ' termini are independently mirror nucleotides, nucleotides joined by a 2' -5 ' phosphodiester linkage, 2' sugar modified nucleotides, or bicyclic nucleotides. In one embodiment, the modifications at the 5 ' and 3 ' ends of (N ') y are the same. In one embodiment, four consecutive nucleotides at the 5 'end of (N') y are joined by three 2 '-5' phosphodiester bonds, and three consecutive nucleotides at the 3 'end of (N') y are joined by two 2 '-5' phosphodiester bonds. In another embodiment, the modification at the 5 'end of (N') y is different from the modification at the 3 'end of (N') y. In one embodiment, the modified nucleotide at the 5 'end of (N') y is a mirror nucleotide, and the modified nucleotide at the 3 'end of (N') y is joined by a2 '-5' phosphodiester linkage. In another specific embodiment, three consecutive nucleotides at the 5 'end of (N') y are LNA nucleotides and three consecutive nucleotides at the 3 'end of (N') y are joined by two 2 '-5' phosphodiester linkages. In (N) x, nucleotides alternate between modified ribonucleotides and unmodified ribonucleotides, each modified ribonucleotide being modified so as to have a 2' -O-methyl group on its sugar, while the ribonucleotides located in the middle of (N) x are unmodified, or the ribonucleotides in (N) x are unmodified.

In another embodiment of structure a1, the present invention provides a compound wherein x ═ y ═ 19; in (N) x, nucleotides alternate between modified ribonucleotides and unmodified ribonucleotides, each modified ribonucleotide being modified so as to have a 2' -O-methyl group on its sugar, while the ribonucleotide located in the middle of (N) x is unmodified; three nucleotides at the 3 'end of (N') y are joined by two 2 '-5' phosphodiester bonds, and three nucleotides at the 5 'end of (N') y are LNAs, such as ENA; and Z and/or Z' independently comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Pi, C3Pi-C3OH, C3Pi-rAb, C3Pi-dAb, dAb-dAb, and rAb-rAb.

In another embodiment, five consecutive nucleotides at the 5 'end of (N') y or N2- (N ') y comprise a 2' -O-methyl sugar modification, and two consecutive nucleotides at the 3 'end of (N') y are L-DNA.

According to other embodiments, in N ') y or N2- (N') y, the 5 'or 3' terminal nucleotide or2, 3, 4, 5, or 6 consecutive nucleotides at either terminus or 1 to 4 modified nucleotides at each of the 5 'and 3' termini are independently phosphonocarboxylate or phosphinocarboxylate nucleotides (PACE nucleotides). In some embodiments, the PACE nucleotide is a deoxyribonucleotide. In some embodiments, in N ') y or N2- (N') y, 1 or2 consecutive nucleotides at each of the 5 'and 3' termini are PACE nucleotides. Examples of PACE nucleotides and analogs are disclosed in U.S. patent nos. 6,693,187 and 7,067,641, both incorporated by reference.

In one embodiment of structure (a1), x ═ y ═ 19; (N) x comprises unmodified ribonucleotides wherein two consecutive nucleotides at the 3 ' terminus are linked by a 2' -5 ' internucleotide linkage; (N ') y comprises unmodified ribonucleotides wherein two consecutive nucleotides at the 5' terminus are linked by a2 '-5' internucleotide linkage; and Z and/or Z' independently comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb, dAb-dAb, and rAb-rAb, each C3, rAb, dAb covalently linked to the adjacent C3Pi, rAb, dAb by a phosphorus-based bond. In some embodiments, the phosphorus-based linkage is a phosphodiester linkage or a phosphorothioate (phosphorothiophosphate) linkage.

In some embodiments, x ═ y ═ 19; (N) x comprises an unmodified ribonucleotide wherein the three consecutive nucleotides at the 3 ' terminus are joined together by two 2' -5 ' phosphodiester bonds; (N ') y comprises unmodified ribonucleotides wherein four consecutive nucleotides at the 5' terminus are joined together by three 2 '-5' phosphodiester bonds; and Z and/or Z' independently comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb, dAb-dAb, and rAb-rAb, wherein each C3Pi, rAb, dAb is covalently linked to the adjacent C3Pi, rAb, dAb by a phosphorus-based bond. In some embodiments, the phosphorus-based linkage is a phosphodiester linkage or a phosphophosphorothioate linkage.

According to one embodiment of structures a1 or a2, four consecutive nucleotides at the 5 ' end of (N ') y or (N ') y-N2, respectively, are joined by three 2' -5 ' phosphodiester bonds; (N ') three consecutive nucleotides at the 3' end of x are joined by two 2 '-5' phosphodiester bonds; and Z and/or Z' independently comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb, C3-dAb, dAb-dAb, and rAb-rAb. The three nucleotides at the 5 ' end of (N ') y and the two nucleotides at the 3 ' end of (N ') x may also comprise a 3 ' -O-Me sugar modification.

In one embodiment of structures a1 or a2, five consecutive nucleotides at the 5 ' terminus of (N ') y or (N ') y-N2 comprise a 2' -O-Me sugar modification, respectively, and five consecutive nucleotides at the 3 ' terminus of (N ') x comprise a 2' -O-Me sugar modification. In another embodiment, ten consecutive nucleotides at the 5 'end of (N') y comprise a2 '-O-Me sugar modification and five consecutive nucleotides at the 3' end of (N ') x comprise a 2' -O-Me sugar modification. In another embodiment, the thirteen consecutive nucleotides at the 5 ' end of (N ') y comprise a 2' -O-Me sugar modification; (N ') x five consecutive nucleotides at the 3 ' terminus comprise a 2' -O-Me sugar modification; and Z and/or Z' independently comprises one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb, dAb-dAb, and rAb-rAb.

In a specific embodiment, five consecutive nucleotides at the 5 'end of (N') y or (N ') y-N2 each comprise a 2' -O-Me sugar modification, and two consecutive nucleotides at the 3 'end of (N') y are L-DNA. In addition, the compound may further comprise five consecutive 2 '-O-methyl modified nucleotides at the 3' terminus of (N ') x, and Z and/or Z' may independently comprise one or more non-nucleotide moieties selected from the group consisting of: an abasic moiety, an inverted abasic moiety, a hydrocarbon moiety, and an inorganic phosphate, or a combination of one or more non-nucleotide moieties and one or more nucleotides. In some embodiments, Z is selected from C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3Pi, C3Pi-rAb, C3Pi-dAb, dAb-dAb, and rAb-rAb.

In various embodiments of structures a1 or a2, the modified nucleotides in (N) x are different from the modified nucleotides in (N') y. For example, the modified nucleotide in (N) x is a2 'sugar modified nucleotide, and the modified nucleotide in (N') y is a nucleotide linked by a2 '-5' internucleotide linkage. In another example, the modified nucleotides in (N) x are mirror nucleotides, and the modified nucleotides in (N ') y are nucleotides linked by 2' -5 ' internucleotide linkages. In another example, the modified nucleotides in (N) x are nucleotides linked by 2' -5 ' internucleotide linkages, and the modified nucleotides in (N ') y are mirror nucleotides.

In some embodiments, provided herein are compounds having the structure shown below:

(X1)5 '(N) X-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

Wherein each of N and N' is a ribonucleotide which may be unmodified or modified, or is an unconventional moiety;

wherein Z consists of two non-nucleotide moieties or a combination of non-nucleotide moieties and nucleotides;

wherein Z' may be present or absent, but when present comprises a non-nucleotide moiety;

wherein z "may be present or absent, but when present is a capping moiety covalently attached to the 5 'terminus of (N') y;

wherein each of x and y is independently an integer from 18 to 27;

wherein (N ') y comprises at least one mirror nucleotide at the 3 ' terminus or at the 3 ' penultimate position; and is

Wherein the sequence of (N) x comprises an antisense sequence of a mammalian gene.

In some embodiments, x-y-19.

In some embodiments, the mirror nucleotides are selected from L-DNA and L-RNA moieties. In some embodiments, the mirror nucleotide is an L-DNA moiety. In some embodiments, Z or Z' is present and comprises an abasic moiety or a hydrocarbon moiety or a combination thereof. In some embodiments, Z' is absent, Z is present and comprises a hydrophobic moiety.

In some embodiments, the mirror nucleotides are selected from L-DNA and L-RNA moieties. In some embodiments, the mirror nucleotide is an L-DNA moiety.

In some embodiments, N ' (y) comprises two or 3 mirror nucleotides at the 3 ' terminus, and N (x) optionally comprises at least one mirror nucleotide at the 3 ' terminus.

In some embodiments, N '(y) comprises two mirror nucleotides at the 3' penultimate position. In some embodiments, N (x) comprises one or two mirror nucleotides at the 3 ' penultimate position, and N ' (y) optionally comprises one or two mirror nucleotides at the 5 ' penultimate position. In some embodiments, (N ') y comprises a mirror nucleotide at the 5 ' terminus or at the 5 ' penultimate position.

In some embodiments, (N) x comprises a 2' OMe sugar modified ribonucleotide. In some embodiments, (N) x comprises a 2' OMe sugar modified pyrimidine ribonucleotide. In some embodiments, (N) x comprises 2' OMe sugar modified ribonucleotides alternating with unmodified ribonucleotides. In some embodiments, x ═ y ═ 19 and (N) x comprises 2' OMe sugar modified ribonucleotides at positions (5 ' > 3 ') 3, 5 and 11, 13, 15, 17 and 19. In some embodiments, (N) x further comprises a mirror nucleotide or a2 '5' nucleotide at position 6 or 7.

In some embodiments, the sequence of (N) x is complementary to the sequence of (N') y; and (N') y has sequence identity to a sequence within mRNA encoded by the target gene.

In another embodiment, provided herein are compounds having the structure shown below:

(X2)5 '(N) X-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

wherein each of x and y is independently an integer from 18 to 27;

wherein (N ') y comprises at least one or more 2' OMe modified pyrimidines and

In some embodiments, x-y-19.

In some embodiments, either Z or Z' is present. In some embodiments, Z and Z' are both present. In some embodiments, Z' is absent, Z is present and comprises a hydrophobic moiety.

(X3)5 '(N) X-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

wherein each of x and y is independently an integer from 18 to 27;

wherein (N ') y comprises at least one nucleotide joined to a neighboring nucleotide by a 2' -5 ' internucleotide linkage; and is

In some embodiments, x-y-19.

In some embodiments, N ' (y) comprises 2, 3, 4, 5,6, 7, or 8 nucleotides joined to an adjacent nucleotide by a 2' -5 ' internucleotide linkage.

In some embodiments, Z or Z' is present and comprises an abasic moiety or a hydrocarbon moiety or a combination thereof. In some embodiments, Z' is absent, Z is present and comprises a hydrophobic moiety.

In some embodiments, N '(y) comprises 2, 3, 4, 5,6, 7, or 8 nucleotides at the 3' terminus joined to an adjacent nucleotide by a2 '-5' internucleotide linkage. In some embodiments, N '(y) comprises 2, 3, 4, 5,6, 7, or 8 nucleotides joined to an adjacent nucleotide at the 3' penultimate position by a2 '-5' internucleotide linkage. In some embodiments, x ═ y ═ 19 and N '(y) comprises 2, 3, 4, or 5 nucleotides at the 3' terminus joined to adjacent nucleotides by 2 '-5' internucleotide linkages. In some embodiments, x ═ y ═ 19 and N '(y) comprises at the 3' end 5 nucleotides joined to adjacent nucleotides by 2 '-5' internucleotide linkages, i.e. at positions 15, 16, 17, 18 and 19 (5 '> 3'). In some embodiments, (N) x comprises a 2' OMe sugar modified ribonucleotide. In some embodiments, (N) x comprises a 2' OMe sugar modified pyrimidine ribonucleotide. In some embodiments, (N) x comprises 2' OMe sugar modified ribonucleotides alternating with unmodified ribonucleotides. In some embodiments, x ═ y ═ 19 and (N) x comprises 2' OMe sugar modified ribonucleotides at positions (5 ' > 3 ') 3, 5 and 11, 13, 15, 17 and 19. In some embodiments, (N) x further comprises a mirror nucleotide or a2 '5' nucleotide at position 6 or 7.

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

3 'HOC 3-N' N 'N' N 'N' N 'N' N 'N' N 'N' N '-z' or

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

wherein each N is linked to an adjacent N by a covalent bond;

wherein each N 'is linked to an adjacent N' by a covalent bond;

wherein 1 to 10N are 2' -OMe sugar modified ribonucleotides;

wherein N at position 5,6, 7, 8 or 9 (5 ' > 3 ') is a 2' 5 nucleotide or a mirror nucleotide;

wherein N ' at positions 15-19 (5 ' > 3 ') is a 2' 5 ' ribonucleotide;

wherein z "is a capping moiety covalently attached to the 5' terminus of the sense strand.

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

or

5′NNNNNNNNNNNNNNNNNNN-C3Pi-C3OH

wherein each N is linked to an adjacent N by a covalent bond;

wherein each N 'is linked to an adjacent N' by a covalent bond;

wherein 1 to 10N are 2' -OMe sugar modified ribonucleotides;

wherein N 'comprises one or more 2' -OMe sugar modified pyrimidine ribonucleotides;

wherein N at position 9 or 10 (5 ' > 3 ') is a 2' 5 nucleotide; and is

In some embodiments of structure (X1-X3), either the sense strand or the antisense strand or both the antisense strand and the sense strand comprise one or two inorganic phosphate moieties at the 3' terminus.

In some embodiments of structure (X1-X3), in (N) X, the N at the 3' terminus is a modified ribonucleotide, and (N) X comprises at least 8 modified ribonucleotides. In some embodiments, the modified ribonucleotide comprises a 2' OMe sugar modified ribonucleotide. In other embodiments, at least 5 of the at least 8 modified ribonucleotides alternate starting at the 3' end.

In various embodiments of structure (X1-X3), z "is present and is selected from the group consisting of an abasic ribose moiety, a deoxyribose moiety, an inverted abasic ribose moiety, a deoxyribose moiety, a C6-amino-Pi, a mirror nucleotide.

In various embodiments of structures (X1-X3), in (N ') y there is at least one additional non-regular moiety, which can be an abasic ribose moiety, an abasic deoxyribose moiety, a modified or unmodified deoxyribonucleotide, a mirror nucleotide, a non-base-pairing nucleotide analog, or a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide phosphate linkage. In some embodiments, (N) x and (N ') y are not phosphorylated at both the 3 ' and 5 ' ends. In other embodiments, either or both of (N) x and (N ') y are phosphorylated at the 3' terminus. In another embodiment, either or both of (N) x and (N ') y are phosphorylated at the 3' end with a non-cleavable phosphate group. In another embodiment, either or both of (N) x and (N ') y are phosphorylated at the 2' terminal position with a cleavable or non-cleavable phosphate group.

In certain embodiments of all of the above structures, Z is present. In other embodiments, Z' is present. In other embodiments, both Z and Z' are present. In some embodiments, Z and Z' are both present and the same. In other embodiments, Z and Z' are both present and different. In some embodiments, Z and Z' are independently 1, 2, 3, 4, or 5 non-nucleotide moieties or a combination of non-nucleotide moieties and nucleotides.

