Patent Application
20240200063
In many species, introduction of double-stranded RNA (dsRNA) induces potent and specific gene silencing. This phenomenon occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. Short interfering RNAs (siRNAs), which are generally much shorter than the target gene, have been shown to be effective at gene silencing.
Microglia are a type of glial cell found in the central nervous system (CNS). Microglia are an essential component of the CNS immune system; however, microglia with dysregulated genes can also be a source of disease. For example, a disease state may precipitate as a result of overactive microglial genes or genes with reduced expression and/or activity in microglia. Therefore, silencing of effector genes or pathway regulatory genes may be needed to restore normal gene network function and ameliorate the disease state. Thus, there remains a need for new and improved therapeutics capable of permeating microglial cells and silencing microglial genes in order to restore genetic and biochemical pathway activity in microglia from a disease state towards a normal healthy state.
In an aspect, the invention features a method of delivering a branched small interfering RNA (siRNA) molecule to a microglial cell in a subject in need of microglial gene silencing. The method may include administering the branched siRNA molecule to the subject (e.g., to the central nervous system of the subject).
In some embodiments, the subject has been diagnosed as having a disease associated with expression of a dysregulated microglial gene or dysregulated microglial gene pathway. In some embodiments, the subject has been diagnosed as having a disease associated with expression and/or activity of a dysregulated microglial gene (e.g., altered expression and/or activity of a wild-type or mutated microglial gene).
In some embodiments, the dysregulated microglial gene exhibits increased expression and/or activity in microglial cells of the subject as compared to the expression and/or activity of the microglial gene in microglial cells of a reference subject. In some embodiments, the dysregulated microglial gene exhibits reduced expression and/or activity in microglial cells of the subject as compared to the expression and/or activity of the microglial gene in microglial cells of a reference subject.
In some embodiments, the microglial gene is a positive regulator of a gene for which increased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, the microglial gene is a negative regulator of a gene for which decreased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, the microglial gene is a splice isoform of a gene for which overexpression of the splice isoform relative to the expression of the splice isoform in a reference subject is associated with a disease state.
In some embodiments, the disease is a neuroinflammatory disease or a neurodegenerative disease. In some embodiments, the disease is Alzheimer's disease. In some embodiments, the disease is Amyotrophic Lateral Sclerosis. In some embodiments, the disease is Parkinson's disease. In some embodiments, the disease is frontotemporal dementia. In some embodiments, the disease is Huntington's disease. In some embodiments, the disease is multiple sclerosis. In some embodiments, the disease is progressive supranuclear palsy.
In some embodiments, the dysregulated microglial gene is selected from the group consisting of ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF1, IL10RA, ILIA, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, LILRB4, LPL, MEF2C, MMP12, MS4A4A, MS4A6A, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PTK2B, SCIMP, SLC24A4, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1.
In some embodiments, the subject is a mammal (e.g., a human).
In some embodiments, the branched siRNA is administered to the subject intrathecally, intracerebroventricularly, or intrastriatally.
In some embodiments, the siRNA molecule is di-branched. In some embodiments, the siRNA molecule is tri-branched. In some embodiments, the siRNA molecule is tetra-branched.
In some embodiments, the siRNA comprises (i) an antisense strand having complementarity to a portion of one or more of genes selected from the group consisting of APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU/APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, ILIA, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF and (ii) a sense strand having complementarity to the antisense strand.
In some embodiments, the siRNA includes (i) an antisense strand having complementarity to a portion of a gene encoding a positive regulator of a gene for which increased expression and/or activity (relative, e.g., to the level of expression and/or activity observed in a reference subject) is associated with a disease state.
In some embodiments, the siRNA includes (i) an antisense strand having complementarity to a portion of a gene encoding a negative regulator of a gene for which decreased expression and/or activity (relative, e.g., to the level of expression and/or activity observed in a reference subject) is associated with a disease state.
In some embodiments, the siRNA includes (i) an antisense strand having complementarity to a splice isoform of a gene for which overexpression of the splice isoform relative to the expression of the splice isoform in a reference subject is associated with a disease state.
In any of the foregoing embodiments, the siRNA may also include (ii) a sense strand having complementarity to the antisense strand.
In some embodiment, the antisense strand has complementarity (e.g., at least 85% complementarity, such as 85% complementarity, 86% complementarity, 87% complementarity, 88% complementarity, 89% complementarity, 90% complementarity, 91% complementarity, 92% complementarity, 93% complementarity, 94% complementarity, 95% complementarity, 96% complementarity, 97% complementarity, 98% complementarity, 99% complementarity, or 100% complementarity) to a portion of at least 10 contiguous nucleotides of an mRNA molecule encoding one or more of the above genes. For example, the antisense strand may have complementarity to a portion of 10 contiguous nucleotides, 11 contiguous nucleotides, 12 contiguous nucleotides, 13 contiguous nucleotides, 14 contiguous nucleotides, 15 contiguous nucleotides, 16 contiguous nucleotides, 17 contiguous nucleotides, 18 contiguous nucleotides, 19 contiguous nucleotides, 20 contiguous nucleotides, 21 contiguous nucleotides, 22 contiguous nucleotides, 23 contiguous nucleotides, 24 contiguous nucleotides, 25 contiguous nucleotides, 26 contiguous nucleotides, 27 contiguous nucleotides, 28 contiguous nucleotides, 29 contiguous nucleotides, 30 contiguous nucleotides, 31 contiguous nucleotides, 32 contiguous nucleotides 33 contiguous nucleotides, 34 contiguous nucleotides, contiguous nucleotides, 36 contiguous nucleotides, 37 contiguous nucleotides, 38 contiguous nucleotides, 39 contiguous nucleotides, 40 contiguous nucleotides, 41 contiguous nucleotides, 42 contiguous nucleotides, 43 contiguous nucleotides, 44 contiguous nucleotides, 45 contiguous nucleotides, 46 contiguous nucleotides, 47 contiguous nucleotides, 48 contiguous nucleotides, 49 contiguous nucleotides, or 50 contiguous nucleotides, or more, of an mRNA molecule encoding one or more of the above genes.
In some embodiments, the antisense strand has complementarity (e.g., at least 85% complementarity, such as 85% complementarity, 86% complementarity, 87% complementarity, 88% complementarity, 89% complementarity, 90% complementarity, 91% complementarity, 92% complementarity, 93% complementarity, 94% complementarity, 95% complementarity, 96% complementarity, 97% complementarity, 98% complementarity, 99% complementarity, or 100% complementarity) to a portion of from 10 to 50 contiguous nucleotides of an mRNA molecule encoding one or more of the above genes. For example, the antisense strand may have complementarity to a portion of from 11 contiguous nucleotides to 45 contiguous nucleotides, from 12 contiguous nucleotides to contiguous nucleotides, from 13 contiguous nucleotides to 35 contiguous nucleotides, from 14 contiguous nucleotides to 30 contiguous nucleotides, from 15 contiguous nucleotides to 29 contiguous nucleotides, from 16 contiguous nucleotides to 28 contiguous nucleotides, from 17 contiguous nucleotides to 27 contiguous nucleotides, from 18 contiguous nucleotides to 26 contiguous nucleotides, or from 19 contiguous nucleotides to 22 contiguous nucleotides of an mRNA molecule encoding one or more of the above genes.
In some embodiments, the antisense strand comprises a region represented by the following chemical formula, in the 5′-to-3′ direction:
Z-((A-P-)n(B-P-)m)q;
wherein Z is a 5′ phosphorus stabilizing moiety; each A is, independently, a 2′-O-methyl (2′-O-Me) ribonucleoside; each B is, independently, a 2′-fluoro-ribonucleoside; each P is, independently, an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).
In some embodiments, the antisense strand has a structure represented by Formula A-I, wherein Formula A-I is, in the 5′-to-3′ direction:
A-B-(A′)j-C-P2-D-P1-(C′-P1)k-C′ Formula A-I;
In some embodiments, the antisense strand has a structure represented by Formula A1, wherein Formula A1 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-II, wherein Formula A-II is, in the 5′-to-3′ direction:
A-B-(A′)j-C-P2-D-P1-(C-P1)k-C′ Formula A-I;
In some embodiments, antisense strand has a structure represented by Formula A2, wherein Formula A2 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-A-S-A Formula A2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-III, wherein Formula S-III is, in the 5′-to-3′ direction:
E-(A′)m-F Formula S-III;
In some embodiments, the sense strand has a structure represented by Formula S1, wherein Formula S1 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-A Formula S1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S2, wherein Formula S2 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-A Formula S2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S3, wherein Formula S3 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-B Formula S3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S4, wherein Formula S4 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-B Formula S4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-IV, wherein Formula A-IV is, in the 5′-to-3′ direction:
A-(A′)j-C-P2-B-(C-P1)k-C′ Formula A-IV;
In some embodiments, the antisense strand has a structure represented by Formula A3, wherein Formula A3 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A-S-A Formula A3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-V, wherein Formula S-V is, in the 5′-to-3′ direction:
E-(A′)m-C-P2-F Formula S-V;
In some embodiments, the sense strand has a structure represented by Formula S5, wherein Formula S5 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A Formula S5;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S6, wherein Formula S6 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A Formula S6;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S7, wherein Formula S7 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B Formula S7;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S8, wherein Formula S8 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B Formula S8;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-VI, wherein Formula A-VI is, in the 5′-to-3′ direction:
A-Bj-E-Bk-E-F-Gl-D-P1-C′ Formula A-VI;
In some embodiments, the antisense strand has a structure represented by Formula A4, wherein Formula A4 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-VII, wherein Formula S-VII is, in the 5′-to-3′ direction:
H-Bm-In-A′-Bo-H-C Formula S-VII;
In some embodiments, the sense strand has a structure represented by Formula S9, wherein Formula S9 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A-S-A Formula S9;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand also has a 5′ phosphorus stabilizing moiety at the 5′ end of the antisense strand.
In some embodiments, the sense strand also has a 5′ phosphorus stabilizing moiety at the 5′ end of the sense strand.
In some embodiments, each 5′-phosphorus stabilizing moiety is, independently represented by any one of Formula I-VIII:
In some embodiments, Z is (E)-vinylphosphonate as represented in Formula III.
In some embodiments, n is from 1 to 4. In some embodiments, n is from 1 to 3. In some embodiments, n is from 1 to 2. In some embodiments, n is 1.
In some embodiments, m is from 1 to 4. In some embodiments, m is from 1 to 3. In some embodiments, m is from 1 to 2. In some embodiments, m is 1.
In some embodiments, n and m are each 1.
In some embodiments, 50% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 60% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 70% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 80% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 90% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 10% or less of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
In some embodiments, 100% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
In some embodiments, 9 internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
In some embodiments, the length of the antisense strand is between 10 and 30 nucleotides (e.g., nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the length of the antisense strand is 20 nucleotides. In some embodiments, the length of the antisense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides.
In some embodiments, the siRNA molecules of the branched compound are joined to one another by way of a linker (e.g., an ethylene glycol oligomer, such as tetraethylene glycol). In some embodiments, the siRNA molecules of the branched compound are joined to one another by way of a linker between the sense strand of one siRNA molecule and the sense strand of the other siRNA molecule. In some embodiments, the siRNA molecules are joined by way of linkers between the antisense strand of one siRNA molecule and the antisense strand of the other siRNA molecule. In some embodiments, the siRNA molecules of the branched compound are joined to one another by way of a linker between the sense strand of one siRNA molecule and the antisense strand of the other siRNA molecule.
In some embodiments, the length of the sense strand is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides). In some embodiments, the length of the sense strand is 15 nucleotides. In some embodiments, the length of the sense strand is 16 nucleotides. In some embodiments, the length of the sense strand is 17 nucleotides. In some embodiments, the length of the sense strand is 18 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides. In some embodiments, the length of the sense strand is 20 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides. In some embodiments, the length of the sense strand is 22 nucleotides. In some embodiments, the length of the sense strand is 23 nucleotides. In some embodiments, the length of the sense strand is 24 nucleotides. In some embodiments, the length of the sense strand is 25 nucleotides. In some embodiments, the length of the sense strand is 26 nucleotides. In some embodiments, the length of the sense strand is 27 nucleotides. In some embodiments, the length of the sense strand is 28 nucleotides. In some embodiments, the length of the sense strand is 29 nucleotides.
In some embodiments, the length of the sense strand is 30 nucleotides.
In some embodiments, 4 internucleoside linkages are phosphorothioate linkages.
In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 18 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 19 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 20 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 21 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 22 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 23 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 24 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 25 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 26 nucleotides in length and the sense strand is 26 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 26 nucleotides in length.
In some embodiments, the antisense strand is 27 nucleotides in length and the sense strand is 27 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 26 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 27 nucleotides in length.
In some embodiments, the antisense strand is 28 nucleotides in length and the sense strand is 28 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 26 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 27 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 28 nucleotides in length.
In some embodiments, the antisense strand is 29 nucleotides in length and the sense strand is 29 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 14 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 16 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 17 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 18 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 19 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 21 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 22 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 23 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 24 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 26 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 27 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 28 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is 29 nucleotides in length.
In some embodiments, the antisense strand is 30 nucleotides in length and the sense strand is nucleotides in length.
In another aspect, the invention features a branched siRNA molecule including a sense strand and an antisense strand, wherein the antisense strand includes a region having complementarity to a segment of contiguous nucleotides within a gene selected from the group consisting of APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU/APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, ILIA, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF.
