CORONAVIRUS IRNA COMPOSITIONS AND METHODS OF USE THEREOF

Information

  • Patent Application
  • 20220127615
  • Publication Number
    20220127615
  • Date Filed
    November 10, 2021
    2 years ago
  • Date Published
    April 28, 2022
    2 years ago
Abstract
The present invention relates to RNAi agents, e.g., dsRNA agents, targeting the coronavirus genome. The invention also relates to methods of using such RNAi agents to inhibit expression of a coronavirus genome and to methods of treating or preventing a coronavirus-associated disease in a subject.
Description
SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 29, 2021, is named 121301_12206_SL.txt and is 577,274 bytes in size.


BACKGROUND OF THE INVENTION

Coronaviruses (CoV) are a large family of viruses that cause diseases in mammals and birds. Coronaviruses constitute the subfamily Orthocoronavirinae, in the family Coronaviridae. They are enveloped viruses with a positive-sense single-stranded RNA genome and a nucleocapsid of helical symmetry. The genome size of coronaviruses ranges from approximately 27 to 34 kilobases. The name coronavirus is derived from the Latin corona, meaning “crown” or “halo”, which refers to the characteristic appearance reminiscent of a crown or a solar corona around the virions (virus particles) when viewed under two-dimensional transmission electron microscopy, due to the surface covering in club-shaped protein spikes.


Coronaviruses can cause illness ranging from the common cold to more severe diseases. For example, infections with the human coronavirus strains CoV-229E, CoV-OC43, CoV-NL63 and CoV-HKU1 usually result in mild, self-limiting upper respiratory tract infections, such as a common cold, e.g., runny nose, sneezing, headache, cough, sore throat or fever (Zumla A. et al., Nature Reviews Drug Discovery 15(5): 327-47, 2016; (Cheng V. C., et al., Clin. Microbial. Rev. 20: 660-694, 2007; Chan J. F. et al., Clin. Microbial. Rev. 28: 465-522, 2015). Other infections may result in more severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV), diseases associated with pneumonia, severe acute respiratory syndrome, kidney failure and death.


MERS-CoV and SARS-CoV have received global attention over the past decades owing to their ability to cause community and health-care-associated outbreaks of severe infections in human populations. MERS-CoV is a viral respiratory disease that was first reported in Saudi Arabia in 2012 and has since spread to more than 27 other countries, according to the World Health Organization (de Groot, R. J. et al., J. Virol. 87: 7790-7792, 2013). SARS was first reported in Asia in 2003, and quickly spread to about two dozen countries before being contained after about four months (Lee N. et al., N. Engl. J. Med. 348: 1986-1994, 2003; Peiris J. S. et al., Lancet 36: 1319-1325, 2003). Detailed investigations found that SARS-CoV was transmitted from civet cats to humans and MERS-CoV from dromedary camels to humans (Cheng V. C., et al., Clin. Microbial. Rev. 20: 660-694, 2007; Chan J. F. et al., Clin. Microbial. Rev. 28: 465-522, 2015).


A recent outbreak of respiratory disease caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first identified in Wuhan City, China. This disease, named by the World Health Organization as coronavirus disease 2019 (“COVID-19”), presents a major threat to public health worldwide. As of Feb. 24, 2020, there were more than 79,000 confirmed cases and 2,600 deaths across the world.


Coronaviruses viruses pose major challenges to clinical management because many questions regarding transmission and control remain unanswered. Moreover, there is currently no vaccine to prevent infections by coronavirus, and there are no specific antiviral treatments available or proven to be effective to treat or prevent coronavirus infection in subjects.


Accordingly, there exists an immediate need for therapeutics to treat coronavirus infections.


BRIEF SUMMARY OF THE INVENTION

The present disclosure provides RNAi agent compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA genome and RNA transcipts of coronavirus genes. The coronavirus genome may be within a cell, e.g., a cell within a subject, such as a human. The present disclosure also provides methods of using the RNAi agent compositions of the disclosure for inhibiting the expression of a coronavirus genome or for treating a subject who would benefit from inhibiting or reducing the expression of a coronavirus genome, e.g., a subject having a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV).


Accordingly, in one aspect, the instant disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a coronavirus genome, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.


In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of coronavirus genome in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.


In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of coronavirus genome in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region complementary to part of an mRNA encoding a coronavirus genome (SEQ ID NO:1), wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.