In some embodiments, Z is present and comprises one or more non-nucleotide moieties selected from the group consisting of: abasic moieties, inverted abasic moieties, hydrocarbon moieties such as (CH2)3, and inorganic phosphate moieties.

In further embodiments, Z is present and comprises one or more non-nucleotide moieties selected from the group consisting of: abasic moieties, inverted abasic moieties, hydrocarbon moieties such as (CH2)3, and inorganic phosphate moieties.

In some embodiments, each of Z and/or Z' comprises one or two non-nucleotide moieties and further comprises a nucleotide.

In some embodiments, Z and/or Z' comprises an abasic moiety, optionally a deoxyriboabasic (referred to herein as "dAb") or riboabasic (referred to herein as "rAb") moiety. In some embodiments, each of Z and/or Z' is a dAb-dAb or rAb-rAb.

In some embodiments, Z and/or Z' comprises one or more hydrocarbon moieties, optionally (CH2)3-Pi (referred to herein as "C3 Pi"). In some embodiments, Z and/or Z' is C3Pi-C3Ps, C3Pi-C3OH, or C3Pi-C3 Pi.

In further embodiments, Z and/or Z' comprises a combination of an abasic moiety and an unmodified nucleotide or a combination of a hydrocarbon-modified moiety and an unmodified nucleotide or a combination of an abasic moiety and a hydrocarbon-modified moiety. In such embodiments, Z and/or Z' is optionally C3 Pi-rAb. In particular embodiments, only Z is present and is C3Pi-C3Ps, C3Pi-C3OH, C3Pi-C3 Pi.

In an embodiment of the above structure, the compound comprises at least one 3 'overhang (Z and/or Z'), which overhang comprises at least one non-nucleotide moiety. Z and Z' independently comprise a non-nucleotide moiety and one or more covalently linked modified or unmodified nucleotides or unconventional moieties, such as inverted dT or dA; dT, LNA, mirror nucleotides, etc. The siRNA in which Z and/or Z ' is present has improved activity and/or stability and/or off-target activity and/or reduced immune response when compared to an siRNA in which Z and/or Z ' is absent or in which Z and/or Z ' is dTdT.

In certain embodiments of all of the above structures, the compound comprises one or more phosphonocarboxylate and/or phosphinocarboxylate nucleotides (PACE nucleotides). In some embodiments, the PACE nucleotide is a deoxyribonucleotide and the phosphinocarboxylate nucleotide is a phosphinoacetate nucleotide. Examples of PACE nucleotides and analogs are disclosed in U.S. patent nos. 6,693,187 and 7,067,641, both incorporated herein by reference.

In certain embodiments of all of the above structures, the compound comprises one or more Locked Nucleic Acids (LNAs), also defined as bridge nucleic acids or bicyclic nucleotides. Exemplary locked nucleic acids include 2 '-O, 4' -C-vinyl nucleoside (ENA) or2 '-O, 4' -C-methylene nucleoside. Other examples of LNA and ENA nucleotides are disclosed in WO98/39352, WO00/47599 and WO99/14226, all incorporated herein by reference.

In certain embodiments of all of the above structures, the compound comprises one or more arabitol monomer (nucleotide), also defined as 1,5 anhydro-2-deoxy-D-arabitol (see, e.g., Allart, et al, 1998, Nucleotides & Nucleotides 17: 1523-.

Double stranded compounds in which each of N and/or N' is a deoxyribonucleotide (dA, dC, dG, dT) are specifically excluded from the invention. In certain embodiments, (N) x and (N') y may independently comprise 1, 2, 3, 4, 5,6, 7, 8, 9, or more deoxyribonucleotides. In certain embodiments, provided herein are compounds wherein each N is an unmodified ribonucleotide and the 3 ' terminal nucleotide or2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive nucleotides of the 3 ' terminus of (N ') y are deoxyribonucleotides. In other embodiments, each N is an unmodified ribonucleotide and the 5 ' terminal nucleotide or2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive nucleotides of the 5 ' terminus of (N ') y are deoxyribonucleotides. In further embodiments, the 5 'terminal nucleotide or2, 3, 4, 5,6, 7, 8, or 9 consecutive nucleotides of the 5' terminus and 1, 2, 3, 4, 5, or 6 consecutive nucleotides of the 3 'terminus of (N) x are deoxyribonucleotides, and each N' is an unmodified ribonucleotide. In further embodiments, (N) x comprises unmodified ribonucleotides and 1 or2, 3 or 4 consecutive deoxyribonucleotides independently at each of the 5 'and 3' termini and 1 or2, 3, 4, 5 or 6 consecutive deoxyribonucleotides at an internal position; and each N' is an unmodified ribonucleotide. In certain embodiments, the 3 ' terminal nucleotide of (N ') y or2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive nucleotides of the 3 ' terminus and the 5 ' terminal nucleotide of (N) x or2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, or 14 consecutive nucleotides of the 5 ' terminus are deoxyribonucleotides. In some embodiments, the 5 'terminal nucleotide of N or consecutive 2 or 3 of N and 1, 2 or 3 of N' are deoxyribonucleotides. Some examples of active DNA/RNAsiRNA chimeras are disclosed in U.S. patent publication 2005/0004064 and Ui-Tei, 2008(NAR36 (7): 2136-.

Covalent bonds refer to internucleotide linkages linking one nucleotide monomer to an adjacent nucleotide monomer. Covalent bonds include, for example, phosphodiester bonds, phosphorothioate bonds, P-alkoxy bonds, P-carboxy bonds, and the like. The normal internucleoside linkage of RNA and DNA is a 3 'to 5' phosphodiester linkage. In certain embodiments, the covalent bond is a phosphodiester bond. Covalent bonds encompass non-phosphorus containing internucleoside linkages, such as those disclosed in WO2004/041924, inter alia. In the embodiments of structures discussed herein, the covalent bond between each successive N or N' is a phosphodiester bond, unless otherwise indicated.

For all of the above structures, in some embodiments, the oligonucleotide sequence of (N) x is fully complementary to the oligonucleotide sequence of (N') y. In other embodiments, (N) x and (N') y are substantially complementary. In certain embodiments, (N) x is fully complementary to the target sequence. In other embodiments, (N) x is substantially complementary to the target sequence.

Definition of

For convenience, certain terms employed in the specification, examples, and claims will be described herein.

It should be noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless expressly stated to the contrary.

While aspects or embodiments of the invention are described in terms of Markush groups or other alternative groupings, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the group.

What is referred to herein as a "sense" or "sense strand" or "passenger strand" of a double-stranded or duplex siRNA compound refers to an oligonucleotide that has identity to a target nucleic acid (e.g., a target RNA, including a target mRNA). The term "antisense" or "antisense strand" or "guide strand" refers to an oligonucleotide that is complementary to a target nucleic acid, e.g., a target mRNA. Without wishing to be bound by theory, the incorporation of antisense or guide strands into the RNA-induced silencing complex (RISC) and direct post-transcriptional gene silencing, a phenomenon that occurs when the guide strand base-pairs with the complementary sequence of the messenger RNA molecule and mediates cleavage of mRNA by the catalytic component Argonaute of the RISC complex.

"pro-apoptotic polypeptide" refers to a polypeptide encoded by any of the above-listed genes, including splice variants, isoforms, orthologs or paralogs, and the like.

An "inhibitor" is a compound that is capable of reducing the expression of a gene or the activity of the gene product to a degree sufficient to achieve a desired biological or physiological effect. The term "inhibitor" as used herein refers to one or more of an oligonucleotide inhibitor, including siRNA, shRNA, miRNA, and ribozyme. Inhibition may also be referred to as down-regulation or, for RNAi, silencing.

The term "inhibit" as used herein refers to a reduction in the expression of a gene or the activity of the product of such a gene to a degree sufficient to achieve a desired biological or physiological effect. Inhibition may be complete inhibition or partial inhibition.

As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to nucleotide sequences that include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is also to be understood that these terms equally include analogs of either RNA or DNA made from nucleotide analogs. Throughout this application, mRNA sequences are shown as targets representing their corresponding genes. The terms "mRNA polynucleotide sequence" and mRNA are used interchangeably.

"oligonucleotide" or "oligomer" refers to a deoxyribonucleotide or ribonucleotide sequence of about 2 to about 50 nucleotides. Each DNA or RNA nucleotide may independently be natural or synthetic, and/or modified or unmodified. Modifications include altering the linkage between sugar moieties, base moieties and/or nucleotides in the oligonucleotide. The compounds disclosed herein encompass the following molecules, including: deoxyribonucleotides, ribonucleotides, modified deoxyribonucleotides, modified ribonucleotides, and combinations thereof. As used herein, the term "unpaired nucleotide analog" refers to a nucleotide analog comprising non-base-pairing moieties, including, but not limited to: 6 deaminated adenosine (Nebuline), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-MeriboU, N3-MeriboT, N3-MedC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-MedC. In some embodiments, the non-base-pairing nucleotide analog is a ribonucleotide. In other embodiments, it is a deoxyribonucleotide.

Provided herein are methods and compositions for inhibiting expression of a target gene in vivo. Generally, the method comprises administering oligoribonucleotides, particularly small interfering RNAs (i.e., sirnas) or nucleic acid agents that generate sirnas in cells, to target mammalian mrnas in an amount sufficient to down-regulate expression of a target gene by an RNA interference mechanism. In particular, the methods can be used to inhibit gene expression to treat a subject having a disease associated with the gene expression. As disclosed herein, siRNA molecules or inhibitors of target genes are useful as drugs for the treatment of various pathologies.

"siRNA compound" and "nucleic acid molecule" are used interchangeably.

"nucleotide" is intended to encompass compounds consisting of a nucleoside (a sugar, typically a ribose or deoxyribose, and a purine or pyrimidine base) and a phosphorus linker; such as deoxyribonucleotides and ribonucleotides, which may be natural or synthetic and modified or unmodified. Modifications include changes and substitutions of sugar moieties, base moieties and/or internucleotide linkages.

The "phosphate-based" moiety includes inorganic phosphates (Pi) and phosphorothioates (Ps).

All analogs or modifications of the nucleotides/oligonucleotides having the molecules disclosed herein can be employed, provided that the analogs or modifications do not significantly adversely affect the function of the nucleotides/oligonucleotides. Acceptable modifications include modifications of the sugar moiety, modifications of the base moiety, modifications in the internucleotide linkage and combinations thereof.

Sometimes referred to as "abasic nucleotides" or "abasic nucleotide analogs" are more suitably referred to as pseudonucleotides or unconventional moieties. Nucleotides are monomeric units of nucleic acids, consisting of ribose or deoxyribose, phosphate, and bases (adenine, guanine, thymine or cytosine in DNA and adenine, guanine, uracil or cytosine in RNA). Modified nucleotides comprise modifications in one or more of the sugar, phosphate and/or base. An abasic pseudonucleotide contains no bases and is therefore not a nucleotide in the strict sense. Abasic deoxyribose moieties include, for example, abasic deoxyribose-3' -phosphate; 1, 2-dideoxy-D-ribofuranose-3-phosphate; 1, 4-anhydro-2-deoxy-D-ribitol-3-phosphate. The inverted abasic deoxyribose moiety comprises an inverted deoxyribose abasic; 3 ', 5 ' inverted deoxyabasic 5 ' -phosphate. Generally, the inverted abasic moiety is covalently linked to the 3 ' terminal nucleotide by a 3 ' -3 ' linkage; the inverted abasic moiety is covalently linked to the 5 ' terminal nucleotide by a 5 ' -5 ' linkage; the inverted abasic moiety is covalently linked to the inverted abasic moiety, typically by a 5 '-3' linkage.

The term "capping moiety" (z ") as used herein includes moieties that can be covalently attached to the 5 ' terminus of (N ') y and includes abasic ribose moieties, abasic deoxyribose moieties, modified abasic ribose and abasic deoxyribose moieties that comprise 2' O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and combinations thereof; C6-imino-Pi; mirror nucleotides including L-DNA and L-RNA; a 5' OMe nucleotide; and nucleotide analogs comprising 4 ', 5' -methylene nucleotides; 1- (. beta. -D-erythrofuranosyl) nucleotide; 4' -thio nucleotides, carbocyclic nucleotides; 5' -aminoalkyl phosphates; 1, 3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1, 5-anhydrohexitol nucleotide; an alpha-nucleotide; threo-pentofuranosyl nucleotides; acyclic 3 ', 4' -seco nucleotides; 3, 4-dihydroxybutyl nucleotide; 3, 5-dihydroxypentylnucleotide, a 5 '-5' -inverted abasic moiety; 1, 4-butanediol phosphate; 5' -amino; and bridged or unbridged methylphosphonates and 5' -mercapto moieties.

Some of the capping moieties are abasic ribose or abasic deoxyribose moieties; an inverted abasic ribose or abasic deoxyribose moiety; C6-amino-Pi, mirror nucleotides including L-DNA and L-RNA. The compounds of the invention can be synthesized using one or more inverted nucleotides, such as inverted thymidine or inverted adenine (see, e.g., Takei, et al, 2002.JBC277 (26): 23800-06).

The term "non-nucleotide moiety" refers to a moiety that is not a nucleotide, i.e., does not contain all components of a nucleotide: sugar, base, and linker.

The term "non-conventional moiety" as used herein refers to a non-nucleotide moiety comprising an abasic moiety, an inverted abasic moiety, a hydrocarbon (alkyl) moiety, and an inorganic phosphate, and further includes deoxyribonucleotides, modified deoxyribonucleotides, mirror nucleotides (L-DNA or L-RNA), non-base-pairing nucleotide analogs, and nucleotides that are joined to adjacent nucleotides by 2 '-5' internucleotide phosphate linkages (also referred to as 2 '5' nucleotides), bridge nucleic acids including LNA and vinyl bridge nucleic acids, linkage-modified (e.g., PACE), and base-modified nucleotides, as well as additional moieties specifically disclosed herein as non-conventional moieties.

When used with respect to an highlighting application, "alkyl moiety" or "hydrocarbon moiety" refers to a C2, C3, C4, C5, or C6 straight or branched chain alkyl moiety, including, for example, C2 (ethyl), C3 (propyl). When used with respect to an outstanding use, an "derivative" of an alkyl or hydrocarbon moiety refers to a C2, C3, C4, C5, or C6 linear or branched alkyl moiety that contains a functional group that may be selected from, inter alia, alcohols, phosphodiesters, phosphorothioates, phosphoacetates, amines, carboxylic acids, esters, amides, and aldehydes.

When used in relation to the modification of the ribose or deoxyribose moiety, "alkyl" is intended to include straight chain, branched chain, or cyclic saturated hydrocarbon structures, and combinations thereof. When it comes toWhen modifications of the ribose or deoxyribose moiety are used, "lower alkyl" specifically refers to an alkyl group having 1 to 6 carbon atoms. Examples of lower alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, and the like. Preferred alkyl groups are C₂₀Or those below. Cycloalkyl groups are a subset of alkyl groups and include cyclic saturated hydrocarbon groups having 3 to 8 carbon atoms. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, norbornyl (norbonyl), adamantyl, and the like.