In some embodiments, the antisense strand has complementarity to a portion of a gene encoding a positive regulator of a gene for which increased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, the antisense strand has complementarity to a portion of a gene encoding a negative regulator of a gene for which decreased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, the antisense strand has complementarity to a splice isoform of a gene for which overexpression of the splice isoform relative to the expression of the splice isoform in a reference subject is associated with a disease state.
In some embodiments, the sense strand has complementarity to the antisense strand.
In some embodiments, the siRNA molecule is di-branched. In some embodiments, the siRNA molecule is tri-branched. In some embodiments, the siRNA molecule is tetra-branched.
In some embodiments, the antisense strand of the branched siRNA has the following Formula in the 5′-to-3′ direction:
Z-((A-P-)n(B-P-)m)q;
wherein Z is a 5′ phosphorus stabilizing moiety; each A is, independently, a 2′-O-Me ribonucleoside; each B is, independently, a 2′-fluoro-ribonucleoside; each P is, independently, an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 15 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).
In some embodiments, the antisense strand has a structure represented by Formula A-I, wherein Formula A-I is, in the 5′-to-3′ direction:
A-B-(A′)j-C-P2-D-P1-(C′-P1)k-C′ Formula A-I;
In some embodiments, the antisense strand has a structure represented by Formula A1, wherein Formula A1 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-II, wherein Formula A-II is, in the 5′-to-3′ direction:
A-B-(A′)j-C-P2-D-P1-(C-P1)k-C′ Formula A-I;
In some embodiments, antisense strand has a structure represented by Formula A2, wherein Formula A2 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-A-S-A Formula A2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-III, wherein Formula S-III is, in the 5′-to-3′ direction:
E-(A′)m-F Formula S-III;
In some embodiments, the sense strand has a structure represented by Formula S1, wherein Formula S1 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-A Formula S1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S2, wherein Formula S2 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-A Formula S2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S3, wherein Formula S3 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-B Formula S3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S4, wherein Formula S4 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-B Formula S4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-IV, wherein Formula A-IV is, in the 5′-to-3′ direction:
A-(A′)j-C-P2-B-(C-P1)k-C′ Formula A-IV;
In some embodiments, the antisense strand has a structure represented by Formula A3, wherein Formula A3 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A-S-A Formula A3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-V, wherein Formula S-V is, in the 5′-to-3′ direction:
E-(A′)m-C-P2-F Formula S-V;
In some embodiments, the sense strand has a structure represented by Formula S5, wherein Formula S5 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A Formula S5;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S6, wherein Formula S6 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A Formula S6;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S7, wherein Formula S7 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B Formula S7;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S8, wherein Formula S8 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B Formula S8;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand has a structure represented by Formula A-VI, wherein Formula A-VI is, in the 5′-to-3′ direction:
A-Bj-E-Bk-E-F-Gl-D-P1-C′ Formula A-VI;
In some embodiments, the antisense strand has a structure represented by Formula A4, wherein Formula A4 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the sense strand has a structure represented by Formula S-VII, wherein Formula S-VII is, in the 5′-to-3′ direction:
H-Bm-In-A′-Bo-H-C Formula S-VII;
In some embodiments, the sense strand has a structure represented by Formula S9, wherein Formula S9 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A-S-A Formula S9;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments, the antisense strand also has a 5′ phosphorus stabilizing moiety at the 5′ end of the antisense strand.
In some embodiments, the sense strand also has a 5′ phosphorus stabilizing moiety at the 5′ end of the sense strand.
In some embodiments, each 5′-phosphorus stabilizing moiety is, independently, represented by any one of Formula I-VIII:
wherein Nuc represents a nucleobase, such as adenine, uracil, guanine, thymine, or cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl), phenyl, benzyl, hydroxy, or hydrogen.
In some embodiments, Z is (E)-vinylphosphonate as represented in Formula III.
In some embodiments, each P is independently selected from phosphodiester and phosphorothioate.
In some embodiments, n is from 1 to 4 (e.g., 1, 2, 3, or 4), 1 to 3 (e.g., 1, 2, or 3), or 1 to 2. In some embodiments, n is 1.
In some embodiments, m is from 1 to 4 (e.g., 1, 2, 3, or 4), 1 to 3 (e.g., 1, 2, or 3), or 1 to 2. In some embodiments, m is 1.
In some embodiments, n and m are each 1.
In some embodiments, 50% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 60% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 70% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 80% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 90% or more of the ribonucleotides in the antisense strand are 2′-O-Me ribonucleotides (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the ribonucleotides in the antisense strand may be 2′-O-Me ribonucleotides).
In some embodiments, 10% or less of the internucleoside linkages are phosphodiester linkages or phosphorothioate. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester linkages or phosphorothioate. In some embodiments, 100% of the internucleoside linkages are phosphodiester linkages or phosphorothioate.
In some embodiments, the length of the antisense strand is between 10 and 30 nucleotides (e.g., nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the length of the antisense strand is 21 nucleotides. In some embodiments, the length of the antisense strand is 22 nucleotides. In some embodiments, the length of the antisense strand is 23 nucleotides. In some embodiments, the length of the antisense strand is 24 nucleotides. In some embodiments, the length of the antisense strand is 25 nucleotides. In some embodiments, the length of the antisense strand is 26 nucleotides. In some embodiments, the length of the antisense strand is 27 nucleotides. In some embodiments, the length of the antisense strand is 28 nucleotides. In some embodiments, the length of the antisense strand is 29 nucleotides. In some embodiments, the length of the antisense strand is 30 nucleotides.
In some embodiments, 9 internucleoside linkages are phosphorothioate.
In some embodiments, the sense strand of the branched siRNA has the following formula in the 5′-to-3′ direction:
Y-((A-P-)n(B-P-)m)qL-((B-P-)m(A-P-)n)q;
wherein Y is a hydrophobic moiety (e.g., cholesterol, vitamin D, or tocopherol); Lisa linker; each A is, independently, a 2′-O-Me ribonucleoside; each B is, independently, a 2′-fluoro-ribonucleoside; each P is, independently, an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (1, 2, 3, 4, or 5); M is an integer from 1 to 5 (1, 2, 3, 4, or 5); and q is an integer between 1 and 15 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15).
In some embodiments, Y is cholesterol.
In some embodiments, Y tocopherol.
In some embodiments, L is an ethylene glycol oligomer.
In some embodiments, L is tetraethylene glycol.
In some embodiments, each P is independently selected from phosphodiester and phosphorothioate.
In some embodiments, n is from 1 to 4 (e.g., 1, 2, 3, or 4), 1 to 3 (e.g., 1, 2, or 3), or 1 to 2. In some embodiments, n is 1.
In some embodiments, m is from 1 to 4 (e.g., 1, 2, 3, or 4), 1 to 3 (e.g., 1, 2, or 3), or 1 to 2. In some embodiments, m is 1.
In some embodiments, n and m are each 1.
In some embodiments, 10% or less of the ribonucleosides are 2′-O-Me ribonucleoside.
In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the ribonucleosides are 2′-O-Me ribonucleoside.
In some embodiments, 10% or less of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages. In some embodiments, 100% of the internucleoside linkages are phosphodiester linkages or phosphorothioate linkages.
In some embodiments, the length of the sense strand is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21, nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides). In some embodiments, the length of the sense strand is 16 nucleotides. In some embodiments, the length of the sense strand is 17 nucleotides. In some embodiments, the length of the sense strand is 18 nucleotides. In some embodiments, the length of the sense strand is 19 nucleotides. In some embodiments, the length of the sense strand is 20 nucleotides. In some embodiments, the length of the sense strand is 21 nucleotides. In some embodiments, the length of the sense strand is 22 nucleotides. In some embodiments, the length of the sense strand is 23 nucleotides. In some embodiments, the length of the sense strand is 24 nucleotides. In some embodiments, the length of the sense strand is 25 nucleotides. In some embodiments, the length of the sense strand is 26 nucleotides. In some embodiments, the length of the sense strand is 27 nucleotides. In some embodiments, the length of the sense strand is 28 nucleotides. In some embodiments, the length of the sense strand is 29 nucleotides. In some embodiments, the length of the sense strand is 30 nucleotides.
In some embodiments, 4 internucleoside linkages are phosphorothioate.
In another aspect, the invention features a method of treating a subject diagnosed as having a disease associated with expression of a dysregulated microglial gene (e.g., wild-type or mutated microglial gene), the method includes administering to the subject the branched siRNA molecule of any one of the above aspects or embodiments.
In some embodiments, the dysregulated microglial gene is selected from the group consisting of ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF1, IL10RA, ILIA, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, LILRB4, LPL, MEF2C, MMP12, MS4A4A, MS4A6A, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PTK2B, SCIMP, SLC24A4, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCWPW1.
In some embodiments, the dysregulated microglial gene exhibits increased expression and/or activity in microglial cells of the subject as compared to the expression and/or activity of the same gene in microglial cells of a reference subject.
In some embodiments, the dysregulated microglial gene exhibits reduced expression and/or activity in microglial cells of the subject as compared to the expression and/or activity of the same gene in microglial cells of a reference subject.
In some embodiments, the administering of the branched siRNA molecule to the subject results in silencing of gene in the subject.
In some embodiments, the silencing of a gene comprises silencing any one of the genes selected from the group consisting of APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU/APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, ILIA, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF.
In some embodiments, silencing of a gene comprises silencing of a positive regulator of a gene for which increased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, silencing of a gene comprises silencing of a negative regulator of a gene for which decreased expression and/or activity relative to the level of expression and/or activity observed in a reference subject is associated with a disease state.
In some embodiments, silencing of a gene comprises silencing of a splice isoform of a gene for which overexpression of the splice isoform relative to the expression of the splice isoform in a reference subject is associated with a disease state.
In some embodiments, the subject is a human.
Unless otherwise defined herein, scientific, and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.
As used herein, the term “nucleic acids” refers to RNA or DNA molecules consisting of a chain of ribonucleotides or deoxyribonucleotides, respectively. As used herein, the term “therapeutic nucleic acid” refers to a nucleic acid molecule (e.g., ribonucleic acid) that has partial or complete complementarity to, and interacts with, a disease-associated target mRNA and mediates silencing of expression of the mRNA.
As used herein, the term “carrier nucleic acid” refers to a nucleic acid molecule (e.g., ribonucleic acid) that has sequence complementarity with, and hybridizes with, a therapeutic nucleic acid. As used herein, the term “3′ end” refers to the end of the nucleic acid that contains an unmodified hydroxyl group at the 3′ carbon of the ribose ring.
As used herein, the term “nucleoside” refers to a molecule made up of a heterocyclic base and its sugar.
As used herein, the term “nucleotide” refers to a nucleoside having a phosphate group on its 3′ or 5′ sugar hydroxyl group.
As used herein, the term “siRNA” refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules can vary in length (generally, between 18-30 base pairs) and contain varying degrees of complementarity to their target mRNA. The term “siRNA” includes duplexes of two separate strands, as well as single strands that optionally form hairpin structures comprising a duplex region.
As used herein, the term “antisense strand” refers to the strand of the siRNA duplex that contains some degree of complementarity to the target gene.
As used herein, the term “sense strand” refers to the strand of the siRNA duplex that contains complementarity to the antisense strand.
As used herein, the terms “chemically modified nucleotide” or “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refer to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function.
As used herein, the term “metabolically stabilized” refers to RNA molecules that contain ribonucleotides that have been chemically modified from 2′-hydroxyl groups to 2′-O-methyl groups.
As used herein, the term “phosphorothioate” refers to the phosphate group of a nucleotide that is modified by substituting one or more of the oxygens of the phosphate group with sulfur.
As used herein, the term “ethylene glycol chain” refers to a carbon chain with the formula ((CH2OH)2).
As used herein, “alkyl” refers to a saturated hydrocarbon group. Alkyl groups may be acyclic or cyclic and contain only C and H when unsubstituted. When an alkyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “butyl” is meant to include n-butyl, sec-butyl, and iso-butyl. Examples of alkyl include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. In some embodiments, alkyl may be substituted. Suitable substituents that may be introduced into an alkyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
As used herein, “alkenyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C═C). Alkenyl groups contain only C and H when unsubstituted. When an alkenyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “butenyl” is meant to include n-butenyl, sec-butenyl, and iso-butenyl. Examples of alkenyl include —CH═CH2, —CH2—CH═CH2, and —CH2—CH═CH—CH═CH2. In some embodiments, alkenyl may be substituted. Suitable substituents that may be introduced into an alkenyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
As used herein, “alkynyl” refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). Alkynyl groups contain only C and H when unsubstituted. When an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described; thus, for example, “pentynyl” is meant to include n-pentynyl, sec-pentynyl, iso-pentynyl, and ted-pentynyl. Examples of alkynyl include —C≡CH and —C≡C—CH3. In some embodiments, alkynyl may be substituted. Suitable substituents that may be introduced into an alkynyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.
As used herein the term “phenyl” denotes a monocyclic arene in which one hydrogen atom from a carbon atom of the ring has been removed. A phenyl group can be unsubstituted or substituted with one or more suitable substituents, wherein the substituent replaces an H of the phenyl group.