"terminal functional groups" include halogens, alcohols, amines, carboxyl groups, esters, amides, aldehydes, ketones, ether groups.

In the context of the present invention, a "mirror" nucleotide (also referred to as spiegelmer) is a nucleotide analogue which has the opposite chirality to the naturally occurring or commonly used nucleotide, i.e. is a mirror image of the naturally occurring or commonly used nucleotide. The mirror nucleotides are ribonucleotides (L-RNA) or deoxyribonucleotides (L-DNA) and may further comprise at least one sugar or base modification and/or backbone modification, such as a phosphorothioate or phosphonate moiety. U.S. Pat. No.6,602,858 discloses nucleic acid catalysts comprising at least one L-nucleotide substitution. Mirror nucleotides include, for example, L-DNA (L-deoxyriboadenosine-3 '-phosphate (mirror dA), L-deoxyribocytidine-3' -phosphate (mirror dC), L-deoxyriboguanosine-3 '-phosphate (mirror dG), L-deoxyribothymidine-3' -phosphate (mirror dT)) and L-RNA (L-riboadenosine-3 '-phosphate (mirror rA), L-ribocytidine-3' -phosphate (mirror rC), L-riboguanosine-3 '-phosphate (mirror rG), L-ribouracil-3' -phosphate (mirror dU)).

Modified deoxyribonucleotides include, for example: 5 ' OMeDNA (5-methyl-deoxyriboguanosine-3 ' -phosphate) which can be used as the nucleotide at the 5 ' terminal position (position No. 1); PACE (deoxyriboadenine 3 'phosphoacetate, deoxycytidine 3' phosphoacetate, deoxyriboguanosine 3 'phosphoacetate, deoxyribothymidine 3' phosphoacetate).

Unconventional moieties include bridge nucleic acids, including LNA (2 '-O, 4' -C-methylene bridge nucleic acid adenosine 3 'monophosphate, 2' -O, 4 '-C-methylene bridge nucleic acid 5-methyl-cytidine 3' monophosphate, 2 '-O, 4' -C-methylene bridge nucleic acid guanosine 3 'monophosphate, 5-methyl-uridine (or thymidine) 3' monophosphate); and ENA (2 '-O, 4' -C-vinyl bridge nucleic acid adenosine 3 'monophosphate, 2' -O, 4 '-C-vinyl bridge nucleic acid 5-methyl-cytidine 3' monophosphate, 2 '-O, 4' -C-vinyl bridge nucleic acid guanosine 3 'monophosphate, 5-methyl-uridine (or thymidine) 3' monophosphate).

In some embodiments of the invention, the non-canonical moieties are abasic ribose moieties, abasic deoxyribose moieties, deoxyribonucleotides, mirror nucleotides, and nucleotides that are joined to adjacent nucleotides by 2 '-5' internucleotide phosphate linkages.

The nucleotide is selected from naturally occurring or synthetic modified bases. Naturally occurring bases include adenine, guanine, cytosine, thymine and uracil. Modified bases of nucleotides include inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl 1, 2-propyl and other alkyl adenine, 5-halogen uracil, 5-halogen cytosine, 6-azacytosine and 6-azathymine, pseudouracil, 4-thiouracil, 8-halogen adenine, 8-amino adenine, 8-thioalkyl adenine, 8-hydroxyadenine and other 8-substituted adenine, 8-halo guanine, 8-aminoguanine, 8-thioalkyl guanine, 8-hydroxyguanine and other substituted guanines, other aza and deaza adenine, other aza and deaza guanine, 5-trifluoromethyl uracil and 5-trifluorocytosine. The present invention encompasses siRNA compounds comprising one or more abasic pseudonucleotides. Nucleotide monomers comprising modified bases, including abasic pseudonucleotide monomers, may be substituted with one or more ribonucleotides of an oligonucleotide. The abasic pseudonucleotide monomer may comprise one or more at a terminal position or as a 5' terminal cap. The 5' end cap may also be selected from the group consisting of inverted abasic pseudonucleotide analogs, L-DNA nucleotides, and C6-iminophosphates.

In addition, analogs of polynucleotides are prepared in which the structure of one or more nucleotides is fundamentally altered and are more suitable as therapeutic or experimental agents. An example of a nucleotide analogue is a Peptide Nucleotide (PNA), in which the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA) comprises a polyamide backbone similar to that present in the peptide. PNA analogs have been shown to be resistant to enzymatic degradation and have a longer in vivo and in vitro lifetime.

Possible modifications of sugar residues are diverse and include 2' -O alkyl, 2' -halo (e.g., 2' deoxy fluoro), Locked Nucleic Acid (LNA), diol nucleic acid (GNA), Threose Nucleic Acid (TNA), arabinoside; altritol (ANA) and other 6-membered sugars, including morpholinos and cyclohexenes (cyclohexenes). Possible modifications on the 2' portion of the sugar residue include amino, fluoro, methoxyalkoxy, alkyl, amino, fluoro, chloro, bromo, CN, CF, imidazole, carboxylate, thioester (thioate), C₁To C₁₀Lower alkyl, substituted lower alkyl, alkaryl or aralkyl, OCF₃OCN, O-, S-or N-alkyl; o-, S-or N-alkenyl; SOCH₃SO₂CH₃；ONO₂；NO₂、N₃(ii) a A heterocycloalkyl group; a heterocycloalkylaryl group; an aminoalkylamino group; polyalkylamino groups or substituted silyl groups, as described inter alia in european patent EP0586520B1 or EP0618925B 1. One or more deoxyribonucleotides may also be tolerated in the compounds of the invention. In some embodiments, (N') comprises 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 DNA moieties.

LNA compounds are disclosed in International patent publication Nos. WO00/47599, WO99/14226, and WO 98/39352. Examples of siRNA compounds comprising LNA nucleotides are disclosed in Elmen et al, (NAR2005.33 (1): 439-447) and International patent publication No. WO2004/083430. Six-membered ring nucleotide analogs are disclosed in Allan, et al, (Nucleotides & Nucleotides, 1998, 17: 1523-. Oligonucleotides comprising 6-membered cyclic nucleotide analogs containing both hexol and altritol nucleotide monomers are disclosed in international patent application publication No. wo 2006/047842.

Backbone modifications (also referred to as internucleotide linkage modifications) such as ethyl (to produce phospho-ethyl triester), propyl (to produce phospho-propyl triester) and butyl (to produce phospho-butyl triester) are also possible. Other backbone modifications include polymer backbones, cyclic backbones, acyclic backbones, phosphorothioate-D-ribose backbones, amidates, phosphoacetate derivatives. Certain structures include siRNA compounds having one or more 2 '-5' internucleotide linkages (bridges or backbones).

In some embodiments, (N) x and (N ') y are not phosphorylated at both the 3 ' and 5 ' ends. In other embodiments, either or both of (N) x and (N ') y are phosphorylated at the 3 ' terminus (3 ' Pi). In another embodiment, either or both of (N) x and (N ') y are phosphorylated at the 3' end with a non-cleavable phosphate group. In another embodiment, either or both of (N) x and (N ') y are phosphorylated at the 2' terminal position using a phosphate group, which may or may not be cleavable. In addition, the inhibitory core molecules of the invention may comprise one or more clefts and/or one or more gaps and/or one or more mismatches. Without wishing to be bound by theory, clefts, gaps, and mismatches have the advantage of destabilizing the nucleic acid/siRNA portion so that it can be more easily processed into its inhibitory component by endogenous cellular mechanisms such as DICER, DROSHA, or RISC.

In the context of the present invention, a nick in a nucleic acid refers to the absence of one or more internal nucleotides in one strand, whereas a nick in a nucleic acid refers to the absence of an internucleotide linkage between two adjacent nucleotides of one strand. Any molecule of the invention may comprise one or more clefts and/or one or more gaps.

Oligonucleotides

In a non-limiting example, table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo2009/044392, assigned to the assignee of the present invention and incorporated by reference in its entirety, comprise nucleic acid sequences useful for preparing sense and corresponding antisense oligomers of siRNA compounds according to the present invention. These compounds are used as chemically and/or structurally modified compounds.

Selection and synthesis of sirnas corresponding to known genes has been widely reported; see, e.g., Ui-Tei et al, JBiomedBiotechnol.2006; 65052; chalk et al, BBRC.2004, 319 (1): 264-74; sioud & Leirdal, met.molbiol.2004, 252: 457-69; levenkova et al, Bioinformam.2004, 20 (3): 430-2; Ui-Tei et al, NAR.2004, 32 (3): 936-48. For examples of the use and preparation of modified sirnas, see, e.g., Braasch et al, biochem.2003, 42 (26): 7967-75; chiu et al, RNA.2003, 9 (9): 1034-48; PCT publication Nos. WO2004/015107 and WO02/44321 and U.S. Pat. Nos. 5,898,031 and 6,107,094.

The present invention provides double-stranded oligonucleotides (e.g., sirnas) that down-regulate expression of a desired gene. The sirnas of the invention are duplex oligoribonucleotides wherein the sense strand is derived from the mRNA sequence of the desired gene and the antisense strand is at least substantially complementary to the sense strand. In general, some deviation from the target mRNA sequence can be tolerated without decreasing siRNA activity (see, e.g., Czauderna et al, NAR.2003, 31 (11): 2705-. The siRNA of the present invention inhibits gene expression at the post-transcriptional level with or without disruption of mRNA. Without being bound by theory, siRNA can target specific cleavage and degradation of mRNA and/or can inhibit translation of the targeted message.

In other embodiments, at least one of the two strands may have an overhang of at least one nucleotide at the 5' -end; the overhang may be comprised of at least one deoxyribonucleotide. The length of the RNA duplex is from about 16 to about 40 ribonucleotides, preferably 19 ribonucleotides. Additionally, each chain may independently have a length selected from the group consisting of: from about 16 to about 40 bases, preferably 18 to 23 bases, more preferably 19 ribonucleotides.

In certain embodiments, the first strand is perfectly complementary to the target nucleic acid. In some embodiments, the strands are substantially complementary, i.e., have one, two, or up to five mismatches between the first strand and the target mRNA or between the first and second strands. Substantially complementary refers to greater than about 70% and less than 100% complementarity to another sequence. For example, in a duplex region consisting of 19 base pairs, one mismatch results in 94.7% complementarity, two mismatches results in about 89.5% complementarity, 3 mismatches results in about 84.2% complementarity, 4 mismatches results in about 79% complementarity and 5 mismatches results in about 74% complementarity, resulting in substantial complementarity of the duplex region. Thus, "substantially identical" refers to greater than about 70% identity to another sequence.

The first and second strands may be connected by a cyclic structure, which may be composed of a non-nucleic acid polymer such as polyethylene glycol, among others. Alternatively, the loop structure may be comprised of nucleic acids, including modified and unmodified ribonucleotides and modified and unmodified deoxyribonucleotides.

In addition, the 5 '-end of the first strand of the siRNA can be linked to the 3' -end of the second strand, or the 3 '-end of the first strand can be linked to the 5' -end of the second strand via a nucleic acid linker that typically has a length of 2-100 nucleobases, preferably about 2 to about 30 nucleobases.

In some embodiments of compounds of the invention having modified alternating ribonucleotides in at least one of the antisense and sense strands of the compound, for 19mer and 23mer oligomers, the ribonucleotides at the 5 'and 3' ends of the antisense strand are modified in their sugar residues, while the ribonucleotides at the 5 'and 3' ends of the sense strand are unmodified in their sugar residues. For the 21mer oligomer, the ribonucleotides at the 5 ' and 3 ' ends of the sense strand are modified in their sugar residues, while the ribonucleotides at the 5 ' and 3 ' ends of the antisense strand are unmodified in their sugar residues or may have optional additional modifications at the 3 ' end. As described above, in some embodiments, the intermediate nucleotide of the antisense strand is unmodified.

According to one embodiment of the invention, the antisense and sense strands of the oligonucleotide/siRNA are phosphorylated only at the 3 '-terminus and not at the 5' -terminus. According to another embodiment of the invention, the antisense and sense strands are not phosphorylated. According to yet another embodiment of the invention, the 5 '-most ribonucleotide in the sense strand is modified to eliminate any possibility of 5' -phosphorylation in vivo.

Preparing any of the siRNA sequences disclosed herein having any of the modifications/structures disclosed herein. The combination of sequences and structures is novel and useful for treating the disorders disclosed herein.

Pharmaceutical composition

Although it is possible to administer the compounds of the present invention as the original chemical, it is preferred that they be present as a pharmaceutical composition. Accordingly, the present invention provides pharmaceutical compositions comprising one or more of the compounds of the present invention and a pharmaceutically acceptable carrier. The composition may comprise a mixture of two or more different oligonucleotides/sirnas.

The present invention further provides a pharmaceutical composition comprising at least one compound of the invention covalently or non-covalently bound to one or more compounds of the invention in an amount effective to inhibit one or more genes disclosed above and a pharmaceutically acceptable carrier. The compounds can be processed intracellularly by endogenous cellular complexes to produce one or more oligoribonucleotides of the invention.

The invention further provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one or more compounds of the invention comprising a compound of formula (I) in combination with (N) in an amount effective to down-regulate the expression of a target RNA, including a target gene and a target mRNA and/or a target protein, in a cell_xSequences having complementarity. In certain embodiments, the target gene is a viral, bacterial, or mammalian gene. In various embodiments, the target gene is a mammalian gene, preferably a human gene.

In addition, the invention provides a method of inhibiting expression of a target gene by at least 50% as compared to a control comprising contacting an mRNA transcript of the target gene with one or more of the compounds of the invention. In some embodiments, the active siRNA compound inhibits gene expression at a level of at least 50%, 60%, or 70% compared to a control. In certain embodiments, the level of inhibition is at least 75%, 80%, or 90% as compared to a control. In some embodiments, the target gene is a pro-apoptotic gene as disclosed herein.

In one embodiment, the oligoribonucleotide inhibits one or more of the pro-apoptotic genes of the invention, thus inhibiting a protein selected from the group consisting of: inhibition of gene function, inhibition of polypeptides and inhibition of mRNA expression.

In one embodiment, the compound inhibits expression of a polypeptide encoded by a target gene, thereby inhibiting a polypeptide selected from the group consisting of: inhibition of function (which can be examined, inter alia, by enzymatic assays or binding assays to known effectors of the native gene/polypeptide), inhibition of protein (which can be examined, inter alia, by Western blot, ELISA, or immunoprecipitation), and inhibition of mRNA expression (which can be examined, inter alia, by Northern blot, quantitative RT-PCR, in situ hybridization, or microarray hybridization).

In additional embodiments, the invention provides a method of treating a subject having a disease associated with an elevated level of a pro-apoptotic gene of the invention, the method comprising administering to the subject a therapeutically effective dose of a compound of the invention, thereby treating the subject.

Delivery of

In some embodiments, the siRNA molecules of the invention are delivered to a target tissue by direct application of naked molecules prepared with a carrier or diluent.