As used herein, the term “benzyl” refers to monovalent radical obtained when a hydrogen atom attached to the methyl group of toluene is removed. A benzyl generally has the formula of phenyl-CH2—. A benzyl group can be unsubstituted or substituted with one or more suitable substituents. For example, the substituent may replace an H of the phenyl component and/or an H of the methylene (—CH2—) component.
As used herein, the term “amide” refers to an alkyl or aromatic group that is attached to an amino-carbonyl functional group.
As used herein, the term “internucleoside” and “internucleotide” refer to the bonds between nucleosides and nucleotides, respectively.
As used herein, the term “triazol” refers to heterocyclic compounds with the formula (C2H3N3), having a five-membered ring of two carbons and three nitrogens, the positions of which can change resulting in multiple isomers.
As used herein, the term “terminal group” refers to the group at which a carbon chain or nucleic acid ends.
As used herein, the term “lipophilic amino acid” refers to an amino acid comprising a hydrophobic moiety (e.g., an alkyl chain or an aromatic ring).
As used herein, the term “antagomiRs” refers to nucleic acids that can function as inhibitors of miRNA activity.
As used herein, the term “gapmers” refers to chimeric antisense nucleic acids that contain a central block of deoxynucleotide monomers sufficiently long to induce RNase H cleavage. The deoxynucleotide block is flanked by ribonucleotide monomers or ribonucleotide monomers containing modifications.
As used herein, the term “mixmers” refers to nucleic acids that are comprised of a mix of locked nucleic acids (LNAs) and DNA.
As used herein, the term “guide RNAs” refers to nucleic acids that have sequence complementarity to a specific sequence in the genome immediately or 1 base pair upstream of the protospacer adjacent motif (PAM) sequence as used in CRISPR/Cas9 gene editing systems. Alternatively, “guide RNAs” may refer to nucleic acids that have sequence complementarity (e.g., are antisense) to a specific messenger RNA (mRNA) sequence. In this context, a guide RNA may also have sequence complementarity to a “passenger RNA” sequence of equal or shorter length, which is identical or substantially identical to the sequence of mRNA to which the guide RNA hybridizes.
As used herein, the term “target of delivery” refers to the organ or part of the body that is desired to deliver the branched oligonucleotide compositions to.
As used herein, the term “branched siRNA” refers to a compound containing two or more double-stranded siRNA molecules covalently bound to one another. Branched siRNA molecules may be “di-branched,” also referred to herein as “di-siRNA,” wherein the siRNA molecule comprises 2 siRNA molecules covalently bound to one another, e.g., by way of a linker. Branched siRNA molecules may be “tri-branched,” also referred to herein as “tri-siRNA,” wherein the siRNA molecule comprises 3 siRNA molecules covalently bound to one another, e.g., by way of a linker. Branched siRNA molecules may be “tetra-branched,” also referred to herein as “tetra-siRNA,” wherein the siRNA molecule comprises 4 siRNA molecules covalently bound to one another, e.g., by way of a linker.
As used herein, the term “5′ phosphorus stabilizing moiety” refers to a terminal phosphate group that includes phosphates as well as modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety can be located at either terminus but is preferred at the 5′-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula —O—P(═O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R′), or alkyl where R′ is H, an amino protecting group, or unsubstituted or substituted alkyl. In some embodiments, the 5′ and or 3′ terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified (di- or tri-phosphates) or modified.
As used herein, the term “between X and Y” is inclusive of the values of X and Y. For example, “between X and Y” refers to the range of values between the value of X and the value of Y, as well as the value of X and the value of Y.
As used herein, an “amino acid” refers to a molecule containing amine and carboxyl functional groups and a side chain specific to the amino acid:
In some embodiments the amino acid is chosen from the group of proteinogenic amino acids. In other embodiments, the amino acid is an L-amino acid or a D-amino acid. In other embodiments, the amino acid is a synthetic amino acid (e.g., a beta-amino acid).
It is understood that certain internucleotide linkages provided herein, including, e.g., phosphodiester and phosphorothioate, comprise a formal charge of −1 at physiological pH, and that said formal charge will be balanced by a cationic moiety, e.g., an alkali metal such as sodium or potassium, an alkali earth metal such as calcium or magnesium, or an ammonium or guanidinium ion.
The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) preferably decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.
As used herein, the term “complementary” refers to two nucleotides that form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
As used herein, the term “percent (%) sequence complementarity” with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs. For the avoidance of doubt, Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. A proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.” Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared. As an illustration, the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, (which can alternatively be phrased as a given nucleic acid sequence, A that has a certain percent complementarity to a given nucleic acid sequence, B) is calculated as follows:
100 multiplied by (the fraction X/Y)
where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program's alignment of A and B, and where Y is the total number of nucleic acids in B. It will be appreciated that where the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, the percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.
The term “gene silencing” refers to the suppression of gene expression, e.g., transgene, heterologous gene and/or endogenous gene expression, which may be mediated through processes that affect transcription and/or through processes that affect post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an RNAi molecule initiates the inhibition or degradation of the mRNA transcribed from a gene of interest in a sequence-specific manner via RNA interference, thereby preventing translation of the gene's product.
The phrase “overactive disease driver gene,” as used herein, refers to a microglial gene having increased activity and/or expression that contributes to or causes a disease state in a subject (e.g., a human). The disease state may be caused or exacerbated by the overactive disease driver gene directly or by way of an intermediate gene(s).
The term “negative regulator,” as used herein, refers to a microglial gene that negatively regulates (e.g., reduces or inhibits) the expression and/or activity of another microglial gene or set of genes (e.g., dysregulated microglial gene or dysregulated microglial gene pathway).
The term “positive regulator,” as used herein, refers to a microglial gene that positively regulates (e.g., increases or saturates) the expression and/or activity of another microglial gene or set of microglial genes (e.g., dysregulated microglial gene or dysregulated microglial gene pathway).
The term “phosphate moiety” as used herein, refers to a terminal phosphate group that includes phosphates as well as modified phosphates. The phosphate moiety can be located at either terminus but is preferred at the 5′-terminal nucleoside. In one aspect, the terminal phosphate is unmodified having the formula —O—P(═O)(OH)OH. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R′) or alkyl where R′ is H, an amino protecting group or unsubstituted or substituted alkyl. In some embodiments, the 5′ and or 3′ terminal group can comprise from 1 to 3 phosphate moieties that are each, independently, unmodified (di or tri-phosphates) or modified.
In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases.
As used herein, the term “reference subject” refers to a healthy control subject of the same or similar, e.g., age, sex, geographical region, and/or education level as a subject treated with a composition of the disclosure. A healthy reference subject is one that does not suffer from a disease associated with expression of a dysregulated microglial gene or a dysregulated microglial gene pathway. Moreover, a healthy reference subject is one that does not suffer from a disease associated with altered (e.g., increased or decreased) expression and/or activity of a microglial gene.
As used herein, the terms “treat,” “treated,” or “treating” mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.
As used herein, the term “ABCA7” refers to the gene encoding Phospholipid-transporting ATPase ABCA7. The terms “ABCA7” and “Phospholipid-transporting ATPase ABCA7” include wild-type forms of the ABCA7 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ABCA7. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ABCA7 nucleic acid sequence (e.g., SEQ ID NO: 1, European Nucleotide Archive (ENA) accession number AF250238). SEQ ID NO: 1 is a wild-type gene sequence encoding ABCA7 protein, and is shown below:
As used herein, the term “ABI3” refers to the gene encoding ABI gene family member 3. The terms “ABI3” and “ABI gene family member 3” include wild-type forms of the ABI3 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ABI3. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ABI3 nucleic acid sequence (e.g., SEQ ID NO: 2, ENA accession number AF037886). SEQ ID NO: 2 is a wild-type gene sequence encoding ABI3 protein, and is shown below:
As used herein, the term “ADAM10” refers to the gene encoding ADAM Metallopeptidase Domain 10. The terms “ADAM10” and “ADAM Metallopeptidase Domain 10” include wild-type forms of the ADAM10 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ADAM10. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ADAM10 nucleic acid sequence (e.g., SEQ ID NO: 3, NCBI Reference Sequence: NM_001110.3). SEQ ID NO: 3 is a wild-type gene sequence encoding ADAM10 protein, and is shown below:
The terms “APOC1” and “Apolipoprotein C1” include wild-type forms of the APOC1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type APOC1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type APOC1 nucleic acid sequence (e.g., SEQ ID NO: 4, NCBI Reference Sequence: NM_001645). SEQ ID NO: 4 is a wild-type gene sequence encoding APOC1 protein, and is shown below:
As used herein, the term “APOE” refers to the gene encoding Apolipoprotein E. The terms “APOE” and “Apolipoprotein E” include wild-type forms of the APOE gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type APOE. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type APOE nucleic acid sequence (e.g., SEQ ID NO: 5, ENA accession number M12529). SEQ ID NO: 5 is a wild-type gene sequence encoding APOE protein, and is shown below:
As used herein, the term “AXL” refers to the gene encoding Tyrosine-protein kinase receptor UFO. The terms “AXL” and “Tyrosine-protein kinase receptor UFO” include wild-type forms of the AXL gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type AXL. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type AXL nucleic acid sequence (e.g., SEQ ID NO: 6, ENA accession number M76125). SEQ ID NO: 6 is a wild-type gene sequence encoding AXL protein, and is shown below:
As used herein, the term “BIN1” refers to the gene encoding Myc box-dependent-interacting protein 1. The terms “BIN1” and “Myc box-dependent-interacting protein 1” include wild-type forms of the BIN1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type BIN1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type BIN1 nucleic acid sequence (e.g., SEQ ID NO: 7, ENA accession number AF004015). SEQ ID NO: 7 is a wild-type gene sequence encoding BIN1 protein, and is shown below:
As used herein, the term “C1QA” refers to the gene encoding Complement C1q A Chain. The terms “C1QA” and “Complement C1q A Chain” include wild-type forms of the C1QA gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type C1QA. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type C1QA nucleic acid sequence (e.g., SEQ ID NO: 8, NCBI Reference Sequence: NM_015991.3). SEQ ID NO: 8 is a wild-type gene sequence encoding C1QA protein, and is shown below:
As used herein, the term “C3” refers to the gene encoding Complement C3. The terms “C3” and “Complement C3” include wild-type forms of the C3 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type C3. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type C3 nucleic acid sequence (e.g., SEQ ID NO: 9, NCBI Reference Sequence: NM_000064.3). SEQ ID NO: 9 is a wild-type gene sequence encoding C3 protein, and is shown below:
As used herein, the term “C9orf72” refers to the gene encoding Guanine nucleotide exchange C9orf72. The terms “C9orf72” and “Guanine nucleotide exchange C9orf72” include wild-type forms of the C9orf72 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type C9orf72. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type C9orf72 nucleic acid sequence (e.g., SEQ ID NO: 10, ENA accession number JN681271). SEQ ID NO: 10 is a wild-type gene sequence encoding C9orf72 protein, and is shown below:
As used herein, the term “CASS4” refers to the gene encoding Cas scaffolding protein family member 4. The terms “CASS4” and “Cas scaffolding protein family member 4” include wild-type forms of the CASS4 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CASS4. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CASS4 nucleic acid sequence (e.g., SEQ ID NO: 11, ENA accession number AJ276678). SEQ ID NO: 11 is a wild-type gene sequence encoding CASS4 protein, and is shown below:
As used herein, the term “CCL5” refers to the gene encoding C-C motif chemokine 5. The terms “CCL5” and “C-C motif chemokine 5” include wild-type forms of the CCL5 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CCL5. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CCL5 nucleic acid sequence (e.g., SEQ ID NO: 12, ENA accession number M21121). SEQ ID NO: 12 is a wild-type gene sequence encoding CCL5 protein, and is shown below:
As used herein, the term “CD2AP” refers to the gene encoding CD2-associated protein. The terms “CD2AP” and “CD2-associated protein” include wild-type forms of the CD2AP gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CD2AP. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CD2AP nucleic acid sequence (e.g., SEQ ID NO: 13, ENA accession number AF146277). SEQ ID NO: 13 is a wild-type gene sequence encoding CD2AP protein, and is shown below:
As used herein, the term “CD33” refers to the gene encoding Myeloid cell surface antigen CD33. The terms “CD33” and “Myeloid cell surface antigen CD33” include wild-type forms of the CD33 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CD33. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CD33 nucleic acid sequence (e.g., SEQ ID NO: 14, ENA accession number M23197). SEQ ID NO: 14 is a wild-type gene sequence encoding CD33 protein, and is shown below:
As used herein, the term “CD68” refers to the gene encoding CD68 Molecule. The terms “CD68” and “CD68 molecule” include wild-type forms of the CD68 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CD68. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CD68 nucleic acid sequence (e.g., SEQ ID NO: 15, NCBI Reference Sequence: NM_001251.2). SEQ ID NO: 15 is a wild-type gene sequence encoding CD68 protein, and is shown below:
As used herein, the term “CLPTM1” refers to the gene encoding CLPTM1 Regulator of GABA Type A Receptor Forward Trafficking. The terms “CLPTM1” and “CLPTM1 Regulator of GABA Type A Receptor Forward Trafficking” include wild-type forms of the CLPTM1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CLPTM1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CLPTM1 nucleic acid sequence (e.g., SEQ ID NO: 16, NCBI Reference Sequence: NM_001294.3). SEQ ID NO: 16 is a wild-type gene sequence encoding CLPTM1 protein, and is shown below:
As used herein, the term “CLU” refers to the gene encoding Clusterin. The terms “CLU” and “Clusterin” include wild-type forms of the CLU gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CLU. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CLU nucleic acid sequence (e.g., SEQ ID NO: 17, ENA accession number M25915). SEQ ID NO: 17 is a wild-type gene sequence encoding CLU protein, and is shown below:
As used herein, the term “CR1” refers to the gene encoding Complement receptor type 1. The terms “CR1” and “Complement receptor type 1” include wild-type forms of the CR1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CR1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CR1 nucleic acid sequence (e.g., SEQ ID NO: 18, ENA accession number Y00816). SEQ ID NO: 18 is a wild-type gene sequence encoding CR1 protein, and is shown below:
As used herein, the term “CSF1” refers to the gene encoding Macrophage colony-stimulating factor 1. The terms “CSF1” and “Macrophage colony-stimulating factor 1” include wild-type forms of the CSF1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CSF1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CSF1 nucleic acid sequence (e.g., SEQ ID NO: 19, ENA accession number M37435). SEQ ID NO: 19 is a wild-type gene sequence encoding CSF1 protein, and is shown below:
As used herein, the term “CST7” refers to the gene encoding Cystatin-F. The terms “CST7” and “Cystatin-F” include wild-type forms of the CST7 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CST7. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CST7 nucleic acid sequence (e.g., SEQ ID NO: 20, ENA accession number AF031824). SEQ ID NO: 20 is a wild-type gene sequence encoding CST7 protein, and is shown below:
As used herein, the term “CTSB” refers to the gene encoding Cathepsin B. The terms “CTSB” and “Cathepsin B” include wild-type forms of the CTSB gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CTSB. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CTSB nucleic acid sequence (e.g., SEQ ID NO: 21, ENA accession number M14221). SEQ ID NO: 21 is a wild-type gene sequence encoding CTSB protein, and is shown below:
As used herein, the term “CTSD” refers to the gene encoding Cathepsin D. The terms “CTSD” and “Cathepsin D” include wild-type forms of the CTSD gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CTSD. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CTSD nucleic acid sequence (e.g., SEQ ID NO: 22, ENA accession number M11233). SEQ ID NO: 22 is a wild-type gene sequence encoding CTSD protein, and is shown below:
As used herein, the term “CTSL” refers to the gene encoding Cathepsin L1. The terms “CTSL” and “Cathepsin L1” include wild-type forms of the CTSL gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CTSL. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CTSL nucleic acid sequence (e.g., SEQ ID NO: 23, ENA accession number X12451). SEQ ID NO: 23 is a wild-type gene sequence encoding CTSL protein, and is shown below:
As used herein, the term “CXCL10” refers to the gene encoding C—X-C motif chemokine 10. The terms “CXCL10” and “C—X-C motif chemokine 10” include wild-type forms of the CXCL10 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CXCL10. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CXCL10 nucleic acid sequence (e.g., SEQ ID NO: 24, ENA accession number X02530). SEQ ID NO: 24 is a wild-type gene sequence encoding CXCL10 protein, and is shown below:
As used herein, the term “CXCL13” refers to the gene encoding C—X-C motif chemokine 13. The terms “CXCL13” and “C—X-C motif chemokine 13” include wild-type forms of the CXCL13 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type CXCL13. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type CXCL13 nucleic acid sequence (e.g., SEQ ID NO: 25, ENA accession number AF044197). SEQ ID NO: 25 is a wild-type gene sequence encoding CXCL13 protein, and is shown below:
As used herein, the term “DSG2” refers to the gene encoding Desmoglein 2. The terms “DSG2” and “Desmoglein 2” include wild-type forms of the DSG2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type DSG2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type DSG2 nucleic acid sequence (e.g., SEQ ID NO: 26, NCBI Reference Sequence: NM_001943.4). SEQ ID NO: 26 is a wild-type gene sequence encoding DSG2 protein, and is shown below:
As used herein, the term “ECHDC3” refers to the gene encoding Enoyl-CoA Hydratase Domain Containing 3. The terms “ECHDC” and “Enoyl-CoA Hydratase Domain Containing 3” include wild-type forms of the ECHDC gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ECHDC. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ECHDC nucleic acid sequence (e.g., SEQ ID NO: 27, NCBI Reference Sequence: NM_024693.4). SEQ ID NO: 27 is a wild-type gene sequence encoding ECHDC protein, and is shown below:
As used herein, the term “EPHA1” refers to the gene encoding Ephrin type-A receptor 1. The terms “EPHA1” and “Ephrin type-A receptor 1” include wild-type forms of the EPHA1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type EPHA1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type EPHA1 nucleic acid sequence (e.g., SEQ ID NO: 28, ENA accession number M18391). SEQ ID NO: 28 is a wild-type gene sequence encoding EPHA1 protein, and is shown below:
As used herein, the term “FABP5” refers to the gene encoding Fatty acid-binding protein 5. The terms “FABP5” and “Fatty acid-binding protein 5” include wild-type forms of the FABP5 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type FABP5. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type FABP5 nucleic acid sequence (e.g., SEQ ID NO: 29, ENA accession number M94856). SEQ ID NO: 29 is a wild-type gene sequence encoding FABP5 protein, and is shown below:
As used herein, the term “FERMT2” refers to the gene encoding Fermitin family homolog 2. The terms “FERMT2” and “Fermitin family homolog 2” include wild-type forms of the FERMT2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type FERMT2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type FERMT2 nucleic acid sequence (e.g., SEQ ID NO: 30, ENA accession number Z24725). SEQ ID NO: 30 is a wild-type gene sequence encoding FERMT2 protein, and is shown below:
As used herein, the term “FTH1” refers to the gene encoding Ferritin heavy chain. The terms “FTH1” and “Ferritin heavy chain” include wild-type forms of the FTH1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type FTH1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type FTH1 nucleic acid sequence (e.g., SEQ ID NO: 31, ENA accession number X00318). SEQ ID NO: 31 is a wild-type gene sequence encoding FTH1 protein, and is shown below:
As used herein, the term “GNAS” refers to the gene encoding Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas. The terms “GNAS” and “Guanine nucleotide-binding protein G(s) subunit alpha isoforms XLas” include wild-type forms of the GNAS gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type GNAS. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type GNAS nucleic acid sequence (e.g., SEQ ID NO: 32, ENA accession number X04408). SEQ ID NO: 32 is a wild-type gene sequence encoding GNAS protein, and is shown below:
As used herein, the term “GRN” refers to the gene encoding Progranulin. The terms “GRN” and “Progranulin” include wild-type forms of the GRN gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type GRN. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type GRN nucleic acid sequence (e.g., SEQ ID NO: 33, ENA accession number X62320). SEQ ID NO: 33 is a wild-type gene sequence encoding GRN protein, and is shown below:
As used herein, the term “HBEGF” refers to the gene encoding Heparin Binding EGF Like Growth Factor. The terms “HBEGF” and “Heparin Binding EGF Like Growth Factor” include wild-type forms of the HBEGF gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type HBEGF. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type HBEGF nucleic acid sequence (e.g., SEQ ID NO: 34, NCBI Reference Sequence: NM_001945.2). SEQ ID NO: 34 is a wild-type gene sequence encoding HBEGF protein, and is shown below:
As used herein, the term “HLA-DRB1” refers to the gene encoding HLA class II histocompatibility antigen, DRB1 beta chain. The terms “HLA-DRB1” and “HLA class II histocompatibility antigen, DRB1 beta chain” include wild-type forms of the HLA-DRB1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type HLA-DRB1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type HLA-DRB1 nucleic acid sequence (e.g., SEQ ID NO: 35, ENA accession number X00699). SEQ ID NO: 35 is a wild-type gene sequence encoding HLA-DRB1 protein, and is shown below:
As used herein, the term “HLA-DRB5” refers to the gene encoding HLA class II histocompatibility antigen, DR beta 5 chain. The terms “HLA-DRB5” and “HLA class II histocompatibility antigen, DR beta 5 chain” include wild-type forms of the HLA-DRB5 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type HLA-DRB5. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type HLA-DRB5 nucleic acid sequence (e.g., SEQ ID NO: 36, ENA accession number M20429). SEQ ID NO: 36 is a wild-type gene sequence encoding HLA-DRB5 protein, and is shown below:
As used herein, the term “IFIT1” refers to the gene encoding Interferon-induced protein with tetratricopeptide repeats 1. The terms “IFIT1” and “Interferon-induced protein with tetratricopeptide repeats 1” include wild-type forms of the IFIT1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IFIT1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IFIT1 nucleic acid sequence (e.g., SEQ ID NO: 37, ENA accession number X03557). SEQ ID NO: 37 is a wild-type gene sequence encoding IFIT1 protein, and is shown below:
As used herein, the term “IFIT3” refers to the gene encoding Interferon-induced protein with tetratricopeptide repeats 3. The terms “IFIT3” and “Interferon-induced protein with tetratricopeptide repeats 3” include wild-type forms of the IFIT3 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IFIT3. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IFIT3 nucleic acid sequence (e.g., SEQ ID NO: 38, ENA accession number AF026939). SEQ ID NO: 38 is a wild-type gene sequence encoding IFIT3 protein, and is shown below:
As used herein, the term “IFITM3” refers to the gene encoding Interferon Induced Transmembrane Protein. The terms “IFITM3” and “Interferon Induced Transmembrane Protein” include wild-type forms of the IFITM3 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IFITM3. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IFITM3 nucleic acid sequence (e.g., SEQ ID NO: 39, NCBI Reference Sequence: NM_021034.2). SEQ ID NO: 39 is a wild-type gene sequence encoding IFITM3 protein, and is shown below:
As used herein, the term “IFNAR1” refers to the gene encoding Interferon alpha/beta receptor 1. The terms “IFNAR1” and “Interferon alpha/beta receptor 1” include wild-type forms of the IFNAR1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IFNAR1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IFNAR1 nucleic acid sequence (e.g., SEQ ID NO: 40, ENA accession number J03171). SEQ ID NO: 40 is a wild-type gene sequence encoding IFNAR1 protein, and is shown below:
As used herein, the term “IFNAR2” refers to the gene encoding Interferon alpha/beta receptor 2. The terms “IFNAR2” and “Interferon alpha/beta receptor 2” include wild-type forms of the IFNAR2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IFNAR2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IFNAR2 nucleic acid sequence (e.g., SEQ ID NO: 41, ENA accession number X77722). SEQ ID NO: 41 is a wild-type gene sequence encoding IFNAR2 protein, and is shown below:
As used herein, the term “IGF1” refers to the gene encoding Insulin-like growth factor I. The terms “IGF1” and “Insulin-like growth factor I” include wild-type forms of the IGF1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IGF1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IGF1 nucleic acid sequence (e.g., SEQ ID NO: 42, ENA accession number X00173). SEQ ID NO: 42 is a wild-type gene sequence encoding IGF1 protein, and is shown below:
As used herein, the term “IL10RA” refers to the gene encoding Interleukin-10 receptor subunit alpha. The terms “IL10RA” and “Interleukin-10 receptor subunit alpha” include wild-type forms of the MORA gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type MORA. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IL10RA nucleic acid sequence (e.g., SEQ ID NO: 43, ENA accession number U00672). SEQ ID NO: 43 is a wild-type gene sequence encoding MORA protein, and is shown below:
As used herein, the term “ILIA” refers to the gene encoding Interleukin-1 alpha. The terms “ILIA” and “Interleukin-1 alpha” include wild-type forms of the ILIA gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ILIA. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ILIA nucleic acid sequence (e.g., SEQ ID NO: 44, ENA accession number X02531). SEQ ID NO: 44 is a wild-type gene sequence encoding ILIA protein, and is shown below:
As used herein, the term “IL1B” refers to the gene encoding Interleukin-1 beta. The terms “IL1B” and “Interleukin-1 beta” include wild-type forms of the IL1B gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IL1B. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IL1B nucleic acid sequence (e.g., SEQ ID NO: 45, ENA accession number X02770). SEQ ID NO: 45 is a wild-type gene sequence encoding IL1B protein, and is shown below:
As used herein, the term “IL1RAP” refers to the gene encoding Interleukin-1 receptor accessory protein. The terms “IL1 RAP” and “Interleukin-1 receptor accessory protein” include wild-type forms of the IL1 RAP gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type IL1 RAP. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type IL1RAP nucleic acid sequence (e.g., SEQ ID NO: 46, ENA accession number AF029213). SEQ ID NO: 46 is a wild-type gene sequence encoding IL1 RAP protein, and is shown below:
As used herein, the term “INPP5D” refers to the gene encoding Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1. The terms “INPP5D” and “Phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase 1” include wild-type forms of the INPP5D gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type INPP5D. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type INPP5D nucleic acid sequence (e.g., SEQ ID NO: 47, ENA accession number X98429). SEQ ID NO: 47 is a wild-type gene sequence encoding INPP5D protein, and is shown below:
As used herein, the term “ITGAM” refers to the gene encoding Integrin Subunit Alpha M. The terms “ITGAM” and “Integrin Subunit Alpha M” include wild-type forms of the ITGAM gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ITGAM. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ITGAM nucleic acid sequence (e.g., SEQ ID NO: 48, NCBI Reference Sequence: NM_000632.3). SEQ ID NO: 48 is a wild-type gene sequence encoding ITGAM protein, and is shown below:
As used herein, the term “ITGAX” refers to the gene encoding Integrin alpha-X. The terms “ITGAX” and “Integrin alpha-X” include wild-type forms of the ITGAX gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ITGAX. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ITGAX nucleic acid sequence (e.g., SEQ ID NO: 49, ENA accession number M81695). SEQ ID NO: 49 is a wild-type gene sequence encoding ITGAX protein, and is shown below:
As used herein, the term “LILRB4” refers to the gene encoding Leukocyte immunoglobulin-like receptor subfamily B member 4. The terms “LILRB4” and “Leukocyte immunoglobulin-like receptor subfamily B member 4” include wild-type forms of the LILRB4 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type LILRB4. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type LILRB4 nucleic acid sequence (e.g., SEQ ID NO: 50, ENA accession number U91925). SEQ ID NO: 50 is a wild-type gene sequence encoding LILRB4 protein, and is shown below:
As used herein, the term “LPL” refers to the gene encoding Lipoprotein lipase. The terms “LPL” and “Lipoprotein lipase” include wild-type forms of the LPL gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type LPL. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type LPL nucleic acid sequence (e.g., SEQ ID NO: 51, ENA accession number M15856). SEQ ID NO: 51 is a wild-type gene sequence encoding LPL protein, and is shown below:
As used herein, the term “MEF2C” refers to the gene encoding Myocyte-specific enhancer factor 2C. The terms “MEF2C” and “Myocyte-specific enhancer factor 2C” include wild-type forms of the MEF2C gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type MEF2C. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type MEF2C nucleic acid sequence (e.g., SEQ ID NO: 52, ENA accession number L08895). SEQ ID NO: 52 is a wild-type gene sequence encoding MEF2C protein, and is shown below:
As used herein, the term “MMP12” refers to the gene encoding Macrophage metalloelastase. The terms “MMP12” and “Macrophage metalloelastase” include wild-type forms of the MMP12 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type MMP12. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type MMP12 nucleic acid sequence (e.g., SEQ ID NO: 53, ENA accession number L23808). SEQ ID NO: 53 is a wild-type gene sequence encoding MMP12 protein, and is shown below:
As used herein, the term “MS4A4A” refers to the gene encoding Membrane Spanning 4-Domains A4A. The terms “MS4A4A” and “Membrane Spanning 4-Domains A4A” include wild-type forms of the MS4A4A gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type MS4A4A. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type MS4A4A nucleic acid sequence (e.g., SEQ ID NO: 54, NCBI Reference Sequence: NM_148975.2). SEQ ID NO: 54 is a wild-type gene sequence encoding MS4A4A protein, and is shown below:
As used herein, the term “MS4A6A” refers to the gene encoding Membrane-spanning 4-domains subfamily A member 6A. The terms “MS4A6A” and “Membrane-spanning 4-domains subfamily A member 6A” include wild-type forms of the MS4A6A gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type MS4A6A. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type MS4A6A nucleic acid sequence (e.g., SEQ ID NO: 55, ENA accession number AB013104). SEQ ID NO: 55 is a wild-type gene sequence encoding MS4A6A protein, and is shown below:
As used herein, the term “NLRP3” refers to the gene encoding NACHT, LRR and PYD domains-containing protein 3. The terms “NLRP3” and “NACHT, LRR and PYD domains-containing protein 3” include wild-type forms of the NLRP3 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type NLRP3. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type NLRP3 nucleic acid sequence (e.g., SEQ ID NO: 56, ENA accession number AF410477). SEQ ID NO: 56 is a wild-type gene sequence encoding NLRP3 protein, and is shown below:
As used herein, the term “NME8” refers to the gene encoding Thioredoxin domain-containing protein 3. The terms “NME8” and “Thioredoxin domain-containing protein 3” include wild-type forms of the NME8 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type NME8. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type NME8 nucleic acid sequence (e.g., SEQ ID NO: 57, ENA accession number AF202051). SEQ ID NO: 57 is a wild-type gene sequence encoding NME8 protein, and is shown below:
As used herein, the term “NOS2” refers to the gene encoding Nitric oxide synthase, inducible. The terms “NOS2” and “Nitric oxide synthase, inducible” include wild-type forms of the NOS2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type NOS2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type NOS2 nucleic acid sequence (e.g., SEQ ID NO: 58, ENA accession number L24553). SEQ ID NO: 58 is a wild-type gene sequence encoding NOS2 protein, and is shown below:
As used herein, the term “PICALM” refers to the gene encoding Phosphatidylinositol-binding clathrin assembly protein. The terms “PICALM” and “Phosphatidylinositol-binding clathrin assembly protein” include wild-type forms of the PICALM gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type PICALM. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type PICALM nucleic acid sequence (e.g., SEQ ID NO: 59, ENA accession number U45976). SEQ ID NO: 59 is a wild-type gene sequence encoding PICALM protein, and is shown below:
As used herein, the term “PILRA” refers to the gene encoding Paired Immunoglobin Like Type 2 Receptor Alpha. The terms “PILRA” and “Paired Immunoglobin Like Type 2 Receptor Alpha” include wild-type forms of the PILRA gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type PILRA. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type PILRA nucleic acid sequence (e.g., SEQ ID NO: 60, NCBI Reference Sequence: NM_013439.2). SEQ ID NO: 60 is a wild-type gene sequence encoding PILRA protein, and is shown below:
As used herein, the term “PLCG2” refers to the gene encoding 1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2. The terms “PLCG2” and “1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-2” include wild-type forms of the PLCG2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type PLCG2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type PLCG2 nucleic acid sequence (e.g., SEQ ID NO: 61, ENA accession number M37238). SEQ ID NO: 61 is a wild-type gene sequence encoding PLCG2 protein, and is shown below:
As used herein, the term “PTK2B” refers to the gene encoding Protein-tyrosine kinase 2-beta. The terms “PTK2B” and “Protein-tyrosine kinase 2-beta” include wild-type forms of the PTK2B gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type PTK2B. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type PTK2B nucleic acid sequence (e.g., SEQ ID NO: 62, ENA accession number U33284). SEQ ID NO: 62 is a wild-type gene sequence encoding PTK2B protein, and is shown below:
As used herein, the term “SCIMP” refers to the gene encoding SLP Adaptor and CSK Interacting Membrane Protein. The terms “SCIMP” and “SLP Adaptor and CSK Interacting Membrane Protein” include wild-type forms of the SCIMP gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SCIMP. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SCIMP nucleic acid sequence (e.g., SEQ ID NO: 63, NCBI Reference Sequence: NM_207103.3). SEQ ID NO: 63 is a wild-type gene sequence encoding SCIMP protein, and is shown below:
As used herein, the term “SLC24A4” refers to the gene encoding Solute Carrier Family 24 Member 4. The terms “SLC24A4” and “Solute Carrier Family 24 Member 4” include wild-type forms of the SLC24A4 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SLC24A4. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SLC24A4 nucleic acid sequence (e.g., SEQ ID NO: 64, NCBI Reference Sequence: NM_153646.3). SEQ ID NO: 64 is a wild-type gene sequence encoding SLC24A4 protein, and is shown below:
As used herein, the term “SORL1” refers to the gene encoding Sortilin-related receptor. The terms “SORL1” and “Sortilin-related receptor” include wild-type forms of the SORL1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SORL1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SORL1 nucleic acid sequence (e.g., SEQ ID NO: 65, ENA accession number Y08110). SEQ ID NO: 65 is a wild-type gene sequence encoding SORL1 protein, and is shown below:
As used herein, the term “SPI1” refers to the gene encoding Transcription factor PU.1. The terms “SPI1” and “Transcription factor PU.1” include wild-type forms of the SPI1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SPI1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SPI1 nucleic acid sequence (e.g., SEQ ID NO: 66, ENA accession number X52056). SEQ ID NO: 66 is a wild-type gene sequence encoding SPI1 protein, and is shown below:
As used herein, the term “SPP1” refers to the gene encoding Secreted Phosphoprotein 1. The terms “SPP1” and “Secreted Phosphoprotein 1” include wild-type forms of the SPP1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SPP1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SPP1 nucleic acid sequence (e.g., SEQ ID NO: 67, NCBI Reference Sequence: NM_001040058.1). SEQ ID NO: 67 is a wild-type gene sequence encoding SPP1 protein, and is shown below:
As used herein, the term “SPPL2A” refers to the gene encoding Signal Peptide Peptidase Like 2A. The terms “SPPL2A” and “Signal Peptide Peptidase Like 2A” include wild-type forms of the SPPL2A gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type SPPL2A. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type SPPL2A nucleic acid sequence (e.g., SEQ ID NO: 68, NCBI Reference Sequence: NM_001040058.1). SEQ ID NO: 68 is a wild-type gene sequence encoding SPPL2A protein, and is shown below:
As used herein, the term “TBK1” refers to the gene encoding Serine/threonine-protein kinase TBK1. The terms “TBK1” and “Serine/threonine-protein kinase TBK1” include wild-type forms of the TBK1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type TBK1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type TBK1 nucleic acid sequence (e.g., SEQ ID NO: 69, ENA accession number AF191838). SEQ ID NO: 69 is a wild-type gene sequence encoding TBK1 protein, and is shown below:
As used herein, the term “TNF” refers to the gene encoding Tumor necrosis factor. The terms “TNF” and “Tumor necrosis factor” include wild-type forms of the TNF gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type TNF. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type TNF nucleic acid sequence (e.g., SEQ ID NO: 70, ENA accession number X01394). SEQ ID NO: 70 is a wild-type gene sequence encoding TNF protein, and is shown below:
As used herein, the term “TREM2” refers to the gene encoding Triggering receptor expressed on myeloid cells 2. The terms “TREM2” and “Triggering receptor expressed on myeloid cells 2” include wild-type forms of the TREM2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type TREM2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type TREM2 nucleic acid sequence (e.g., SEQ ID NO: 71, ENA accession number AF213457). SEQ ID NO: 71 is a wild-type gene sequence encoding TREM2 protein, and is shown below:
As used herein, the term “TREML2” refers to the gene encoding Triggering Receptor Expressed on Myeloid Cells Like 2. The terms “TREML2” and “Triggering Receptor Expressed on Myeloid Cells Like 2” include wild-type forms of the TREML2 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type TREML2. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type TREML2 nucleic acid sequence (e.g., SEQ ID NO: 72, NCBI Reference Sequence: NM_024807.3). SEQ ID NO: 72 is a wild-type gene sequence encoding TREML2 protein, and is shown below:
As used herein, the term “TYROBP” refers to the gene encoding TYRO protein tyrosine kinase-binding protein. The terms “TYROBP” and “TYRO protein tyrosine kinase-binding protein” include wild-type forms of the TYROBP gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type TYROBP. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type TYROBP nucleic acid sequence (e.g., SEQ ID NO: 73, ENA accession number AF019562). SEQ ID NO: 73 is a wild-type gene sequence encoding TYROBP protein, and is shown below:
As used herein, the term “ZCWPW1” refers to the gene encoding Zinc finger CW-type PWWP domain protein 1. The terms “ZCWPW1” and “Zinc finger CW-type PWWP domain protein 1” include wild-type forms of the ZCWPW1 gene, as well as variants (e.g., splice variants and polymorphisms) of wild-type ZCWPW1. Examples of such variants are nucleic acids having at least 70% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identity, or more) to a wild-type ZCWPW1 nucleic acid sequence (e.g., SEQ ID NO: 74, ENA accession number AL136735). SEQ ID NO: 74 is a wild-type gene sequence encoding ZCWPW1 protein, and is shown below:
The present invention provides new forms of siRNA, including single- and double-stranded short interfering RNA (ds-siRNA), and methods for their use in treating a patient in need of microglial gene silencing (e.g., a patient having dysregulated microglial gene expression, such as a patient with, e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, frontotemporal dementia, Huntington's disease, multiple sclerosis, or progressive supranuclear palsy). The branched siRNA in the present invention has shown a surprising ability to permeate the cell. The branched compositions described herein may employ a variety of modifications known and previously unknown in the art. The siRNA of the invention may contain an antisense strand including a region that is represented by Formula IX:
Z-((A-P-)n(B-P-)m)q; (IX)
wherein Z is a 5′ phosphorus stabilizing moiety; each A is, independently, a 2′-modified-ribonucleoside of a first type; each B is, independently, a 2′-modified-ribonucleoside of a second type; each P is, independently, an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5; m is an integer from 1 to 5; and q is an integer between 1 and 15. The embodiments of each part of Formula IX and the methods of use for the molecules Formula IX represents are described herein.
In some embodiments, the siRNA of the invention may have a sense strand represented by Formula X:
Y-((A-P-)n(B-P-)m)qL-((B-P-)m(A-P-)n)q; (X)
wherein Y is a hydrophobic moiety (e.g., cholesterol, vitamin D, or tocopherol); Lisa linker; each A is, independently, a 2′-modified-ribonucleoside of a first type; each B is, independently, a 2′-modified-ribonucleoside of a second type; each P is, independently, an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5; m is an integer from 1 to 5; and q is an integer between 1 and 15. The embodiments of each part of Formula X and the methods of use for the molecules Formula X represents are described herein.
siRNA Structure
The simplest siRNAs consist of a ribonucleic acid comprising a single- or double-stranded structure, formed by a first strand, and in the case of a double-stranded siRNA, a second strand. The first strand comprises a stretch of contiguous nucleotides that is at least partially complementary to a target nucleic acid. The second strand also comprises a stretch of contiguous nucleotides where the second stretch is at least partially identical to a target nucleic acid. The first strand and said second strand may be hybridized to each other to form a double-stranded structure. The hybridization typically occurs by Watson Crick base pairing.