The term "naked siRNA" refers to an siRNA molecule that is free of any delivery vehicle that facilitates, or facilitates entry into a cell, including viral sequences, viral particles, liposomal formulations, Lipofectin, or precipitating agents, and the like. For example, the siRNA in PBS is "naked siRNA".

However, in some embodiments, the siRNA molecules of the invention are delivered in liposomes or Lipofectin formulations, or the like, and prepared by methods well known to those skilled in the art. Such processes are disclosed, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are incorporated herein by reference.

Delivery systems have been developed specifically for enhancing and improving the delivery of siRNA to mammalian cells (see, e.g., Shen et al FEBSLet.2003, 539: 111-114; Xia et al, nat. Biotech.2002, 20: 1006-1010; Reich et al, mol. Vision2003, 9: 210-216; Sorensen et al, J. mol. biol. 2003.327: 761-766; Lewis et al, nat. Gen.2002, 32: 107-108 and Simeoni et al, NAR2003, 31, 11: 2717-2724). siRNA has recently been successfully used to inhibit gene expression in primates (see, e.g., Tolentino et al, Retina24 (4): 660).

Pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles, as well as implant carriers, generally refer to inert, non-toxic solid or liquid fillers, diluents or encapsulating materials that do not react with the active ingredients of the invention, and they include liposomes and microspheres. Examples of delivery systems useful in the present invention include U.S. Pat. Nos. 5,225,182, 5,169,383, 5,167,616, 4,959,217, 4,925,678, 4,487,603, 4,486,194, 4,447,233, 4,447,224, 4,439,196, and 4,475,196. Many other such implants, delivery systems, and modules are well known to those skilled in the art. In one embodiment of the invention, topical and transdermal formulations may be selected. The siRNA or pharmaceutical composition of the present invention is administered and administered according to good medical practice in consideration of the following aspects: the clinical condition of the individual patient, the disease to be treated, the site and method of administration, the administration schedule, the age, sex, weight of the patient and other factors known to the physician.

Thus, for purposes herein, a "therapeutically effective dose" is determined by such considerations as are known in the art. The dosage must be effective to achieve an improvement, including but not limited to improving survival or faster recovery, or ameliorating or eliminating symptoms, and other indicators selected as appropriate by one of skill in the art.

Generally, the dose can be 0.01mg to 1g per kilogram body weight (e.g., 0.1mg, 0.25mg, 0.5mg, 0.75mg, 1mg, 2.5mg, 5mg, 10mg, 25mg, 50mg, 100mg, 250mg, 500mg, 1mg, 2.5mg, 5mg, 10mg, 25mg, 50mg, 100mg, 250mg, or 500mg per kilogram).

Suitable dosage units of the nucleic acid molecule may be in the range of 0.001 to 0.25mg per kg body weight of the recipient per day, or in the range of 0.01 to 20 micrograms per kg body weight per day, or in the range of 0.01 to 10 micrograms per kg body weight per day, or in the range of 0.10 to 5 micrograms per kg body weight per day, or in the range of 0.1 to 2.5 micrograms per kg body weight per day.

Appropriate amounts of the nucleic acid molecule can be administered, and these amounts can be determined empirically using standard methods. An effective concentration of each nucleic acid molecule species in a cellular environment can be about 1 femolar (fmolar), about 50 femolar, 100 femolar, 1 picomolar, 1.5 picomolar, 2.5 picomolar, 5 picomolar, 10 picomolar, 25 picomolar, 50 picomolar, 100 picomolar, 500 picomolar, 1 nanomolar, 2.5 nanomolar, 5 nanomolar, 10 nanomolar, 25 nanomolar, 50 nanomolar, 100 nanomolar, 500 nanomolar, 1 micromolar, 2.5 micromolar, 5 micromolar, 10 micromolar, 100 micromolar, or higher.

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Dosage unit forms typically contain from about 1mg to about 500mg of the active ingredient.

It will be understood that the specific dose level for any particular subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

Pharmaceutical compositions comprising a nucleic acid molecule disclosed herein can be administered once daily, four times daily, three times daily, twice daily, once daily, or at any interval and for any length of time medically appropriate. However, the therapeutic agent may also be administered in dosage units comprising two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day. In this case, the nucleic acid molecules contained in each sub-dose may be correspondingly smaller in order to achieve a total daily dosage unit. Dosage unit units may also be compounded in a single dose over several days, for example using conventional sustained release formulations, which provide sustained and consistent dsRNA release over a period of several days. Sustained release formulations are well known in the art. A dosage unit may contain a corresponding plurality of daily doses. The compositions can be compounded in such a way that the sum of the multiple units of nucleic acid together comprise a sufficient dosage.

Kit and container

Kits, containers, and formulations comprising a nucleic acid molecule (e.g., a siNA molecule) for reducing expression of a target gene as provided herein are also provided for administering the nucleic acid molecule to a subject. In some embodiments, a kit comprises at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container may be formed from a variety of materials such as glass, metal or plastic. The kit may further comprise relevant indications and/or instructions; reagents and other compositions or means for this purpose may also be included.

Alternatively, the container may contain a composition effective in treating, diagnosing, prognosing or preventing a condition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition can be a nucleic acid molecule capable of specifically binding to a target gene and/or modulating the function of a target gene.

The kit may further comprise a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, ringer's solution, and/or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, agitators, needles, syringes, and/or package inserts with instructions for indication and/or use.

Unit-dose ampoules or multi-dose containers for packaging nucleic acid molecules prior to use may comprise a hermetically sealed container enclosing an amount of the nucleic acid molecule or a solution containing the nucleic acid molecule which is suitable for one pharmaceutically effective dose or a plurality of effective doses thereof. The nucleic acid molecules are packaged as sterile preparations and the hermetically sealed container is designed to ensure sterility of the preparation prior to use.

The container holding the nucleic acid molecule may comprise a package to which the label is affixed, and the label may have a notice in the form prescribed by a governmental agency, such as the food and drug administration, which notice reflects approval by the governmental agency under federal regulations for the manufacture, use or sale of polynucleotide materials therein for human administration.

Federal or state law requires that pharmaceutical compositions for use in human therapy must be approved by federal or state government agencies. In the united states, the administration is under the responsibility of the food and drug administration, which issues a specification for obtaining such approval, as described in detail in 21u.s.c. § 301-. Procedures unique to most foreign countries require similar approval as is well known to those skilled in the art, and the compositions and methods provided herein preferably conform to the approval accordingly.

The dose to be administered depends to a large extent on the condition and size of the subject being treated, as well as the frequency of treatment and route of administration. The regimen (including dose and frequency) of continuous treatment may be determined based on initial response and clinical judgment.

The nucleic acid compounds disclosed herein are administered by any conventional route of administration. It should be noted that the compounds are administered as such or as pharmaceutically acceptable salts, as well as alone or as active ingredients in combination with pharmaceutically acceptable carriers, solvents, diluents, excipients, adjuvants and vehicles. Routes of administration of the compounds are oral, topical, subcutaneous or parenteral (including intravenous, intraarterial, intramuscular, intraperitoneal and intranasal), inhalation, intratympanic administration and intrathecal injection and infusion techniques. Implants of the compounds are also useful. Liquid injectable dosage forms can be prepared, and the term includes subcutaneous, transdermal, intravenous, intramuscular, intrathecal and other parenteral routes of administration. Liquid compositions include aqueous solutions with and without organic co-solvents, aqueous or oily suspensions, emulsions with edible oils and similar pharmaceutical carriers. In particular embodiments, administering comprises intravenous administration. In another embodiment, administering comprises topical or topical administration. In some embodiments, topically applying comprises external application to the ear canal of the mammal. In some embodiments, topical application comprises external application to the ocular surface of a mammal.

Furthermore, in certain embodiments, the compositions for use in the novel treatments of the present invention may be formed as an aerosol, e.g., for intranasal administration.

In certain embodiments, oral compositions (such as tablets, suspensions, solutions) are effective for topical delivery to the oral cavity, such as oral compositions suitable for use in mouthwashes for the treatment of oral mucositis.

In embodiments, the subject undergoing treatment is a warm-blooded animal and particularly a mammal including a human.

In further embodiments, the modification is a modification of a phosphate moiety, such that the modified phosphate moiety is selected from the group consisting of phosphorothioates or contains no phosphate groups.

The molecules of the invention include siRNA, siNA, synthetic siRNA, synthetic shRNA and miRNA in addition to other nucleic acid sequences or molecules encoding such molecules or other inhibitory nucleotide molecules.

In some embodiments, the nucleic acid compounds are useful for diagnosis. Without wishing to be bound by theory, double-stranded nucleic acid molecules comprising a 3' terminal non-nucleotide can be efficiently delivered to a particular cell or tissue and can be used to diagnose a condition with respect to the particular cell or tissue. Thus, the terminal modifications include detectable moieties, including chromogenic agents (colorgenigent), radiolabelled moieties, and enzymatic reagents. In some embodiments, the detectable agent is a biotin group. This biotin group may preferably be attached to either or both of the 5 'endmost nucleotide of the sense strand or the 3' endmost nucleotide of the antisense strand. The various end modifications disclosed herein are preferably located in the ribose moiety of the nucleotides of the nucleic acids according to the invention. More specifically, the terminal modification may be attached to or replace any OH-group of the ribose moiety, including but not limited to the 2 'OH, 3' OH and 5 'OH positions, provided that the nucleotide thus modified is a terminal nucleotide, preferably the 5' terminal nucleotide of the sense strand. It is understood that the nucleic acid molecules disclosed herein, or any long double stranded RNA molecule (typically 25-500 nucleotides in length) processed by endogenous cellular complexes (such as DICER, see above) to form the siRNA molecules disclosed herein, or molecules comprising the siRNA molecules disclosed herein, are incorporated into the molecules of the invention to form additional novel molecules and used to treat the diseases or conditions described herein.

In particular, it is contemplated that long oligonucleotides may be delivered in a carrier, preferably a pharmaceutically acceptable carrier, and may be processed intracellularly by endogenous cellular complexes (e.g., by DROSHA and DICER as described above) to form one or more smaller double-stranded oligonucleotides (sirnas) that are oligonucleotides of the invention. This oligonucleotide is referred to as a tandem shRNA construct. It is contemplated that the long oligonucleotide is a single stranded oligonucleotide comprising one or more stem-loop structures, wherein each stem region comprises a sense and corresponding antisense siRNA sequence. Any molecule such as, for example, an antisense DNA molecule comprising an inhibitory sequence (with appropriate nucleic acid modifications) as disclosed herein is particularly desirable and can be used for all uses and methods disclosed herein with the same capabilities as their corresponding RNA/siRNA.

Main chain

The nucleoside subunits of the nucleic acid molecules disclosed herein can be linked to each other by phosphodiester bonds. The standard 5 '3' phosphodiester bond may optionally be substituted with other linkages. For example, phosphorothioate-D-ribose entities, triesters, thioesters, 2 '-5' bridged backbones (also referred to as 5 '-2' or2 '5'), PACE, 3 '- (or-5') deoxy-3 '- (or-5') thio-phosphorothioate, phosphorodithioate, phosphoroselenoate, 3 '- (or-5') deoxyphosphite, boranophosphate, 3 '- (or-5') deoxy-3 '- (or-5') phosphoramidate, hydrogenphosphonate, phosphonate, boranophosphate, alkyl or aryl phosphonate, and phosphotriester modifications such as alkyl phosphotriesters, phosphotriester phosphorous linkages, 5 '-ethoxy phosphodiesters, P-alkoxy phosphotriesters, methyl phosphonates, and linkages other than phosphorus, e.g., carbonates, phosphoesters, thioesters, 2' -5 'bridged backbones (also referred to as 5' -2 'or 2' 5 '), PACE, 3' - (or, Carbamate, silyl, sulfur, sulfonate, sulfonamide, formacetal, thioformacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazine, methylenedimethylhydrazine, and methyleneoxymethylimino linkages. In addition, analogs of polynucleotides can be prepared in which the structure of the nucleotide is fundamentally altered and more suitable as therapeutic or experimental agents. An example of a nucleotide analogue is a Peptide Nucleotide (PNA), in which the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA) comprises a polyamide backbone similar to that present in the peptide. PNA analogs have been shown to be resistant to enzymatic degradation and have extended in vivo and in vitro lifetimes. In addition, PNAs have been shown to bind stronger to complementary DNA sequences than DNA molecules. This finding is attributed to the absence of charge repulsion between the PNA strand and the DNA strand. Other modified monomers that may be used in the synthesis of oligonucleotides include moieties having a polymeric backbone, a cyclic backbone, or an acyclic backbone.

Method of treatment

In another aspect, the invention relates to a method of treating a subject in need of treatment for a disease or condition associated with aberrant expression of a target gene, comprising administering to the subject an amount of an inhibitor that reduces or inhibits expression of the gene.

In certain embodiments, the subject undergoing treatment is a warm-blooded animal and particularly a mammal including a human.

The methods of the invention comprise administering to a subject one or more inhibitory compounds that down-regulate gene expression; and particularly a therapeutically effective dose of siRNA, to treat the subject.

The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (alleviate) the conditions listed herein. Those in need of treatment include those already with the disease or condition, those predisposed to developing the disease or condition, and those in which the disease or condition is to be prevented. The compounds of the invention are administered before, during or after the onset of the disease or disorder or symptoms associated therewith. Where treatment is used for the purposes of the present invention, then the present invention relates to methods of delaying the onset or preventing the development of a disease or condition.

The present invention relates to the use of compounds that down-regulate the expression of the pro-apoptotic genes of the invention in the treatment of the following diseases or conditions in which inhibition of the expression of the pro-apoptotic genes is beneficial, in particular to novel small interfering rnas (sirnas): hearing loss, Acute Renal Failure (ARF), glaucoma, Acute Respiratory Distress Syndrome (ARDS) and other acute lung and respiratory tract injuries, ischemia-reperfusion injury after lung transplantation, injury in organ transplantation (including lung, liver, heart, bone marrow, pancreas, cornea and kidney transplantation), including DGF; spinal cord injury, decubitus ulcers, age-related macular degeneration (AMD), dry eye syndrome, ocular ischemic conditions (including ION and NAION); oral mucositis and Chronic Obstructive Pulmonary Disease (COPD). Other indications include chemically induced nephrotoxicity and chemically induced neurotoxicity, such as toxicity induced by cisplatin and cisplatin-like compounds, by aminoglycosides, by loop diuretics, and by hydroquinones and analogs thereof.

Methods, molecules, and compositions for inhibiting the pro-apoptotic genes of the present invention are discussed in detail herein, and any such molecule and/or composition can be advantageously used to treat a subject having any such disorder.

In another aspect, an article of manufacture is provided that includes packaging material containing an oligonucleotide composition according to the invention that is therapeutically effective to treat a subject having any of the indications disclosed herein, and instructions for use.