Depending on the sequence of the first and second strand, the hybridization or base pairing is not necessarily complete or perfect, which means that the first and second strand are not 100% base-paired due to mismatches. One or more mismatches may also be present within the duplex without necessarily impacting the siRNA activity.
The first strand contains a stretch of contiguous nucleotides which is essentially complementary to a target nucleic acid. Typically, the target nucleic acid sequence is, in accordance with the mode of action of interfering ribonucleic acids, a single-stranded RNA, preferably an mRNA. Such hybridization occurs most likely through Watson Crick base pairing but is not necessarily limited thereto. The extent to which the first strand has a complementary stretch of contiguous nucleotides to a target nucleic acid sequence can be between 80% and 100%, e.g., 80%, 85%, 90%, 95%, or 100% complementary.
siRNAs described herein may employ modifications to the nucleobase, phosphate backbone, ribose core, 5′- and 3′-ends, and branching, wherein multiple strands of siRNA may be covalently linked.
Length of siRNA Molecules
It is within the scope of the invention that any length, known and previously unknown in the art, may be employed for the current invention. As described herein, potential lengths for an antisense strand of the branched siRNA of the present invention is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides). In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.
In some embodiments, the sense strand of the branched siRNA of the present invention is between 12 and 30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or 14 and 18 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, or 18 nucleotides). In some embodiments, the sense strand is 16 nucleotides. In some embodiments, the sense strand is 17 nucleotides. In some embodiments, the sense strand is 18 nucleotides. In some embodiments, the sense strand is 19 nucleotides. In some embodiments, the sense strand is 20 nucleotides. In some embodiments, the sense strand is 21 nucleotides. In some embodiments, the sense strand is 22 nucleotides. In some embodiments, the sense strand is 23 nucleotides. In some embodiments, the sense strand is 24 nucleotides. In some embodiments, the sense strand is 25 nucleotides. In some embodiments, the sense strand is 26 nucleotides. In some embodiments, the sense strand is 27 nucleotides. In some embodiments, the sense strand is 28 nucleotides. In some embodiments, the sense strand is 29 nucleotides. In some embodiments, the sense strand is 30 nucleotides.
2′ Modifications
The present invention includes single- and double-stranded compositions comprising at least one alternating motif. Alternating motifs of the present invention may have the formula ((A-P-)n(B-P-)m) q where A is a nucleoside of a first type, B is a nucleoside of a second type, n is from 1 to 5, m is from 1 to 5, and q is from 1 to 15, and P is an internucleoside linkage. The result may include a regular or irregular pattern of alternating nucleosides of the first and second types. Each of the types of nucleosides may be identical with the exception that at least the 2′-substituent groups are different.
Possible 2′-modifications comprise all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. In some embodiments, the modification includes a 2′-O-methyl (2′-O-Me) modification. Some embodiments use O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3]2, where n and m are from 1 to about 10. Other potential sugar substituent groups include: C1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. In some embodiments, the modification includes 2′ methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE). In some embodiments, the modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylamino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH2OCH2N(CH3)2. Other potential sugar substituent groups include aminopropoxy (—OCH2CH2CH2NH2), allyl (—CH2—CH═CH2), —O-allyl (—O—CH2—CH═CH2) and fluoro (F). 2′-sugar substituent groups may be in the arabino (up) position or ribo (down) position. In some embodiments, the 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligomeric compound, particularly the 3′ position of the sugar on the 3′ terminal nucleoside or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
Nucleobase Modifications
Oligomeric compounds may also include nucleosides or other surrogate or mimetic monomeric subunits that include a nucleobase (often referred to in the art simply as “base” or “heterocyclic base moiety”). The nucleobase is another moiety that has been extensively modified or substituted and such modified and or substituted nucleobases are amenable to the present invention. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases also referred herein as heterocyclic base moieties include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C═C-CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and, Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Oligomeric compounds of the present invention can also include polycyclic heterocyclic compounds in place of one or more heterocyclic base moieties. A number of tricyclic heterocyclic compounds have been previously reported. These compounds are routinely used in antisense applications to increase the binding properties of the modified strand to a target strand.
Representative cytosine analogs that make 3 hydrogen bonds with a guanosine in a second strand include 1,3-diazaphenoxazine-2-one (Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one (Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874), and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998, 39, 8385-8388). Incorporated into oligonucleotides these base modifications were shown to hybridize with complementary guanine and the latter was also shown to hybridize with adenine and to enhance helical thermal stability by extended stacking interactions (also see U.S. patent application entitled “Modified Peptide Nucleic Acids” filed May 24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled “Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser. No. 10/013,295, both of which are herein incorporated by reference in their entirety). Further helix-stabilizing properties have been observed when a cytosine analog/substitute has an aminoethoxy moiety attached to the rigid 1,3-diazaphenoxazine-2-one scaffold (Lin, K.-Y.; Matteucci, M. J. 25 Am. Chem. Soc. 1998, 120, 8531-8532).
Internucleoside Linkage Modifications
Another variable in the design of the present invention are the internucleoside linkages making up the phosphate backbone. Although the natural RNA phosphate backbone may be employed here, derivatives thereof, known and yet unknown in the art, may be used which enhance desirable characteristics of a siRNA. Although not limiting, of particular importance in the present invention is protecting parts, or the whole, of the siRNA from hydrolysis. One example of a modification that decreases the rate of hydrolysis is phosphorothioates. Any portion or the whole of the backbone may contain phosphate substitutions (e.g., phosphorothioates, phosphodiesters, etc.). For instance, the internucleoside linkages may be between 0 and 100% phosphorothioate, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100% 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70% 40 and 60%, 10 and 40%, 20 and 50%, and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphorothioate linkages. Similarly, the internucleoside linkages may be between 0 and 100% phosphodiester linkages, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100% 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40 and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphodiester linkages.
Specific examples of some potential oligomeric compounds useful in this invention include oligonucleotides containing modified e.g. non-naturally occurring internucleoside linkages. As defined in this specification, oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom and internucleoside linkages that do not have a phosphorus atom. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In the C. elegans system, modification of the internucleotide linkage (phosphorothioate) did not significantly interfere with RNAi activity. Based on this observation, it is suggested that some compositions of the invention can also have one or more modified internucleoside linkages. A preferred phosphorus containing modified internucleoside linkage is the phosphorothioate internucleoside linkage. In some embodiments, the modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. In some embodiments, the modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.
siRNA Patterning
Nucleosides used in the invention tolerate a range of modifications in the nucleobase and sugar. A complete siRNA, single-stranded or double-stranded, may have 1, 2, 3, 4, 5, or more different nucleosides that each appear in the siRNA strand or strands once or more. The nucleosides may appear in a repeating pattern (e.g., alternating between two modified nucleosides) or may be a strand of one type of nucleoside with substitutions of a second type of nucleoside. Similarly, internucleoside linkages may be of one or more type appearing in a single- or double-stranded siRNA in a repeating pattern (e.g., alternating between two internucleoside linkages) or may be a strand of one type of internucleoside linkage with substitutions of a second type of internucleoside linkage. Though the siRNAs of the invention tolerate a range of substitution patterns, the following exemplify some preferred patterns in which A and B represent nucleosides of two types, and T and P represent internucleoside linkages of two types:
In some embodiments, T represents phosphorothioate, and P represents phosphodiester.
In some embodiments, the siRNA molecule of the disclosure features any one of the siRNA nucleotide modification patterns and/or internucleoside linkage modification patterns described in International Patent Application Publication Nos. WO 2016/161388 and WO 2020/041769, the disclosures of which are incorporated in their entirety herein. In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula A-I, wherein Formula A-I is, in the 5′-to-3′ direction
A-B-(A′)j-C-P2-D-P1-(C′-P1)k-C′ Formula A-I;
wherein A is represented by the formula C-P1-D-P1; each A′ is represented by the formula C-P2-D-P2; B is represented by the formula C-P2-D-P2-D-P2-D-P2; each C is a 2′-O-methyl (2′-O-Me) ribonucleoside; each C′, independently, is a 2′-O-Me ribonucleoside or a 2′-fluoro (2′-F) ribonucleoside; each D is a 2′-F ribonucleoside; each P1 is a phosphorothioate internucleoside linkage; each P2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.
In some embodiments, the antisense strand includes a structure represented by Formula A1, wherein Formula A1 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula A-II, wherein Formula A-II is, in the 5′-to-3′ direction:
A-B-(A),-C-P2-D-P1-(C-P1)k-C′ Formula A-II;
wherein A is represented by the formula C-P1-D-P1; each A′ is represented by the formula C-P2-D-P2; B is represented by the formula C-P2-D-P2-D-P2-D-P2; each C is a 2′-O-methyl (2′-O-Me) ribonucleoside; each C′, independently, is a 2′-O-Me ribonucleoside or a 2′-fluoro (2′-F) ribonucleoside; each D is a 2′-F ribonucleoside; each P1 is a phosphorothioate internucleoside linkage; each P2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.
In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A2, wherein Formula A2 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-A-S-A Formula A2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S-III, wherein Formula S-III is, in the 5′-to-3′ direction:
E-(A′)m-F Formula S-III;
wherein E is represented by the formula (C-P1)2; F is represented by the formula (C-P2)3-D-P1-C-P1-C, (C-P2)3-D-P2-C-P2-C, (C-P2)3-D-P1-C-P1-D, or (C-P2)3-D-P2-C-P2-D; A′, C, D, P1, and P2 are as defined in Formula I; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S1, wherein Formula S1 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-A Formula S1;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S2, wherein Formula S2 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-A Formula S2;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S3, wherein Formula S3 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-S-A-S-B Formula S3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S4, wherein Formula S4 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A-O-A-O-B-O-A-O-B Formula S4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula A-IV, wherein Formula A-IV is, in the 5′-to-3′ direction:
A-(A)j-C-P2-B-(C-P1)k-C′ Formula A-IV;
wherein A is represented by the formula C-P1-D-P1; each A′ is represented by the formula C-P2-D-P2; B is represented by the formula D-P1-C-P1-D-P1; each C is a 2′-O-Me ribonucleoside; each C′, independently, is a 2′-O-Me ribonucleoside or a 2′-F ribonucleoside; each D is a 2′-F ribonucleoside; each P1 is a phosphorothioate internucleoside linkage; each P2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 6. In some embodiments, k is 4. In some embodiments, j is 6 and k is 4. The antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid. In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A3, wherein Formula A3 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A-S-A Formula A3;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA of the disclosure may have a sense strand represented by Formula S-V, wherein Formula S-V is, in the 5′-to-3′ direction:
E-(A′)m-C-P2-F Formula S-V;
wherein E is represented by the formula (C-P1)2; F is represented by the formula D-P1-C-P1-C, D-P2-C-P2-C, D-P′-C-P′-D, or D-P2-C-P2-D; A′, C, D, P1, and P2 are as defined in Formula IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S5, wherein Formula S5 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A Formula S5;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S6, wherein Formula S6 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-A Formula S6;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S7, wherein Formula S7 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-B Formula S7;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S8, wherein Formula S8 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B Formula S8;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region represented by Formula A-VI, wherein Formula A-VI is, in the 5′-to-3′ direction:
A-Bj-E-Bk-E-F-Gl-D-P1-C′ Formula A-VI;
wherein A is represented by the formula C-P1-D-P1; each B is represented by the formula C-P2; each C is a 2′-O-Me ribonucleoside; each C′, independently, is a 2′-O-Me ribonucleoside or a 2′-F ribonucleoside; each D is a 2′-F ribonucleoside; each E is represented by the formula D-P2-C-P2; F is represented by the formula D-P1-C-P1; each G is represented by the formula C-P1; each P1 is a phosphorothioate internucleoside linkage; each P2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and I is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 3. In some embodiments, k is 6. In some embodiments, I is 2. In some embodiments, j is 3, k is 6, and I is 2. The antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.
In some embodiments of the disclosure, the antisense strand includes a structure represented by Formula A4, wherein Formula A4 is, in the 5′-to-3′ direction:
A-S-B-S-A-O-A-O-A-O-B-O-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA may contain a sense strand including a region represented by Formula S-VII, wherein Formula S-VII is, in the 5′-to-3′ direction:
H-Bm-In-A′-Bo-H-C Formula S-VII;
wherein A′ is represented by the formula C-P2-D-P2; each H is represented by the formula (C-P1)2; each I is represented by the formula (D-P2); B, C, D, P1, and P2 are as defined in Formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.
In some embodiments of the disclosure, the sense strand includes a structure represented by Formula S9, wherein Formula S9 is, in the 5′-to-3′ direction:
A-S-A-S-A-O-A-O-A-O-B-O-B-O-B-O-A-O-B-O-A-O-A-O-A-O-A-S-A-S-A Formula S9;
wherein A represents a 2′-O-Me ribonucleoside, B represents a 2′-F ribonucleoside, 0 represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.
In some embodiments of the disclosure, the siRNA may contain an antisense strand including a region that is represented by Formula VIII:
5′ Phosphorus Stabilizing Moiety
To further protect the siRNA from degradation a 5′-phosphorus stabilizing moiety may be employed. A 5′-phosphorus stabilizing moiety replaces the 5′-phosphate to prevent hydrolysis of the phosphate. Hydrolysis of the 5′-phosphate prevents binding to RISC, a necessary step in gene silencing. Any replacement for phosphate that does not impede binding to RISC is contemplated in this disclosure. In some embodiments, the replacement for the 5′-phosphate is also stable to in vivo hydrolysis. Each siRNA strand may independently and optionally employ any suitable 5′-phosphorus stabilizing moiety.
Some exemplary endcaps are demonstrated in Formula I-VIII. Nuc in Formula I-VIII represents a nucleobase or nucleobase derivative or replacement as described herein. X in Formula I-VIII represents a 2′-modification as described herein. Some embodiments employ hydroxy as in Formula I, phosphate as in Formula II, vinylphosphonates as in Formula III, and VI, 5′-methylsubstitued phosphates as in Formula IV, VI, and VIII, or methylenephosphonates as in Formula VII, vinyl 5′-vinylphosphonate as a 5′-phosphorus stabilizing moiety as demonstrated in Formula III.
siRNA Branching
Branching of the siRNA molecules is a key feature in the present invention. The siRNA molecule may not be branched, or may be dibranched, tribranched, or tetrabranched, connected through a linker. Each main branch may be further branched to allow for 2, 3, 4, 5, 6, 7, or 8 separate RNA single- or double-strands. The branch points on the linker may stem from the same atom, or separate atoms along the linker. Some exemplary embodiments are listed in Table 1, where L represent a linker, and X represents any atom suitable to the siRNA molecule branch points:
Linkers
Multiple strands of siRNA described herein may be covalently attached by way of a linker. The effect of this branching improves, inter alia, cell permeability allowing better access into microglia in the CNS. Any linking moiety may be employed which is not incompatible with the siRNAs of the present invention. Exemplary linkers include ethylene glycol chains of 2 to 10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others. In some embodiments, any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. In some embodiments, the linker is a poly-ethylene glycol (PEG) linker. The PEG linkers suitable for use with the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEGs, linear forked PEGs, or branched forked PEGs.
PEG linkers of various weights may be used with the disclosed compositions and methods. For example, the PEG linker may have a weight that is between 5 and 500 Daltons. In some embodiments, a PEG linker having a weight that is between 500 and 1,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 1,000 and 10,000 Dalton may be used. In some embodiments, a PEG linker having a weight that is between 200 and 20,000 Dalton may be used. In some embodiments, the linker is covalently attached to a sense strand of the siRNA. In some embodiments, the linker is covalently attached to an antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene linker (TEG).
In some embodiments, the linker is an alkyl chain linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a RNA linker. In some embodiments, the linker is a DNA linker.
Linkers may covalently link 2, 3, 4, or 5 unique siRNA strands. The linker may covalently bind to any part of the siRNA oligomer. In some embodiments, the linker attaches to the 3′ end of nucleosides of each siRNA strand. In some embodiments, the linker attaches to the 5′ end of nucleosides of each siRNA strand. In some embodiments, the linker attaches to a nucleoside of an siRNA strand (e.g., sense or antisense strand) by way of a covalent bond-forming moiety. In some embodiments, the covalent-bond-forming moiety is selected from the group consisting of an alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivative (e.g., phosphorothioate, phosphoramidate, etc.).
In some embodiments, the linker has a structure of Formula L1, as is shown below:
In some embodiments, the linker has a structure of Formula L2, as is shown below:
In some embodiments, the linker has a structure of Formula L3, as is shown below:
In some embodiments, the linker has a structure of Formula L4, as is shown below:
In some embodiments, the linker has a structure of Formula L5, as is shown below:
In some embodiments, the linker has a structure of Formula L6, as is shown below:
In some embodiments, the linker has a structure of Formula L7, as is shown below:
In some embodiments, the linker has a structure of Formula L8, as is shown below:
In some embodiments, the linker has a structure of Formula L9, as is shown below:
In some embodiments, the selection of a linker for use with one or more of the branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, such that, e.g., desirable hydrophobicity is achieved for the one or more branched siRNA molecules of the disclosure. For example, a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule as compared to a branched siRNA molecule having a less hydrophobic linker or a hydrophilic linker.
The invention provides methods of treating a subject in need of gene silencing. The gene silencing may be performed in order to silence defective or overactive microglial genes, silence negative regulators of microglial genes with reduced expression and/or activity, silence wild type microglial genes with an activating role in a pathway(s) that increases expression and/or activity of a disease driver gene, silence splice isoforms of a microglial gene(s) that, when selectively knocked down, may elevate total expression and/or activity of the gene(s), among other reasons, so long as the goal is to restore genetic and biochemical pathway activity from a disease state towards a healthy state. The active compound can be administered in any suitable dose. The actual dosage amount of a composition of the present invention administered to a patient can be determined by physical and physiological factors such as body weight, severity of condition, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. Administration may occur any suitable number of times per day, and for as long as necessary. Subjects may be adult or pediatric humans, with or without comorbid diseases.
The methods of the invention feature delivering a branched siRNA molecule to a microglial cell in a subject in need of microglial gene silencing. Subjects in need of microglial gene silencing may be suffering from neurodegenerative diseases in which neuroinflammation is a primary component of the disease pathology (e.g., Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, frontotemporal dementia, Huntington's disease, multiple sclerosis, or progressive supranuclear palsy).
Alzheimer's Disease
Alzheimer's disease (AD) is a late-onset neurodegenerative disorder responsible for the majority of dementia cases in the elderly. AD patients suffer from a progressive cognitive decline characterized by symptoms including an insidious loss of short- and long-term memory, attention deficits, language-specific problems, disorientation, impulse control, social withdrawal, anhedonia, and other symptoms. Distinguishing neuropathological features of AD are extracellular aggregates of amyloid-6 plaques and neurofibrillary tangles composed of hyperphosphorylated microtubule-associated tau proteins. Accumulation of these aggregates is associated with neuronal loss and atrophy in a number of brain regions including the frontal, temporal, and parietal lobes of the cerebral cortex as well as subcortical structures like the basal forebrain cholinergic system and the locus coeruleus within the brainstem. AD is also associated with increased neuroinflammation characterized by reactive gliosis and elevated levels of pro-inflammatory cytokines.
Amyotrophic Lateral Sclerosis
Amyotrophic Lateral Sclerosis (ALS) is a fast-progressing fatal neurodegenerative disease that affects motor neurons both in the brain and spinal cord, consequently resulting in paralysis of voluntary muscles at later stages of disease. ALS affects about 6 persons per 100,000 people and typically leads to death within 3 to 5 years after the onset of symptoms, with no cure yet available. ALS leads to muscle weakness, atrophy, and muscle spasms as a result of degeneration of upper and lower motor neurons. Cognitive and behavioral dysfunction (e.g., language dysfunction, executive dysfunction, social cognition, and verbal memory dysfunction), and frontotemporal dementia are all possible symptoms of ALS.
Parkinson's Disease
PD is a progressive disorder that affects movement, and it is recognized as the second most common neurodegenerative disease after Alzheimer's disease. Common symptoms of PD include resting tremor, rigidity, and bradykinesia, and non-motor symptoms, such as depression, constipation, pain, sleep disorders, genitourinary problems, cognitive decline, and olfactory dysfunction, are also increasingly being associated with PD. A key feature of PD is the death of dopaminergic neurons in the substantia nigra pars compacta, and, for that reason, most current treatments for PD focus on increasing dopamine. Another well-known neuropathological hallmark of PD is the presence of Lewy bodies containing α-synuclein in brain regions affected by PD, which are thought to contribute to the disease.
PD is thought to result from a combination of genetic and environmental risk factors. There is no single gene responsible for all Parkinson's disease cases, and the vast majority of PD cases seem to be sporadic and not directly inherited. Mutations in the genes encoding parkin, PTEN-induced putative kinase 1 (PINK1), leucine-rich repeat kinase 2 (LRRK2), and Parkinsonism-associated deglycase (DJ-1) have been found to be associated with PD, but they represent only a small subset of the total number of PD cases. Occupational exposure to some pesticides and herbicides has also been proposed as a risk factor for PD. The synthetic neurotoxin MPTP can cause Parkinsonism, but its use is extremely rare.
Frontotemporal Dementia
Frontotemporal dementia (FTD; also known as frontotemporal lobar degeneration (FTLD)) is a clinical syndrome characterized by progressive neurodegeneration in the frontal and temporal lobes of the cerebral cortex. The manifestation of FTD is complex and heterogeneous, and may present as one of three clinically distinct variants including: 1) behavioral-variant frontotemporal dementia (BVFTD), characterized by changes in behavior and personality, apathy, social withdrawal, perseverative behaviors, attentional deficits, disinhibition, and a pronounced degeneration of the frontal lobe; 2) semantic dementia (SD), characterized by fluent, anomic aphasia, progressive loss of semantic knowledge of words, objects, and concepts and a pronounced degeneration of the anterior temporal lobes. Furthermore, SD variant of FTD exhibit a flat affect, social deficits, perseverative behaviors, and disinhibition; or 3) progressive nonfluent aphasia; characterized by motor deficits in speech production, reduced language expression, and pronounced degeneration of the perisylvian cortex. Neuronal loss in brains of FTD patients is associated with one of three distinct neuropathologies: 1) the presence of tau-positive neuronal and glial inclusions; 2) ubiquitin (ub)-positive and TAR DNA-binding protein 43 (TDP43)-positive, but tau-negative inclusions; or 3) ub and fused in sarcoma (FUS)-positive, but tau and TDP-43-negative inclusions. These neuropathologies are considered to be important in the etiology of FTD.
Nearly half of FTD patients have a first-degree family member with dementia, ALS, or Parkinson's disease, suggesting a strong genetic link to the cause of the disease. A number of mutations in chromosome 17q21 have been linked to FTD presentation.
Huntington's Disease
Huntington's Disease (HD) is an example of a trinucleotide repeat expansion disorder. This class of disorders involve the localized expansion of unstable repeats of sets of three nucleotides and can result in loss of function of a gene in which the expanded repeat is found, a gain of toxic function, or both. Trinucleotide repeats can be located in any part of the gene, including coding and non-coding regions. Repeats located within coding regions typically involve a repeated glutamine encoding triplet (CAG) or an alanine encoding triplet (CGA). Expanded repeat regions within non-coding sequences can lead to aberrant expression of the gene, while expanded repeats within coding regions (also known as codon reiteration disorders) may cause protein mis-folding and aggregation. Typically, regions of wild-type genes contain a variable number of repeat sequences in the normal population, but in the afflicted populations, the number of repeats can increase from a doubling to a log order increase in the number of repeats. In HD, repeats are inserted within the N-terminal coding region of the large cytosolic protein Huntingtin (Htt). Normal Htt alleles contain 15-20 CAG repeats, while alleles containing 35 or more repeats can be considered to confer a risk for developing the disease. Alleles containing 36-39 repeats are considered incompletely penetrant, and those individuals harboring those alleles may or may not develop the disease (or exhibit delayed presentation later in life), while alleles containing 40 repeats or more are considered completely penetrant. Those individuals with juvenile onset HD (<21 years of age) are often found to have 60 or more CAG repeats.
Multiple Sclerosis
Multiple sclerosis (MS) is the most common demyelinating disease of the CNS affecting young adults (disease onset between 20 to 40 years of age) and is the third leading cause for disability after trauma and rheumatic diseases in the US.
MS patients present with destruction of myelin, death of oligodendrocytes, and axonal loss. The main pathologic finding in MS is the presence of infiltrating mononuclear cells, predominantly T lymphocytes and macrophages, which breach the blood brain barrier and induce active inflammation within the CNS. The neurological symptoms that characterize MS include complete or partial vision loss, diplopia, sensory symptoms, motor weakness that can progress to complete paralysis, bladder dysfunction, and cognitive deficits. The associated inflammatory foci lead to myelin destruction, plaques of demyelination, gliosis, and axonal loss within the brain and spinal cord and are the primary drivers of the clinical manifestations of neurological disability.
The etiology of MS is not fully understood. The disease develops in genetically predisposed subjects exposed to yet undefined environmental factors and the pathogenesis involves autoimmune mechanisms associated with autoreactive T cells against myelin antigens. It is well established that not one dominant gene determines genetic susceptibility to develop MS, but rather many genes, each with different influence, are involved. The detailed molecular mechanisms underlying MS etiology are still to be elucidated.
Progressive Supranuclear Palsy
Progressive supranuclear palsy (PSP), a progressive and fatal tauopathy, represents ˜10% of all Parkinsonian cases in the US. PSP patients have a variety of motor disorders, including postural instability, falls, abnormalities in gait, bradykinesia, vertical gaze paralysis, pseudobulbar paralysis, and axial stiffness without limb stiffness, in addition to cognitive impairments such as apathy, loss of executive function, and reduced fluency. Neuropathology of PSP is characterized by an accumulation of tau protein, which is associated with abnormal intracellular microtubules, resulting in insoluble filament deposits. The neuropathological presentation of PSP neurodegeneration is located in the subcortical regions, including substantia nigra, globus pallidus, and subthalamic nucleus. PSP neurodegeneration is characterized by the destruction of tissues and cytokine profiles of activated microglia and astrocytes.