Disclosed herein is a method for preparing a double stranded RNA molecule capable of target-specific inhibition or down-regulation of target gene expression, wherein each RNA strand has a length of 19 to 25 nucleotides, wherein at least one strand has a non-nucleotide moiety, the non-nucleotide moiety is covalently attached at the 3 ' or 2' position of the sugar residue at its 3 ' terminus, the method comprising (a) synthesizing two RNA strands each 19 to 25 nucleotides in length, wherein the RNA strands are capable of forming a double-stranded RNA molecule, (b) combining the synthesized RNA strands under conditions, wherein a double-stranded RNA molecule is formed, wherein the double-stranded RNA molecule consists of a single double-stranded region and at least one single-stranded region, the at least one single stranded region comprises a non-nucleotide moiety covalently attached at a 3 ' or 2' position to a sugar residue at the 3 ' terminus of the strand in which it is present; wherein the non-nucleotide moiety is selected from the group consisting of propanol, a C3 alkyl moiety linked to a phosphodiester, a C3 alkyl moiety linked to a phosphorothioate, a deoxyriboabasic moiety, a riboabasic moiety, and combinations thereof.

Provided herein is a process for preparing a pharmaceutical composition comprising:

providing one or more compounds disclosed herein; and is

Compounding the compound with a pharmaceutically acceptable carrier.

The invention also provides a process for preparing a pharmaceutical composition comprising compounding one or more compounds of the invention with a pharmaceutically acceptable carrier.

In one embodiment, the compound used to prepare the pharmaceutical composition is compounded with a carrier in a pharmaceutically effective dose. In particular embodiments, the compounds of the invention are conjugated to a steroid or a lipid or another suitable molecule, such as cholesterol.

Synthesis of modified Compounds

The compounds of the invention can be synthesized by any method known in the art for the synthesis of ribonucleic acid (or deoxyribonucleic acid) oligonucleotides. This synthesis is described, inter alia, in Beaucage and Iyer, Tetrahedron 1992; 48: 2223-2311; beaucage and Iyer, Tetrahedron 1993; 49: 6123-6194 and Caruthers, et al, methods Enzymol.1987; 154: 287-313; the synthesis of thioesters is described, inter alia, in Eckstein, annu, rev, biochem, 1985; 54: 367- > 402; RNA molecules were synthesized at Sproat, humanpress 2005, herdewjnp. master edition; kap.2: 17-31; and corresponding downstream processes are, inter alia, documented in Pingoud et al, IRLPress1989, oliver r.w.a.; kap.7: 183- & 208.

Other synthetic procedures are known in the art, for example, as described in Usman et al, j.am.chem.soc., 1987, 109: 7845 of the total weight of the composition; scaringe et al, NAR, 1990, 18: 5433; wincott et al, NAR1995,. 23: 2677-2684; and Wincott et al, methods mol. bio, 1997, 74: 59, and these procedures may utilize common nucleic acid protecting and coupling groups such as a 5 '-terminal dimethoxytrityl group and a 3' -terminal phosphoramidite. Modified (e.g., 2' -O-methylated) nucleotides and unmodified nucleotides are incorporated as desired.

The oligonucleotides of the invention may be synthesized separately and joined together after synthesis, for example by ligation (ligation) (Moore et al, Science1992, 256: 9923; International patent publication No. WO93/23569; Shabarova et al, NAR1991, 19: 4247; Bellon et al, Nucleotides & Nucleotides, 1997, 16: 951; Bellon et al, bioconjugate technology 1997, 8: 204) or by hybridization after synthesis and/or deprotection.

Notably, commercially available equipment (available especially from applied biosystems) may be used; oligonucleotides were prepared according to the sequences disclosed herein. Overlapping pairs of chemically synthesized fragments can be ligated using methods well known in the art (see, e.g., U.S. Pat. No.6,121,426). The individual strands are synthesized separately and then annealed to one another in a tube. Next, the double stranded siRNA is separated from the non-annealed (e.g., because one of them is in excess) single stranded oligonucleotide by HPLC. With respect to the siRNA or siRNA fragment of the present invention, two or more such sequences may be synthesized and ligated together for use in the present invention.

The compounds of the present invention can also be synthesized by tandem synthesis methods, as described, for example, in U.S. patent publication No.2004/0019001(McSwiggen), in which both siRNA strands are synthesized in the following form: a single contiguous oligonucleotide segment, or strands separated by a cleavable linker (which is subsequently cleaved to provide individual siRNA segments), or strands that hybridize and allow purification of the siRNA duplex. The linker is selected from a polynucleotide linker or a non-nucleotide linker.

The term "covalently bonded" as used herein refers to a chemical bond characterized by a shared pair of electrons between atoms.

The term "non-covalent association" as used herein refers to a variety of interactions between molecules or molecular moieties that are non-covalent in nature, which provide a force to bind the molecules or molecular moieties together, usually in a particular orientation or conformation. These non-covalent interactions include: ionic bonds, hydrophobic interactions, hydrogen bonds, van der waals forces, and dipole-dipole bonds.

siRNA and RNA interference

RNA interference (RNAi) is a phenomenon involving double-stranded (ds) RNA-related gene specific post-transcriptional silencing. Initial efforts to study this phenomenon and experimentally manipulate mammalian cells failed by active, non-specific antiviral defense mechanisms that are activated in response to long dsRNA molecules (Gil et al Apoptosis, 2000.5: 107-114). Later, synthetic 21-nucleotide RNA duplexes were found to mediate gene-specific RNAi in mammalian cells without stimulating the general antiviral defense mechanisms (see Elbashir et al Nature2001, 411: 494-97498 and Caplen et al PNASA 2001, 98: 9742-9747). Therefore, small interfering RNA (siRNA) has become a powerful tool in attempting to understand gene function.

RNA interference (RNAi) in mammals is mediated by small interfering RNA (siRNA) (Fire et al, Nature1998, 391: 806) or microRNA (miRNA) (Ambros, Nature2004, 431 (7006): 350-. The corresponding process in plants is often referred to as specific post-transcriptional gene silencing (PTGS) or RNA silencing, also known as suppression in fungi.

An siRNA is a double-stranded RNA or modified RNA molecule that down-regulates or silences (prevents) the gene/mRNA expression of its endogenous (cellular) counterpart.

Selection and synthesis of sirnas corresponding to known genes has been widely reported; (see, e.g., Ui-Tei et al, JBiomedBiotech.2006; 2006: 65052; Chalk et al, BBRC.2004, 319 (1): 264-74; Sioud & Leirdal, Met.MolBiol.; 2004, 252: 457-69; Levenkova et al, Bioinformam.2004, 20 (3): 430-2; Ui-Tei et al, NAR.2004, 32 (3): 936-48).

For examples of the use and preparation of modified sirnas, see, e.g., Braasch et al, biochem.2003, 42 (26): 7967-75; chiu et al, RNA, 2003, 9 (9): 1034-48; PCT publications WO2004/015107 (atugennA AG) and WO02/44321(Tuschl et al). Chemically modified oligomers are taught in U.S. Pat. Nos. 5,898,031 and 6,107,094. U.S. patent publication nos. 2005/0080246 and 2005/0042647 are directed to oligomeric compounds having alternating motifs and dsRNA compounds having chemically modified internucleoside linkages, respectively.

Other modifications have been disclosed. It has been shown that inclusion of a 5' -phosphate moiety enhances siRNA activity in Drosophila embryos (Boutra, et al, Curr. biol.2001, 11: 1776-1780) and is necessary for siRNA function in human HeLa cells (Schwarz et al, mol. cell, 2002, 10: 537-48). Amarzguioui et al, (NAR, 2003, 31 (2): 589-95) showed that siRNA activity was dependent on the localization of the 2 '-O-methyl (2' OMe) modification. Holen et al (NAR.2003, 31 (9): 2401-07) reported that siRNA with a smaller number of 2'OMe modified nucleosides produced good activity compared to the wild type, but the activity decreased with an increase in the number of 2' OMe modified nucleosides. Chiu and Rana (RNA.2003, 9: 1034-48) teach that incorporation of 2' OMe modified nucleosides in the sense or antisense strand (fully modified strand) severely reduces siRNA activity relative to unmodified siRNAs. It has been reported that placing a 2' OMe group at the 5 ' -end of the sense strand severely limits activity, while allowing placement at the 3 ' -end of the antisense strand as well as at both ends of the sense strand (Czauderna et al, NAR.2003, 31 (11): 2705-16; WO 2004/015107).

Several studies have shown that siRNA therapeutics are effective in vivo in both mammals and humans. Bitko et al have demonstrated that specific siRNA molecules directed against the N gene of the Respiratory Syncytial Virus (RSV) nucleocapsid can be effective in treating mice when administered intranasally (nat. Med.2005, 11 (1): 50-55). Recently, there have been reviews of siRNA therapeutics (Barik, et al, J.mol.Med2005, 83: 764-773; Dallas and Vlassov, Med.Sci.Monitor2006, 12 (4): RA 67-74;Chakraborty，currentdrug targets2007, 8 (3): 469-82; dykxhoorn et al, Gene therapy 2006.13: 541-552).

Mucke (IDrugs200710 (1): 37-41) reviewed current therapeutic agents (including siRNAs against multiple targets) for the treatment of ophthalmic diseases such as age-related macular degeneration (AMD) and glaucoma.

Recently, a number of PCT applications related to the phenomenon of RNAi have been published. These include: PCT publication WO00/44895, PCT publication WO00/49035, PCT publication WO00/63364, PCT publication WO01/36641, PCT publication WO01/36646, PCT publication WO99/32619, PCT publication WO00/44914, PCT publication WO01/29058, and PCT publication WO 01/75164.

RNA interference (RNAi) is based on the ability of dsRNA species to enter the cytoplasmic protein complex, where the dsRNA is subsequently targeted to and specifically degrades complementary cellular RNA. The RNA interference response is characterized by an endonuclease complex comprising the siRNA, commonly referred to as an RNA-induced silencing complex, which mediates cleavage of single-stranded RNA having a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA may occur in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al, GenesDev., 2001, 15 (2): 188-. More specifically, longer dsRNA is digested into short (17-29bp) dsRNA fragments (also known as short inhibitory RNA, "siRNA") by type III RNase (DICER, DROSHA et al; Bernstein et al, Nature, 2001, 409 (6818): 363-6; Lee et al, Nature, 2003, 425 (6956): 415-9). The RISC protein complex recognizes these fragments and the complementary mRNA. The entire process is completed by endonuclease cleavage of the target mRNA (McManus & Sharp, NatureRevGenet, 2002, 3 (10): 737-47; Paddison & Hannon, CurrinMolTher.2003, 5 (3): 217-24). (for details on these terms and the proposed mechanism, see, e.g., Bernstein et al, RNA2001, 7 (11): 1509-21; Nishikura, Cell2001, 107 (4): 415-8 and PCT publication WO 01/36646).

siRNA structure

Selection and synthesis of sirnas corresponding to known genes has been widely reported; (see, e.g., Ui-Tei et al, JBiomedBiotech.2006; 2006: 65052; Chalk et al, BBRC.2004, 319 (1): 264-74; Sioud & Leirdal, Met.MolBio1.; 2004, 252: 457-69; Levenkova et al, Bioinformam.2004, 20 (3): 430-2; Ui-Tei et al, NAR.2004, 32 (3): 936-48).

The double stranded RNA molecules disclosed herein have one 3 'non-nucleotide overhang on either the sense or antisense strand and optionally two 3' overhangs, one on each of the sense and antisense strands.

The molecules disclosed herein offer the advantage that they are non-toxic and can be used to prepare pharmaceutical compositions for the treatment of a variety of diseases and conditions.

PCT patent application No. PCT/IL2007/001278(PCT publication No. wo2008/050329) and U.S. patent No.11/978,089, assigned to the assignee of the present invention, relate to inhibitors of pro-apoptotic genes and are incorporated by reference in their entirety.

The present invention relates generally to compounds that down-regulate the expression of a variety of genes, and in particular to novel small interfering rnas (sirnas), and to the use of these novel sirnas in treating subjects suffering from a variety of medical conditions.

These molecules and compositions are discussed in detail herein, and any such molecule and/or composition can be advantageously used to treat a subject having any such disorder.

The siRNA compounds of the invention have structures and modifications that can, for example, enhance activity, increase stability, and/or minimize toxicity; the novel modifications of the siRNA of the present invention can be advantageously applied to double-stranded RNA that can be used to prevent or attenuate expression of target genes, particularly target genes discussed herein.

According to one aspect, provided herein are inhibitory oligonucleotide compounds comprising unmodified and/or modified nucleotides. One strand of the compound comprises at least one 3' overhang comprising at least one non-nucleotide moiety, preferably two non-nucleotide moieties. The compounds disclosed herein preferably comprise unmodified ribonucleotides and modified ribonucleotides and one or more non-conventional moieties. In some embodiments, at least one of N or N' is selected from the group consisting of: sugar modifications, base modifications, and internucleotide linkage modifications. In some embodiments, the compounds disclosed herein comprise at least one modified nucleotide, including DNA, LNA (locked nucleic acid) including ENA (ethylene bridge nucleic acid); PNA (peptide nucleic acid); arabinoside; PACE (phosphoacetate and its derivatives), mirror nucleotides or nucleotides with 6-membered sugar analogues (such as hexose or morpholino).

In one embodiment, the compound comprises at least one modified ribonucleotide that has a2 'modification on the sugar moiety ("2' sugar modification"). In certain embodiments, the compounds comprise 2 'O-alkyl or 2' -fluoro or2 'O-allyl or any other 2' sugar modification, optionally in alternating positions. One possible 2 'modification is 2' O-methyl (2 'methoxy, 2' OMe).

Other stabilizing modifications are also possible (e.g., modified nucleotides added to the 3 'or 5' end of the oligomer). In some embodiments, the backbone of the oligonucleotide is modified and comprises a phosphate-D-ribose entity, but may also comprise a phosphorothioate-D-ribose entity, a phosphodiester L-ribose entity, a triester, a thioester, a2 '-5' bridge backbone (also referred to as 5 '-2'), a PACE-modified nucleotide linkage, or any other type of modification.

The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

The present invention will be illustrated in detail below according to embodiments, but should not be construed as being limited thereto.

Citation of any document herein is not an admission that the document is pertinent prior art, or that the material is patentable in any claim of the application. Any statement as to the content or date of any document is based on the information available to the applicant at the time of filing and does not constitute an admission as to the correctness of this statement.

Examples

Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following detailed description is, therefore, to be construed as merely illustrative, and not limitative of the invention claimed in any way.

Standard molecular biology protocols known in the art not specifically described herein generally substantially follow the following references: sambrook et al, molecular cloning: alaboryanual, ColdSpringsHarbor laboratory, New-York (1989, 1992); and Ausubel et al, Current protocol molecular biology, John Wiley and Sons, Baltimore, Maryland (1988); and Ausubel et al, Current protocol molecular biology, John Wiley and Sons, Baltimore, Maryland (1989); and Perbal, APracta guiding molecular cloning, John Wiley & Sons, New York (1988); watson et al, Recombinant DNA, scientific American Books, NewYork; birren et al (eds.) genome analysis: ALABORTYRORyManualSeries, ColdSpringHarbor laboratory Press, New York (1998), and methods as shown in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659, and 5,272,057, and incorporated herein by reference. Polymerase Chain Reaction (PCR) is generally described as PCRProtocols: AGuideTomethods AndAPPLICATIONS, academic Press, SanDiego, Calif. (1990). In situ (in cell) PCR coupled with flow cytometry can be used to detect cells containing specific DNA and mRNA sequences (Testoni et al, Blood1996, 87: 3822). Methods for performing RT-PCR are also well known in the art.

Sequence listing

The sequence listing filed herewith in electronic form is hereby incorporated by reference in its entirety (file name: 217pct2st25. txt; creation date: 2011 year 1 month 6 days; file size: 6.00 Kb).

Example 1: production of nucleic acid molecules and in vitro testing of modified siRNA compounds

Sense and antisense sequences of many potential siRNA nucleic acid molecules are generated using proprietary algorithms and known sequences of the target nucleotide, such as mRNA of the target gene. Unless otherwise indicated, the nucleic acid molecules are shown in a 5 'to 3' orientation, and the sense and complementary antisense sequences are shown in the same row of the table.

Table a provides exemplary, non-limiting nucleic acid sequences that can be used to generate the nucleic acid molecules disclosed herein.

Table a:

activity:

single-stranded oligonucleotides (sense and antisense strands) were synthesized using standard synthetic procedures. DMT-propane-diol phosphoramidite (Chemgenes; CLP-9908) coupling was performed at a concentration of 0.05M. Duplexes are generated by annealing complementary single stranded oligonucleotides. About 500. mu.M of single stranded oligonucleotide stock solution was prepared by dilution in WFI (water for injection, Norbrook) in a laminar flow hood. The actual ssRNA concentration was determined by diluting 500. mu. MsRNA each with WFI at a ratio of 1: 200 and measuring OD using NanoDrop. This procedure was repeated 3 times and the average concentration was calculated. The stock solution was then diluted to a final concentration of 250. mu.M. Annealing the complementary single strand by heating to 85 ℃ and cooling to room temperature over at least 45 minutes. The duplexes were tested for complete annealing by testing 5 μ l and staining on a 20% polyacrylamide gel. The duplexes were stored at-80 ℃.

The activity of the double stranded nucleic acid molecules disclosed herein is tested at about 1.5-2 × 10 per well⁵Cell density test cells (HeLa cells and/or 293T cells for siRNA targeting human genes; NRK52 cells (normal rat renal proximal tubule cells) and/or NMuMG cells (mouse mammary epithelial cell line) for siRNA targeting rat/mouse genes) were seeded into 6-well plates (70-80% confluency).

After about 24 hours, cells were transfected with the modified siRNA compound using lipofectamine (tm) 2000 reagent (Invitrogen) at a final concentration of 0.001nM to about 50 nM. Subjecting the cells to CO at 37 ℃₂Incubate in incubator for 72 h.

As a positive control for transfection, a PTEN-Cy3 labeled modified siRNA compound was used. The GFPsiRNA compound was used as a negative control for siRNA activity.

At 72h post-transfection, cells were harvested and RNA was extracted from the cells. Transfection efficiency was tested by fluorescence microscopy.

The percent inhibition of gene expression using a particular preferred siRNA structure was determined using qPCR analysis of the target gene in cells expressing the endogenous gene.

Using the activity results obtained with various final siRNA concentrations, dose response curves were established to determine IC50 values for the RNAi activity tested. Dose response curves were established by plotting the relative amount of residual target mRNA versus the logarithm of transfected siRNA concentration. The curve is calculated by fitting the best sigmoid curve to the measured data. The sigmoid fitting method is also referred to as 3-point curve fitting.

Where Y is the residual target mRNA response, X is the logarithm of the concentration of transfected siRNA, Bot is the Y value for the bottom plateau, Logic50 is the X value for Y at half-way between the bottom and top plateaus, and HillSlope is the steepness of the curve.

Serum stability test

Duplex stability of double stranded nucleic acid molecules was tested in human serum or human tissue extracts by the following method:

siRNA molecules at a final concentration of 7. mu.M were incubated at 37 ℃ in 100% human serum (Sigma Cat. No. H4522). (siRNA stock diluted to 100. mu.M in human serum at a ratio of 1: 14.29, or in human tissue extracts from various tissue types). Five microliters (5 μ l) were added to 15 μ l of 1.5 × TBE loading buffer at different time points (e.g., 0, 30min, 1h, 3h, 6h, 8h, 10h, 16h, and 24 h). The samples were immediately frozen in liquid nitrogen and kept at-20 ℃.

Each sample was loaded onto a non-denaturing 20% acrylamide gel prepared according to methods known in the art. Oligomers were visualized by ethidium bromide under uv light.

Generally, sirnas with specific sequences selected for in vitro testing are specific for human and second species such as rat and rabbit genes.

Stability to exonucleases

To investigate the stabilizing effect of the 3' non-nucleotide moiety on the sense strand of a nucleic acid molecule, the sense strand, antisense strand and annealed siRNA duplexes were incubated in cytoplasmic extracts prepared from different cell types. The protocol for testing stability in HCT116 cells is provided below.

Extracting: HCT116 cytosolic extract (12 mg/ml).

Extracting a buffer solution: 25mM HEPES pH-7.3, 37 ℃; 8mM MgCl; 150mM NaCl with 1mM DTT was added as prepared prior to use.

The method comprises the following steps: 3.5ml of test siRNA (100mM) was mixed with 46.5ml of the solution containing 120mg of cytoplasmic extract of HCT 116. The 46.5ml solution consisted of 12ml of HCT116 extract and 34.5ml of extraction buffer supplemented with DDT and protease inhibitor cocktail/100 (Calbiochem, setIII-539134). The final concentration of siRNA in the incubation tube was 7 mM. Samples were incubated at 37 ℃ and at the indicated time points, 5ml were transferred to a new tube, mixed with 15ml of 1 XTBE-50% glycerol loading buffer, and snap frozen in liquid nitrogen. The final concentration of siRNA in the loading buffer was 1.75mM (21ng siRNA/ml). For analysis by native polyacrylamide gel electrophoresis (native PAGE) and EtBr staining, 50ng was loaded per lane. For Northern analysis, 1ng of siRNA tested was loaded per lane. Other cell types include HeLa and Hepatic Stellate Cells (HSCs).

Applicants have demonstrated that nucleic acid molecules comprising a 3 'terminal alkyl or alkyl derivative overhang exhibit enhanced stability compared to blunt-ended nucleic acid molecules and nucleic acid molecules comprising a 3' nucleotide overhang.

Exemplary Compounds

siRNA compounds comprising a non-nucleotide moiety covalently linked to the 3' terminus were synthesized and tested as described above. Figure 2 provides a table of compounds useful for RNAi comprising the sequences and modifications disclosed herein. The legend of the modifications is as follows: the prefix "z" denotes a moiety (nucleotide or non-nucleotide) covalently attached to the 3 'or 5' terminal nucleotide. For example, zdT refers to a dT protrusion; zdT, respectively; zdT refers to a dTdT protrusion. The prefix "y" denotes nucleotide substitutions, e.g., yLdA refers to L-deoxyriboadenine that replaces a ribonucleotide in the sense strand or the antisense strand; and ydT refers to deoxyribothymidine substituted for the ribonucleotide in the sense or antisense oligonucleotide. The prefix "m" refers to a 2' OMe sugar-modified ribonucleotide. Additional codes are shown in table B below.

TABLE B

In the following tables, the codes for the columns labeled "sense 5- > 3 antisense 5- > 3" are the codes shown in Table B. The columns labeled "sense modification" and "antisense modification" provide a brief description of the positional modifications for each of the sense and antisense strands, e.g., 20-C3; c3 means a 3 'C3 Pi-C3OH terminal overhang, and 20-dTdT means a 3' dTdT terminal overhang (starting at position 20 on the 19-mer); 2, 4,6, 8, 10, 12, 14, 16, 18-2 '-OMe refers to 2' -OMe sugar modified ribonucleotides at positions 2, 4,6, 8, 10, 12, 14, 16 and 18.

TABLE 1

The data shown in Table 1 indicate that C3Pi-C3OH 3' end overhang (2) improves nuclease resistance of siRNA in cell extracts compared to blunt, unmodified siRNA (1). The stabilization by C3-C3 was similar to that by dTdT (3) and 2 'OMeU (4) 3' ends. The RAC1_2 sequence is shown as SEQIDNO: 5 and 6.

TABLE 2

The data in table 2 show that C3-C33 ' overhang (1) and strand (2) comprising 5 ' and 3 ' inverted deoxyabasic moieties improved nuclease resistance of the strand in cell extracts compared to unmodified strand (3). The CASP2_4 sequence is shown as SEQIDNO: 1 and 2.

TABLE 3

The data provided in table 3 indicates that: 1) introduction of C3-C3 moieties (3, 4 and 7), inverted deoxyabasic (2), C3-riboabasic (8) at the 3 'end of the antisense strand or on both the sense and antisense strands improved siRNA activity compared to blunt-ended compounds (1 and 5) and was more active compared to the same compound (6) with a 3' dTdT overhang; and 2) the improved activity of the compound comprising the 3' terminal C3-C3 was not due to enhanced stability in cell extracts, but to the high stability (at least 36h) of both compounds (5 and 7).

TABLE 4

The data in table 4 show that: the most significant activity enhancement was obtained when C3C3 was present at both the 3' end of the antisense and sense strands (2) compared to blunt end (1) and C3-C3(3 or 4) present on only one strand.

TABLE 5

The data in table 5 show that: 1) the presence of a C3OH (2) or C3Pi-C3Pi-C3OH (3) moiety on the antisense strand did not improve activity compared to blunt siRNA (5); and 2) the presence of C3Pi-C3Pi (1) and C3Pi-C3Ps (4) moieties on the antisense strand improved activity compared to blunt-ended compounds and this effect was most pronounced in the case of C3Pi-C3 Pi.

TABLE 6

The data in table 6 show that: compounds (1) with inverted abasic moieties on both the sense and antisense strands were more active than either blunt-ended compounds (4) or compounds (2 or 3) with inverted abasic moieties on one strand.

TABLE 7

The data in table 7 show that: double stranded RNA compounds with end caps (inverted deoxyabasic moieties) at both the 3 'end of the antisense strand and the 3' end of the sense strand showed enhanced activity compared to dsRNA compounds with a 3 'end cap covalently attached to the 3' end of the antisense strand (compare compounds 2 and 3 with compounds 4 and 5).

TABLE 8

The data in table 8 show that: double stranded RNA compounds comprising a 3 'terminal C3Pi-C3OH overhang covalently linked to the 3' terminus of the antisense strand (guide strand) (Z ═ two C3 moieties) showed excellent activity (more than 80% knockdown at 20 nM) regardless of the modification on the complementary sense strand (see compounds 1-7).

Compounds 1-7 utilize the amino acid sequences as shown in seq id no: 3 and 4 and comprises a common antisense strand (seq id no: 4) comprising two 3 'terminal C3 moieties (C3Pi-C3OH) covalently linked to the 3' terminal nucleotide and a different sense strand comprising the various modifications disclosed herein. The sense strand of compound 1 comprises unmodified ribonucleotides at positions 1-14 and 19, and 2 '5' ribonucleotides at positions 15-18. The sense strand of compound 2 comprises unmodified ribonucleotides at positions 1-14 and 2 '5' ribonucleotides at positions 15-19, and a terminal phosphate (p (o) 3). The sense strand of compound 3 comprises unmodified ribonucleotides at positions 1-14, and 2' 5 ' ribonucleotides at positions 15-19, and a 3 ' terminal C3 Pi. The sense strand of compound 4 comprises unmodified ribonucleotides at positions 1-14 and 2 '5' ribonucleotides at positions 15-19, and a 3 'terminal amino C6 moiety covalently linked to the 3' terminal nucleotide. The sense strand of compound 5 comprises unmodified ribonucleotides (each N' is unmodified) at positions 1-19. The sense strand of compound 6 comprises unmodified ribonucleotides at positions 1-14 and 19, and 2 '5' ribonucleotides at positions 15-18, and an inverted 3 'terminal abasic moiety covalently linked to a 3' terminal nucleotide. The sense strand of compound 7 comprises unmodified ribonucleotides at positions 1-3, 5, 7-8, 14-16 and 19, deoxyribonucleotides at positions 4,6, 9, 10-13 and 17-18, and a 3 ' terminal phosphate (Pi) and C3OH moiety covalently linked at the 5 ' end of an inverted abasic moiety covalently linked to the 3 ' terminal nucleotide.

Compound 9, having two C3 moieties covalently attached to the 3 'terminus of the sense and antisense strands, was more active than the analogous compound (8), having two C3 moieties covalently attached to the 3' terminus of the antisense strand.

Example 2: delivery of a modified nucleic acid molecule targeting CASP2 to the retina.

Target cell siRNA delivery, target cell gene knockdown activity and target gene mRNA cleavage Evaluation of specificity

For example, after intravitreal injection into rat retina, target bases are measured in target tissues Due to knock-down.

Background: different structural modifications were performed in sirnas targeting CASP2 gene and tested for their exonuclease resistance. The aim of this study was to examine the in vivo distribution and activity of oligonucleotides comprising these modifications as described below.

S1003inv-dAb-GCCAGAAUGUGGAACUCCU-inv-dAb

AGGAGUUCCACAUUCUGGC-inv-dAb

S800GCCAGAAUGUGGAACUCCU

AGGAGUUCCACAUUCUGGC-C3Pi-C3OH

Description of test material (S1003): an RNA duplex having the structure: the sense strand is an unmodified 19mer with inverted abasic moieties as 5 'and 3' -caps. The antisense strand is a 19-mer with 2 'O-Me at positions 2, 4,6, 8, 11, 13, 15, 17 and 19 and an inverted abasic moiety as a 3' -cap, annealed. The amount provided: 336. mu.g. Storage conditions are as follows: -80 ℃.

Description of test material (S800): an RNA duplex having the structure: the sense strand is a 19-mer with L-DNA at position 18. The antisense strand is a 19-mer with a 2'OMe at positions 2, 4,6, 8, 11, 13, 15, 17 and 19 and two (CH2)3 propanediol (C3Pi-C3OH) at the 3' end. The amount provided: 840 mug. Storage conditions are as follows: -80 ℃.

CNL: an RNA duplex with the same modification as S800 but without a C3Pi-C3OH moiety attached to the 3' terminus.

Animals: age: male rats of 6-8 weeks of age. 180-220g

Group size: n-4/10; total number of animals: 112

Animal feeding management: diet: the animals were provided with ad libitum choking rodent chow (HarlanTeklad rodent chow) and allowed to drink water ad libitum.

Environment: (i) the administration is carried out for at least 5 days.

(ii) All animals were housed in a restricted activity facility with room conditions for environmental control throughout the study and maintained according to Standard Operating Procedures (SOP) approved by HBI. The automatically controlled environmental conditions were set to maintain a temperature of 20-24 ℃, a Relative Humidity (RH) of 30-70%, a 12 hour light/12 hour dark cycle and 15-30 air changes/hour in the study room. The temperature, relative humidity and light cycle were monitored by a control computer.