There are currently no disease-modifying treatments for PSP. The current standard of care is palliative. Patients in the advanced stages of the disease often have feeding tubes inserted to avoid choking hazards and to provide nutrition. Although therapies are available to decrease some symptoms of PSP, none protect the brain from neurodegeneration. Current medications to treat symptoms of PSP include dopamine agonists, tricyclic antidepressants, methysergide, onabotulinumtoxin A (to treat muscle stiffness in the face). However, as the disease progresses and symptoms worsen, medications may fail to adequately decrease symptoms.
The methods of the invention feature delivering a branched siRNA molecule to a microglial cell in a subject in need of microglial gene silencing. Patients in need of microglial gene silencing may have dysregulated expression and/or activity of a gene selected from the group consisting of ABCA7, ABI3, ADAM10, APOC1, APOE, AXL, BIN1, C1QA, C3, C9ORF72, CASS4, CCL5, CD2AP, CD33, CD68, CLPTM1, CLU, CR1, CSF1, CST7, CTSB, CTSD, CTSL, CXCL10, CXCL13, DSG2, ECHDC3, EPHA1, FABP5, FERMT2, FTH1, GNAS, GRN, HBEGF, HLA-DRB1, HLA-DRB5, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IGF1, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, ITGAX, LILRB4, LPL, MEF2C, MMP12, MS4A4A, MS4A6A, NLRP3, NME8, NOS2, PICALM, PILRA, PLCG2, PTK2B, SCIMP, SLC24A4, SORL1, SPI1, SPP1, SPPL2A, TBK1, TNF, TREM2, TREML2, TYROBP, and ZCVVPW1 gene.
In some embodiments, the patient in need of microglial gene silencing may require silencing of any one of the genes selected from the group consisting of APOE, BIN1, C1QA, C3, C9ORF72, CCL5, CD33, CLU/APOJ, CR1, CXCL10, CXCL13, IFIT1, IFIT3, IFITM3, IFNAR1, IFNAR2, IL10RA, IL1A, IL1B, IL1RAP, INPP5D, ITGAM, MEF2C, MMP12, NLRP3, NOS2, PILRA, PLCG2, PTK2B, SLC24A4, TBK1, and TNF.
The branched siRNA molecules in the present invention can be formulated into a pharmaceutical composition for administration to a subject in a biologically compatible form suitable for administration in vivo. Accordingly, in one aspect, the present invention provides a pharmaceutical composition containing a branched siRNA in admixture with a suitable diluent, carrier, or excipient. The siRNA can be administered, for example, orally or by intravenous injection.
Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington: The Science and Practice of Pharmacy (2012, 22 nd ed.) and in The United States Pharmacopeia: The National Formulary (2015, USP 38 NF 33).
Under ordinary conditions of storage and use, a pharmaceutical composition may contain a preservative, e.g., to prevent the growth of microorganisms. Pharmaceutical compositions may include sterile aqueous solutions, dispersions, or powders, e.g., for the extemporaneous preparation of sterile solutions or dispersions. In all cases the form may be sterilized using techniques known in the art and may be fluidized to the extent that may be easily administered to a subject in need of treatment.
A pharmaceutical composition may be administered to a subject, e.g., a human subject, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which may be determined by the solubility and/or chemical nature of the compound, chosen route of administration, and standard pharmaceutical practice.
A physician having ordinary skill in the art can readily determine an effective amount of siRNA for administration to a mammalian subject (e.g., a human) in need thereof. For example, a physician could start prescribing doses of a siRNA of the invention at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician may begin a treatment regimen by administering a siRNA at a high dose and subsequently administer progressively lower doses until a therapeutic effect is achieved (e.g., a reduction in expression of a target gene sequence). In general, a suitable daily dose of a siRNA of the invention will be an amount of the siRNA which is the lowest dose effective to produce a therapeutic effect. A single-strand or double-strand siRNA of the invention may be administered by injection, e.g., intrathecally, intracerebroventricularly, or intrastriatally. A daily dose of a therapeutic composition of a siRNA of the invention may be administered as a single dose or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally, in unit dosage forms. While it is possible for a siRNA of the invention to be administered alone, it may also be administered as a pharmaceutical formulation in combination with excipients, carriers, and optionally, additional therapeutic agents.
The method of the invention contemplates any route of administration tolerated by the therapeutic composition. Some embodiments of the method include injection intrathecally, intracerebroventricularly, or intrastriatally.
Intrathecal injection is the direct injection into the spinal column or subarachnoid space. By injecting directly into the CSF of the spinal column the siRNA molecule of the invention has direct access to microglia in the spinal column and a route to access the microglia in the brain by bypassing the blood brain barrier.
Intracerebroventricular (ICV) injection is a method to directly inject into the CSF of the cerebral ventricles. Similar to intrathecal injection, ICV is a method of injection which bypasses the blood brain barrier. Using ICV allows the advantage of access to the microglia of the brain and spinal column without the danger of the therapeutic being degraded in the blood.
Intrastriatal injection is the direct injection into the striatum, or corpus striatum. The striatum is an area in the subcortical basal ganglia in the brain. Injecting into the striatum bypasses the blood brain barrier and the pharmacokinetic challenges of injection into the blood stream and allows for direct access to the microglia of the brain and spinal column.
The experiments described in this example were conducted to assess the ability of branched siRNA molecules to permeate the central nervous system and internalize within microglial cells. To this end, a branched siRNA compound targeting the huntingtin (HTT) gene and conjugated to a fluorescent dye (Cy3) was first injected into the cerebrospinal fluid via intrathecal injection into non-human primates (NHP; cynomolgus macaque). Central nervous system tissue samples were later obtained from the animals. To assess the extent to which the branched siRNA molecules were internalized by microglial cells, the tissue samples were stained using fluorescent-labeled antibodies that are specific for markers expressed in certain cell types (e.g., microglia). Fluorescence microscopy was then utilized to determine the degree of colocalization of the Cy3-labeled branched siRNA molecules and antibody-labeled microglial cells, which served as an indicator of microglial uptake. These experiments, and their results, are described in further detail below:
Paraffin embedded CNS tissue slides were tested. A dose of fluorescent labeled branched siRNA was administered to a NHP (cynomolgus macaque) via intrathecal injection. 48 hours after injection a distribution study was done. The control was an uninjected NHP. NHP tissues for imaging were post-fixed for 48-72 hours in 4% PFA at 5±3° C., and then transferred to PBS. All tissues were paraffin-embedded and sliced into 4 μm sections and mounted on slides for immunofluorescence staining. Subsequently, sections were deparaffinized and subjected to antigen retrieval. Samples were deparaffanized by two changes of xylene, 5 minutes each, then 50% xylene+50% ethanol (100%) for 5 minutes. Samples were hydrated by two changes of 100% ethanol for 3 minutes each, 90%, 80%, 70% and then 50% ethanol for 3 minutes each, followed by distilled water rinse. Antigen retrieval was carried out using 150 mL of Tris-EDTA buffer (pH9), placing the staining dish in a pressure cooker (containing 1200 mL DDH2O) for 10 minutes, allowing the slides to cool to room temperature, followed by section-wise rinsing with H2O and TBST. Sections were blocked with Background Terminator Blocking Reagent and the slides were then incubated with the primary antibody against the microglial-specific gene, Iba-1, for 1.5 hours at room temperature, followed by treatment with a secondary antibody labeled with Alexa Flour 488 (Alexa-488). Alexa-488 was used to visualize Iba-1 antibody. DAPI was used to visualize cell nuclei. Tissues were washed three times for 5 min with TBS-Tween 20. Fluoromount-G was used to place glass coverslips, and slides were left to dry at 4° C. overnight protected from light. Olympus VS200 slide scanner was used to acquire immunofluorescent images of brain and spinal cord (20× objective). Images within each imaging channel were acquired under the same settings for light intensity and exposure times.
Colocalization of DAPI stained nuclei, Alexa-488-labeled Iba-1 antibody, and Cy3-labeled siRNA was observed across all tested brain and spinal cord tissues of cynomolgus macaques, indicating microglial cell penetration and/or uptake of the branched di-siRNA. Control experiments included uninjected NHP control (no Cy3-siRNA), non-specific primary antibody (isotype antibody control), and no secondary antibody (no Alexa Fluor 488 reagent). Robust colocalization was observed in the cortex (
These results demonstrate that the ds-siRNA agents of the present disclosure are capable of being internalized by microglial cells of CNS tissues, including brain and spinal cord, and support the use of such agents for treatment of neurological conditions, such as Alzheimer's disease or amyotrophic lateral sclerosis.
A subject diagnosed with Alzheimer's disease is treated with a dose and frequency determined by a practitioner (e.g., three times daily, twice daily, once daily, once weekly, once monthly, bi-monthly, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or annually). Dosage and frequency are determined based on the subject's height, weight, age, sex, and other disorders.
The branched siRNA is selected by the practitioner for compatibility with the disease and subject. Single- or double-stranded branched siRNA are available for selection. The siRNA chosen has an antisense strand, and in the case of double-stranded siRNA, a sense strand with a sequence and RNA modifications (e.g., natural and non-natural internucleoside linkages, modified sugars, and 5′-phosphorus stabilizing moieties) best suited to the patient and the disease being targeted (e.g., PSM-A-T-B-T-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-T-A-T-B-T-A-T-B-T-A-T-B-T B-T-A-T-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-T-B-T where A and B are different nucleosides, T is phosphorothioate, P is a phosphodiester, and PSM is a 5′-phosphorus stabilizing moiety).
The branched siRNA is delivered by the route best suited the patient and condition (e.g., intrathecally, intracerebroventricularly, or intrastriatally), at a rate tolerable to the patient until the subject has reached a maximum tolerated dose, or until the symptoms of the disease are ameliorated satisfactorily.
A subject diagnosed with Amyotrophic Lateral Sclerosis is treated with a dose and frequency determined by a practitioner (e.g., three times daily, twice daily, once daily, once weekly, once monthly bi-monthly, once every 4 months, once every 5 months, once every 6 months, once every 7 months, once every 8 months, once every 9 months, once every 10 months, once every 11 months, or annually). Dosage and frequency are determined based on the subject's height, weight, age, sex, and other disorders.
The branched siRNA is selected by the practitioner for compatibility with the disease and subject. Single- or double-stranded branched siRNA are available for selection. The siRNA chosen has an antisense strand, and in the case of double-stranded siRNA, a sense strand with a sequence and RNA modifications (e.g., natural and non-natural internucleoside linkages, modified sugars, and 5′-phosphorus stabilizing moieties) best suited to the patient and the disease being targeted (e.g., PSM-A-T-B-T-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-T-A-T-B-T-A-T-B-T-A-T-B-T B-T-A-T-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-P-B-P-A-T-B-T where A and B are different nucleosides, T is phosphorothioate, P is a phosphodiester, and PSM is a 5′-phosphorus stabilizing moiety).
The branched siRNA is delivered by the route best suited the patient and condition (e.g., intrathecally, intracerebroventricularly, or intrastriatally), at a rate tolerable to the patient until the subject has reached a maximum tolerated dose, or until the symptoms of the disease are ameliorated satisfactorily.
Some specific embodiments are listed below. The below enumerated embodiments should not be construed to limit the scope of the invention, rather, the below are presented as some examples of the utility of the invention.
Z-((A-P-)n(B-P-)m)q;
wherein Nuc represents a nucleobase, such as adenine, uracil, guanine, thymine, or cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl), phenyl, benzyl, hydroxy, or hydrogen.
A-T-B-T-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-B-T-A-T-A-T-A-T-A-T-A-T-A-T Formula A1;
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-A-T-A-P-B-P-B-P-B-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-B-T-A-T-A-T-A-T-A-T-A-T-A-T (Formula A2);
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-B-T-A-P-B-P-B-P-B-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-B-T-A-T-A-T-A-T-A-T-A-T-A-T (Formula A3)
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-B-T-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-B-T-A-T-A-T-A-T-A-T-A-T-A-T (Formula A4)
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-B-T-A-P-A-P-A-P-B-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-B-T-A-T-B-T-A-T-A-T-A-T-A-T (Formula A5)
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
Y-((A-P-)n(B-P-)m)qL-((B-P-)m(A-P-)n)q;
A-T-A-T-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-P-A-T-A-T Formula S1;
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-A-T-A-P-A-P-A-P-A-P-B-P-A-P-A-P-B-P-B-P-A-P-A-P-A-T-A-T Formula S2;
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
A-T-A-T-A-P-A-P-A-P-A-P-B-P-A-P-B-P-B-P-B-P-A-P-A-P-A-T-A-T Formula S3;
wherein A represents a 2′-O-methyl ribonucleoside, B represents a 2′-F ribonucleoside, T represents a phosphorothioate internucleoside linkage, and P represents a phosphodiester internucleoside linkage.
Z-((A-P-)n(B-P-)m)q;
wherein Nuc represents a nucleobase, such as adenine, uracil, guanine, thymine, or cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl), phenyl, benzyl, hydroxy, or hydrogen.
Y-((A-P-)n(B-P-)m)qL-((B-P-)m(A-P-)n)q;
Sclerosis.
Various modifications and variations of the described disclosure will be apparent to those skilled in the art without departing from the scope and spirit of the disclosure. Although the disclosure has been described in connection with specific embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled in the art are intended to be within the scope of the disclosure.
Other embodiments are in the claims.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/021789 | 3/24/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63165518 | Mar 2021 | US |