Experimental design is provided in Table 2-1

Research and design: all animals in experimental groups I-XII were injected unilaterally into the Left Eye (LE) via IVT at a dose of 20 μ g of test or Control (CNL) in 10 μ l PBS vehicle or only 10 μ l PBS vehicle. Experimental group XIII will be used as normal control. The end steps (1, 3 and 7 days post IVT treatment) were completed according to study design.

Anesthesia: animals were anesthetized by isoflurane special loop system (Stoelting, USA). Mydreid (0.5% tropicamide) was used in eye drops to dilate the pupils. For additional local anesthesia, Localin (0.4% oxybuprocaine hydrochloride) will be used. Lacromycin (gentamicin sulfate (equivalent to 0.3% gentamicin base)) eye drops are used to prevent/reduce the post-operative inflammatory process.

Intravitreal injections were performed under a dissecting microscope. A 1mm incision was made posterior to the temporal limbus using an 30/33 gauge needle, and a syringe needle (30/33 insulin syringe 0.3ml, pic0.8mm, Italy) was inserted into the incision 1.5mm deep (viewed through the scattered pupil).

Scheduled euthanasia: all animals were deeply anesthetized (Equisthesine 4ml/kgI.P) and euthanized (decapitation) according to the study design (Table, end).

Tissue collection: eyes were enucleated from all animals and kept on ice. The eyes will be dissected using a microscope and the gross pathology will be rated according to the sample rating scale (see "ocular pathology score" in appendix 5). The cornea would be punctured with an 27/30G needle to remove aqueous humor from the anterior chamber. Using a microsurgical blade, an incision is made along the limbus, and the cornea and lens are removed. The remaining cup will be opened by a sagittal incision through the sclera. The retinas will be extracted from the eye cups, rinsed in PBS and then isolated. Using a fine-tipped forceps, retinas were harvested into appropriate tubes, frozen in liquid nitrogen, and then transferred to a molecular biologyUnit for total RNA extraction.

Evaluation of

Knock-down activity of CASP 2-targeted siRNA in rat retina was quantified by CASP2mRNA expression levels using qPCR method. The CASP2_4siRNA cleavage site on the target gene will be confirmed by RACE and siRNA quantification in retina will be performed by S & LqPCR (stem loop qPCR).

And (3) separating sample RNA: RNA was processed from retinal samples by double extraction according to standard procedures for total RNA isolation using EZRNA. CASP2_4siRNA quantification by qPCR: delivery of CASP2_4siRNA in the retina was measured by qPCRsiRNA quantitation. qPCR was performed according to standard methods on an applied biosystem7300PCR system using the SYBRGreen method. In the corresponding experimental group, CASP2_4 siRNA-directed CASP2mRNA cleavage in rat retina was determined by detecting cleavage products using RACE (rapid amplification of cDNA ends) method. If evidence of the expected cleavage product is shown, the siRNA cleavage site on the target gene will be confirmed by sequence analysis and, optionally, quantification of the cleavage product will be performed using qPCR. CASP2mRNA quantification by qPCR: following cDNA preparation, CASP2 knockdown will be confirmed by CASP2mRNA quantification by qPCR. qPCR will be performed according to standard methods on an applied biosystem7300PCR system using the SYBRGreen method.

Preliminary results

Preliminary results indicate that S800 is more efficiently taken up into retinal cells than S1003. The results are shown in Table 2-2 below.

Tables 2 to 2

Example 3: delivery of a modified nucleic acid molecule targeting MYD88 to the retina.

Evaluation of target cell siRNA delivery. The purpose of this study was:

1.1 determination of delivery of different structural and modified MYD88_11 siRNAs to rat retinas at 4 hours, one day, and three days after unilateral Intravitreal (IVT) injection.

1.2 knockdown activity of MYD88_11 sirnas targeting different structures of MYD88 at 4 hours, one day, and three days post IVT injection in rat eyes was determined by qPCR of MYD88 mRNA.

Background: different structural modifications were performed in sirnas targeting CASP2 gene and tested for their exonuclease resistance. The objective of this study was to examine the in vivo distribution of double stranded RNA oligonucleotides with modifications as disclosed herein, in particular modifications as described below.

MYD88_11S5055′GAAUGUGACUUCCAGACCA

5′UGGUCUGGAAGUCACAUUC

MYD88_11S11595′idAb-GAAUGUGACUUCCAGACCA

5′UGGUCUGGAAGUCACAUUC-C3Pi-C3OH

MYD88_11S12705′OHC3-GAAUGUGACUUCCAGACCA-Pi

5′UGGUCUGGAAGUCACAUUC-C3Pi-C3OH

Description of test material S505: an RNA duplex having the structure: the sense strand is an unmodified 19mer with one L-DNA moiety at position 18 (lower case bold). The antisense strand is a 19-mer with 2' O-Me at positions 2, 4,6, 8, 11, 13, 15, 17 and 19, annealed. Storage conditions are as follows: -80 ℃.

Description of test material S1159: an RNA duplex having the structure: the sense strand is a 19-mer with an inverted abasic moiety as the 5 ' cap and a 2' -5 ' bridge RNA at positions 15-18. The antisense strand is a 19-mer having 2 'O-Me at positions 1, 4-6, 9, 12-13, 15, 17-19 and two 1, 3-propanediol building blocks connected at the 3' end by phosphodiester linkages. And (6) annealing. Storage conditions are as follows: -80 ℃.

Description of test material S1270: an RNA duplex having the structure: the sense strand is a 19-mer with 2 '5' nucleotides at positions 15-19 and a terminal phosphate (Pi). The antisense strand is a 19-mer with 2'OMe at positions 1,4, 5,6, 9, 12, 13, 15, 17-19 and two (CH2)3 propanediol (C3Pi-C3OH) at the 3' terminus. And (6) annealing. Storage conditions are as follows: -80 ℃.

Animals: age: male rats 8-10 weeks old. 180-220g

Group size: n-4/8; total number of animals: 104

Environment: (i) the administration is carried out for at least 5 days.

Research and design: all animals in experimental groups 1-12 were injected unilaterally into the Left Eye (LE) via IVT at a20 μ g test dose or only 10 μ l PBS vehicle. Experimental group 13 was used as a normal control. The end steps (4 hours, 1 day and 7 days post IVT treatment) were done according to study design.

Experimental design is provided in Table 3-1

TABLE 3-1

Anesthesia: animals were anesthetized by the following isoflurane special circuit system (Stoelting, USA) working setup: 3-4.5% of a mixture of oxygen and oxygen₂Isoflurane in (1) is O with the concentration of 600-₂Flow rate. Mydriid (0.5% tropicamide) was used in eye drops to dilate the pupils. For additional local anesthesia, Localin (0.4% oxybuprocaine hydrochloride) will be used. Lacromycin (gentamicin sulfate (equivalent to 0.3% gentamicin base)) eye drops are used to prevent/reduce the post-operative inflammatory process.

Tissue collection: eyes were enucleated from animals and kept on ice. The eyes will be dissected using a microscope and the gross pathology will be rated according to the sample rating scale (see "ocular pathology score" in appendix 5). The cornea would be punctured with an 27/30G needle to remove aqueous humor from the anterior chamber. Using a microsurgical blade, an incision was made along the limbus and the cornea and lens were removed. The remaining cup will be opened by a sagittal incision through the sclera. The retinas will be extracted from the eye cups, rinsed in PBS and then isolated. Using fine-tipped forceps, the retinas were harvested into appropriate tubes, frozen in liquid nitrogen, and total RNA extracted.

Evaluation of

siRNA quantification in the retina will be performed by stem-loop qPCR and knockdown activity of MYD 88-targeting siRNA in rat retina will be determined by MYD88mRNA expression level quantification using qPCR.

RNA isolation: RNA was processed from retinal samples using an EZRNA kit according to standard procedures.

MYDD88siRNA quantification by qPCR: delivery of MYDD88siRNA in the retina was measured by qPCRsiRNA quantification (S & L). qPCR was performed according to standard procedures on an applied biosystem7300PCR system using the SYBRGreen method.

MYDD88mRNA quantification by qPCR: MYDD88 knockdown was confirmed by making cDNA using standard procedures and MYD88mRNA quantification by qPCR. qPCR will be performed on an applied biosystem7300PCR system using the SYBRGreen method.

Preliminary results are provided in Table 3-2 below.

The delivery of the C3C3 modified compounds (S1159 and S1270) to retinal ganglion cells was significantly higher than the delivery of the compound with blunt ends (S505) (see data provided in the "median" column.

TABLE 3-2

Example 4: model system for Acute Renal Failure (ARF)

ARF is a clinical syndrome characterized by a rapid decline in renal function occurring within a few days. Without being bound by theory, acute kidney injury may be the result of renal ischemia-reperfusion injury, such as in patients undergoing major surgery (such as major cardiac surgery). The main feature of ARF is a sharp decrease in Glomerular Filtration Rate (GFR), resulting in the entrapment of nitrogen waste (urea, creatinine). Recent studies have shown that apoptosis of renal tissue is evident in most human ARF cases. The major site of apoptotic cell death is the distal nephron. In the initial phase of ischemic injury, loss of actin cytoskeleton integrity leads to epithelial cell flattening, and loss of brush border, loss of focal cell contact and subsequent detachment of cells from the underlying substrate.

Active siRNA compounds were tested using an animal model of ischemia-reperfusion-induced ARF, as shown in PCT patent application publication No. WO/2009/044392.

Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In a non-limiting example, siRNA compounds utilizing oligonucleotide pairs as shown in table B (B1-B74), table C (C1-C4) and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, in particular, specific pro-apoptotic genes, specifically TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, tybp, CTGF and SPP1 genes, and further comprising at least one 3' highlighted siRNA compound according to the present invention, were tested in the above-described model system and found to prevent ischemia-reperfusion.

Example 5: model system for bedsores or pressure ulcers

Decubitus ulcers, including diabetic ulcers, or pressure ulcers are areas of skin and tissue damage that occur when continuous pressure (usually from a bed or wheelchair) cuts circulation through delicate parts of the body, particularly the skin over the buttocks, hips and heels. Insufficient blood flow leads to ischemic necrosis and ulceration of the affected tissue. Bedsores are most commonly seen in patients with poor or no sensory ability or weakness, haggard, paralysis or long-term bedridden. Tissues on the sacrum, ischial bones, greater trochanter, lateral malleolus, and heel are particularly susceptible; other sites may also be involved depending on the patient's condition.

In a system such as Reid et al, JSurg.Res.116: 172-180, 2004, the activity inhibitors (such as siRNA compounds) of the invention were tested for the treatment of bedsores, ulcers and similar wounds.

Mustoe et al JCI, 1991.87 (2): 694-; ahn and Mustoe, anplsurg, 1991.24 (1): 17-23 describe additional rabbit models and were used to test siRNA compounds designed and synthesized as disclosed herein. Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In some embodiments, testing siRNA compounds utilizing oligonucleotide pairs as set forth in table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, particularly compounds directed against CIQBP, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, or TYROBP genes and further comprising at least one 3' non-nucleotide overhang according to the present invention, in animal models indicates that these siRNA compounds can treat and/or prevent decubitus and ulcers.

Example 6: model system for Chronic Obstructive Pulmonary Disease (COPD)

Chronic Obstructive Pulmonary Disease (COPD) is primarily characterized by emphysema, a permanent disruption of the peripheral airways distal to the terminal bronchioles. Emphysema is also characterized by the accumulation of inflammatory cells such as macrophages and neutrophils in the bronchioles and alveoli. Emphysema and chronic bronchitis may occur as part of COPD or independently.

The activity inhibitors (such as siRNA) of the invention are tested for the treatment of COPD/emphysema/chronic bronchitis in animal models such as those disclosed below:

starcher and Williams, 1989.Lab. animals, 23: 234, 240; peng, et al, 2004; amjrespircritcitcarememed, 169: 1245 and 1251; jeyaseeelan et al, 2004. feed. immunol, 72: 7247-56. Additional models are described in PCT patent publication WO2006/023544, assigned to the assignee of the present application, which is hereby incorporated by reference.

Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In some embodiments, siRNA compounds utilizing oligonucleotide pairs as set forth in table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, particularly siRNA against CIQBP, BNIP3, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, and DUOXl genes and further comprising at least one 3' overhang as disclosed herein, are tested in these animal models, indicating that these siRNA compounds can treat and/or prevent emphysema, chronic bronchitis, and COPD.

Example 7: model system for spinal cord injury

Spinal cord injury or myelopathy is a spinal cord disorder that results in loss of sensation and/or movement. These two common types of spinal cord injuries are caused by trauma and disease. Among the causes of trauma are car accidents, falls, gunshots, diving accidents, and diseases that can affect the spinal cord including polio, spina bifida, tumors, and friedreich's ataxia.

Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In some embodiments, testing siRNA compounds utilizing oligonucleotide pairs as shown in table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, and in particular against LRDD, CYBA, ATF3, CASP2, HRK, CIQBP, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, CX43, TYROBP, CTGF, and RHOA genes in this animal model and further comprising at least one 3' protruding siRNA as disclosed herein, indicates that these siRNA compounds can promote functional recovery following spinal cord injury and thus can be used to treat spinal cord injury.

Example 8: model system for glaucoma

In the case of, for example, pepe et al, j.glaucoma, 2006, 15 (6): 512-9 (Manometricitabionandcomporisonopentanetrostingswithperpendicommunications) the active inhibitors (such as siRNAs) of the present invention are tested for the treatment or prevention of glaucoma.

Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In some embodiments, testing in this animal model using oligonucleotide pairs as shown in table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, particularly siRNA compounds directed against TP53BP2, LRDD, CYBA, ATF3, CASP2, HRK, BNIP3, MAPK8, MAPK14, RAC1, and RHOA genes and further comprising at least one 3' non-nucleotide overhang as disclosed herein indicates that these siRNA compounds can treat and/or prevent glaucoma.

Example 8A: model system for Ischemic Optic Neuropathy (ION)

An animal model of ischemic optic neuropathy was established in adult Wistar rats using an optic nerve clamp injury protocol. Retinal Ganglion Cells (RGCs) were selectively labeled seven days prior to optic nerve clamp injury by applying the retrograde tracer FluoroGold (2%, Fluorochrome, Englewood, CO) to the superior colliculus. The tracer is transported along the RGC axons by retrograde transport, resulting in complete and specific labeling of all RGCs within 1 week after injection of the fluorescent tracer. Animals were subjected to optic nerve pinch injury 7 days after retrograde tracing. The orbital optic nerve was exposed by supraorbital approach and all axons in the optic nerve were severed by grasping with forceps 2mm from the lamina cribrosa for 10 seconds. In optic nerve clamp injury, a single dose of 20 μ g/5 μ l of a siRNA compound according to the present invention in PBS was microinjected into the 2mm vitreous anterior to the nerve head using a micro-glass needle. Survival of RGCs was determined 7 days after optic nerve pinch by counting FluoroGold-labeled RGCs on tiled retinas. Experimental animals were perfused through the heart with 4% paraformaldehyde at 1 week post optic nerve clamp injury. Both retinas were dissected out, fixed for an additional 30 minutes, and then plated on glass slides for ganglion cell layer quantification. Fluorescent RGCs were counted in 16 different regions in each retina and compared to samples taken from rats that did not receive optic nerve clamps at all or from rats that received optic nerve clamps and injected with PBS, control siRNA or GFPsiRNA to determine the percent survival of RGCs. Microglia incorporating FluoroGold, possibly after RGC-stained phagocytosis, were distinguished by their characteristic morphology and excluded from the quantitative analysis.

Another model of optic nerve dissection, in which the entire RGC population is dissected by transecting the optic nerve near the eye, can be used to test the compounds and compositions of the invention (ChengL, et al J.Neurosci.May15, 20022002; 22: 3977-.

Example 9: model system for ischemia/reperfusion injury after rat lung transplantation

In one or more experimental animal models, for example, as described in Mizobuchi et al, 2004.j. heartlungtplant, 23: 889-93; huang, et al, 1995. J.HeartLungTransplant.14: s49; matsumura, et al, 1995.Transplantation 59: 1509-; wilkes, et al, 1999 Transplantation 67: 890-; naka, et al, 1996.circulation research, 79: 773 783 active inhibitors of the invention (such as siRNA) are tested for the treatment or prevention of ischemia/reperfusion injury or hypoxic injury following lung transplantation.

Existing siRNAs can be advantageously modified, and future siRNAs can be designed and prepared to provide active nucleic acid molecules. In some embodiments, siRNA compounds directed against TP53BP2, LRDD, CYBA, CASP2, BNIP3, RAC1, and DUOX1 genes and further comprising at least one 3' non-nucleotide overhang as disclosed herein, using oligonucleotide pairs as shown in table B (B1-B74), table C (C1-C4), and table D (D1-D34) of PCT patent application publication No. wo/2009/044392, are tested in these models, indicating that these siRNA compounds can treat and/or prevent ischemia/reperfusion injury following lung transplantation and thus can be used in conjunction with transplantation surgery.

Example 10: model system for acute respiratory distress syndrome

In a reaction system such as Chen et al (JBiomedSci.2003; 10(6Pt 1): 588-92 inhibitors of activity of the invention (such as siRNA) are tested in animal models for the treatment of acute respiratory distress syndrome. In this animal model, testing of siRNA compounds according to PCT patent application publication No. wo/2009/044392, table B (B1-B74), table C (C1-C4) and table D (D1-D34), particularly against the CYBA, HRK, BNIP3, MAPK8, MAPK14, RAC1, GSK3B, P2RX7, TRPM2, PARG, SPP1 and DUOX1 genes and further comprising at least one 3' overhang as disclosed herein, indicates that these sirnas can treat and/or prevent acute respiratory distress syndrome and are thus useful in treating this condition.

Example 11: animal model of Osteoarthritis (OA)

Collagen-induced arthritis (CIA): trentham et al describe mouse CIA (1977.J.exp. Med.146: 857-868). Adjuvant-induced arthritis (AA): AA is described by Kong et al (1999.Nature, 402: 304-308). Han et al describe the meniscus excision model (1999.NagoyaJMedSci62 (3-4): 115-26).

In addition to in vitro models known in the art, the effects of different siRNA inhibitors (such as siRNA against SSP1) on different parameters associated with OA (such as chondrocyte proliferation, terminal differentiation and arthritis development) were evaluated using one or more of the above models. Testing siRNA compounds directed against a particular pro-apoptotic gene (particularly against SSP1) and further comprising at least one 3' overhang as disclosed herein in these animal models indicates that these sirnas can treat and/or prevent OA and thus can be used to treat this disorder.

Example 12: rat model system for transplant-related acute kidney injury

Warm ischemia-in experimental rats, a left nephrectomy was performed followed by an autograft, which resulted in a 45 minute heat-transplanted kidney shelf life. Following autografting, a right nephrectomy was performed on the same animal. Chemically modified siRNA to target is administered via the femoral vein before harvesting transplanted kidneys (mimicking donor therapy) ("pre") or after renal autotransplantation (mimicking recipient therapy) or before harvest and after transplantation (combined donor and recipient therapy) ("pre-post").

Cold ischemiaLeft nephrectomy was performed on the donor animals, and the harvested kidneys were then cold stored (on ice) for a period of 5 hours. At the end of this period, the recipient rats will undergo bilateral nephrectomy, followed by transplantation of cold-preserved transplanted kidneys. The total warm ischemia time (including the surgical procedure) was about 30 minutes. Chemically modified siRNA is administered to a donor animal via the femoral vein prior to harvesting the kidney ("pre") or injected into a recipient animal 15 minutes ("post 15 min") or 4 hours (post4h) after transplantation.

To evaluate the efficacy of siRNA in improving post-transplant renal function, serum creatinine levels were measured on days 1, 2, and 7 post-transplant in both hot and cold ischemia models.

Claims

1. A double stranded nucleic acid molecule comprising a sense strand and an antisense strand, wherein at least the antisense strand comprises a non-nucleotide overhang covalently attached by a phosphodiester or phosphorothioate linkage at a 3 ' or 2' position of the sugar residue of the 3 ' terminal nucleotide of the strand in which it is present; wherein the non-nucleotide overhang comprises a C3 alkyl moiety selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps, wherein Pi represents an inorganic phosphate, dAb represents a phosphorothioate, rAbP represents a deoxyribo-nucleobase moiety, and dAb represents a ribose-nucleobase-moiety;

or a pharmaceutically acceptable salt of said molecule.

2. The double stranded nucleic acid molecule of claim 1 having the structure (A1)

(A1)5 '(N) x-Z3' (antisense strand)

3 'Z' - (N ') y-Z "5' (sense strand)

wherein Z comprises a non-nucleotide overhang covalently linked at the 3 ' or 2' position of the sugar residue of the 3 ' terminal nucleotide by a phosphodiester or phosphorothioate linkage; wherein the non-nucleotide overhang comprises a C3 alkyl moiety selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps

Wherein Z' is present or absent;

wherein each of x and y is independently an integer between 18 and 40;

wherein the sequence of (N') y is complementary to the sequence of (N) x; and wherein the sequence of (N) x has complementarity to a contiguous sequence in the target RNA;

or a pharmaceutically acceptable salt of said molecule.

3. The molecule of claim 2, wherein (N) x and (N') y are fully complementary.

4. The molecule of claim 2, wherein x-y-19.

5. The molecule of claim 2, wherein Z' is present.

6. The molecule of claim 5, wherein Z' is an inorganic phosphate or a non-nucleotide overhang selected from the group consisting of: an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, and combinations thereof.

7. The molecule of claim 6, wherein Z' is an alkyl moiety selected from the group consisting of C3OH, C3Pi, and C3 Ps.

8. The molecule of claim 6, wherein Z' is an inorganic phosphate.

9. The molecule of claim 6, wherein each of Z and Z' independently comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3 Ps.

10. The molecule of claim 2, wherein Z comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3 Ps.

11. The molecule of claim 6, wherein each of Z and Z' independently comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3 Ps.

12. The molecule of claim 2, wherein Z comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3 Ps.

13. The molecule of claim 5, wherein Z consists of C3Pi-C3Pi or C3Pi-C3 OH.

14. The molecule of claim 13, wherein Z consists of C3Pi-C3 OH.

15. The molecule of claim 13, wherein Z consists of C3Pi-C3 Pi.

16. The molecule of claim 13, wherein Z' is selected from the group consisting of C3OH, C3Pi, and C3 Ps.

17. The molecule of any one of claims 2-16, wherein x-y-19 and (N) x comprises a 2' OMe sugar modified ribonucleotide at each of positions 2, 4,6, 8, 11, 13, 15, 17, and 19.

18. The molecule of any one of claims 2-16, wherein x-y-19 and (N) x comprises a 2' OMe sugar modified ribonucleotide at each of positions 1, 3, 5, 7, 9, 11, 13, 15, 17, and 19.

19. The molecule of any one of claims 2-16, wherein x-y-19 and (N') y comprises L-DNA at position 18.

20. The molecule of claim 17, wherein x-y-19 and (N') y comprises L-DNA at position 18.

21. The molecule of claim 18, wherein x-y-19 and (N') y comprises L-DNA at position 18.

22. The molecule of any one of claims 2-16, wherein x-y-19 and (N ') y comprises a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide linkage.

23. The molecule of claim 17, wherein x-y-19 and (N ') y comprises a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide linkage.

24. The molecule of claim 18, wherein x-y-19 and (N ') y comprises a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide linkage.

25. The molecule of claim 2, wherein the (N) x is not fully complementary to the target RNA.

26. The molecule of claim 1, having the structure (A2) shown below:

(A2)5 'N1- (N) x-Z3' (antisense strand)

3 ' Z ' -N2- (N ') y-Z ' 5 ' (sense strand)

wherein each of x and y is independently an integer between 17 and 39;

wherein N1 is covalently bound to (N) x and forms a Watson-Crick base pair with N2;

wherein N1 is mismatched with the target RNA or is a DNA portion complementary to the target RNA;

Wherein Z comprises a non-nucleotide overhang at the 3 ' or 2' position of the sugar residue covalently linked to the 3 ' terminal nucleotide by a phosphodiester or phosphorothioate linkage, wherein the non-nucleotide overhang is selected from the group consisting of a C3 alkyl moiety: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3 Ps; and is

Wherein Z' is present or absent;

or a pharmaceutically acceptable salt of said molecule.

27. The molecule of claim 26, wherein (N) x has complementarity to a contiguous sequence in a mammalian or non-mammalian RNA.

28. The molecule of claim 26, wherein x-y-18.

29. The molecule of claim 26, wherein N1 is selected from the group consisting of: riboadenosine, modified riboadenosine, deoxyriboadenosine, modified deoxyriboadenosine.

30. The molecule of claim 26, wherein N1 is selected from the group consisting of: ribouridine, deoxyribouridine, modified ribouridine, and modified deoxyribouridine.

31. The molecule of claim 26, wherein N1 is a modified ribose adenosine or a modified ribose uridine.

32. The molecule of claim 31, wherein N1 is a 2'OMe sugar modified ribose uridine or a 2' OMe sugar modified ribose adenosine.

33. The molecule of claim 26, wherein N2 is a 2'OMe sugar modified ribonucleotide or a 2' OMe sugar modified deoxyribonucleotide.

34. The molecule of claim 26, wherein Z' is present.

35. The molecule of claim 34, wherein Z' is an inorganic phosphate or a non-nucleotide overhang selected from the group consisting of: an abasic moiety, an inverted abasic moiety, an alkyl moiety or derivative thereof, and combinations thereof.

36. The molecule of claim 35, wherein Z' is an alkyl moiety selected from the group consisting of C3OH, C3Pi, and C3 Ps.

37. The molecule of claim 35, wherein Z' is an inorganic phosphate.

38. The molecule of claim 34, wherein each of Z and Z' independently comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3 Ps.

39. The molecule of claim 26, wherein Z comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi and C3Ps-C3 Ps.

40. The molecule of claim 34, wherein each of Z and Z' independently comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3 Ps.

41. The molecule of claim 26, wherein Z comprises a non-nucleotide overhang selected from the group consisting of: c3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3 Ps.

42. The molecule of claim 26, wherein Z consists of C3Pi-C3Pi or C3Pi-C3 OH.

43. The molecule of claim 42, wherein Z consists of C3Pi-C3 OH.

44. The molecule of claim 42, wherein Z consists of C3Pi-C3 Pi.

45. The molecule of any one of claims 26-44, wherein x-y-18 and (N') y comprises a mirror nucleotide at position 17.

46. The molecule of claim 45, wherein the mirror nucleotide is L-DNA.

47. The molecule of any one of claims 26-44, wherein x-y-18 and (N ') y comprises a nucleotide joined to an adjacent nucleotide by a 2' -5 ' internucleotide linkage.

48. The molecule of any one of claims 26-44, wherein (N) x comprises at least one 2-OMe sugar-modified ribonucleotide.

49. The molecule of claim 45, wherein (N) x comprises at least one 2-OMe sugar-modified ribonucleotide.

50. The molecule of claim 46, wherein (N) x comprises at least one 2-OMe sugar-modified ribonucleotide.

51. The molecule of claim 47, wherein (N) x comprises at least one 2-OMe sugar-modified ribonucleotide.

52. A pharmaceutical composition comprising the molecule of any one of claims 1-16 and 26-44 and a pharmaceutically acceptable carrier.

53. Use of a molecule of any one of claims 1-16 and 26-44 in the manufacture of a medicament for treating a subject having a disease or condition associated with expression of a target gene.

54. A method for preparing a double stranded RNA molecule that mediates cleavage of a target RNA, the method comprising:

(a) synthesizing an RNA strand having a length of 18 to 27 nucleotides and having identity to a sequence in the target RNA,

(b) synthesizing a second RNA strand having complementarity to the RNA strand of (a); and

(c) annealing the synthesized RNA strand under conditions suitable to form a double-stranded RNA molecule, wherein the double-stranded RNA molecule has a double-stranded region of 14 to 25 nucleotides in length and a non-nucleotide overhang covalently linked by a phosphodiester or phosphorothioate linkage at least the 3 ' or 2' position of the sugar residue at the 3 ' terminus of the second RNA strand; wherein the non-nucleotide overhang comprises a C3 alkyl moiety selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps, wherein Pi represents an inorganic phosphate, dAb represents a phosphorothioate, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps, and dAbPs represent a ribose deoxynucleobase portion.

55. The method of claim 54, wherein the RNA strand has a length of 19-21 nucleotides.

56. A method of making a double stranded RNA molecule capable of down regulating expression of a target gene, wherein each RNA strand has a length of 19 to 25 nucleotides, wherein at least one strand has a non-nucleotide overhang covalently linked via a phosphodiester or phosphorothioate linkage to the 3 ' or 2' position of a sugar residue at its 3 ' terminus, the method comprises (a) synthesizing two RNA strands each 19 to 25 nucleotides in length, wherein the RNA strands are capable of forming a double-stranded RNA molecule, (b) combining the synthesized RNA strands under conditions, wherein a double stranded RNA molecule is formed which mediates down-regulation of a target nucleic acid, wherein the double stranded RNA molecule consists of a single double stranded region and at least one single stranded region, the at least one single stranded region comprises a non-nucleotide overhang covalently linked at the 3 ' or 2' position of a sugar residue at the 3 ' terminus of the strand in which it is present; wherein the non-nucleotide overhang comprises a C3 alkyl moiety selected from the group consisting of: c3Pi-C3OH, C3Pi-C3Pi, C3Pi-C3Ps, C3Ps-C3OH, C3Ps-C3Pi, C3Ps-C3Ps, C3Pi-rAb, C3Pi-dAb, C3Ps-rAb, C3Ps-dAb, rAbPi-C3OH, rAbPs-C3OH, rAbPi-C3Pi, rAbPs-C3Pi, dAbPi-C3OH, dAbPs-C3OH, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps, wherein Pi represents an inorganic phosphate, dAb represents a phosphorothioate, dAbPi-C3Pi, dAbPi-C3Ps, dAbPs-C3Pi and dAbPs-C3Ps, and dAbPs represent a ribose deoxynucleobase portion.