In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of coronavirus genome in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region complementary to part of a reverse complement of an mRNA encoding a coronavirus genome (SEQ ID NO:2), wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.


In yet another aspect, the present invention provides a double stranded RNAi agent for inhibiting expression of a coronavirus genome in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.


In one embodiment, the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-5. In one embodiment, the sense strand or the antisense strand is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1184210, AD-1184270, AD-1184233, AD-1184271, AD-1184212, AD-1184228, AD-1184223, AD-1231490, AD-1231513, AD-1231485, AD-1231507, AD-1231471, AD-1231494, AD-1231496, and AD-1231497. In another embodiment, the sense strand or the antisense strand is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1231490, AD-1231513, AD-1231485, AD-1231471, AD-1231496, and AD-1231497. In one embodiment, the sense strand or the antisense strand is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137 and AD-1184150. In one embodiment, the sense strand and the antisense strand are the sense strand and antisense strand of AD-1184137. In another embodiment, the sense strand and the antisense strand are the sense strand and antisense strand of AD-1184150.


In one embodiment, both the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.


In one embodiment, the lipophilic moiety is conjugated to one or more positions in the double stranded region of the dsRNA agent.


In one embodiment, the lipophilic moiety is conjugated via a linker or a carrier.


In one embodiment, lipophilicity of the lipophilic moiety, measured by log Kow, exceeds 0.


In one embodiment, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.


In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.


In one embodiment, the dsRNA agent comprises at least one modified nucleotide.


In one embodiment, no more than five of the sense strand nucleotides and no more than five of the nucleotides of the antisense strand are unmodified nucleotides


In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.


In one embodiment, at least one of the modified nucleotides is selected from the group a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, a 2′-0 hexadecyl nucleotide, a nucleotide comprising a 2′-phosphate, a cytidine-2′-phosphate nucleotide, a guanosine-2′-phosphate nucleotide, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide, a a 5′-vinyl phosphonate (VP), a 2′-deoxyadenosine-3′-phosphate nucleotide, a 2′-deoxycytidine-3′-phosphate nucleotide, a 2′-deoxyguanosine-3′-phosphate nucleotide, a 2′-deoxythymidine-3′-phosphate nucleotide, a 2′-deoxyuridine nucleotide, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.


In another embodiment, modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.


In another embodiment, the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).


In yet another embodiment, the modifications on the nucleotides are 2′-O-methyl modifications, 2′-deoxy-modifications, and 2′fluoro modifications.


In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.


In one embodiment, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages.


In one embodiment, each strand is no more than 30 nucleotides in length.


In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide.


In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.


The double stranded region may be 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.


Each strand of the dsRNA agent may be has 19-30 nucleotides in length; 19-23 nucleotides in length; or 21-23 nucleotides in length.


In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.


In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.


In one embodiment, the internal positions include all positions except the terminal two positions from each end of the at least one strand.


In another embodiment, the internal positions include all positions except the terminal three positions from each end of the at least one strand.


In another embodiment, the internal positions exclude a cleavage site region of the sense strand.


In yet another embodiment, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand.


In one embodiment, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand.


In one embodiment, the internal positions exclude a cleavage site region of the antisense strand.


In one embodiment, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand.


In one embodiment, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.


In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand.


In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.


In one embodiment, the positions in the double stranded region exclude a cleavage site region of the sense strand.


In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.


In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.


In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, or position 15 of the sense strand.


In one embodiment, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.


In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.


In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.


In one embodiment, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.


In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.


In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.


In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.


In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5′-end of the strand.


In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.


In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.


In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.


In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.


In one embodiment, the lipophilic moiety or a targeting ligand is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.


In one embodiment, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.


In one embodiment, the dsRNA agent further comprises a targeting ligand that targets a liver tissue.


In one embodiment, the targeting ligand is a GalNAc conjugate.


In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.


In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand.


In one embodiment, the phosphate mimic is a 5′-vinyl phosphonate (VP).


In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.


In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.


In one embodiment, the sense strand comprises the nucleotide sequence 5′-UAACAAUGUUGCUUUUCAAAC-3′ (SEQ ID NO: 5) and the antisense strand comprises the nucleotide sequence 5′-GUUUGAAAAGCAACAUUGUUAGU-3′ (SEQ ID NO: 6).


In another embodiment, the sense strand comprises the nucleotide sequence 5′-ACUGUACAGUCUAAAAUGUCA-3′ (SEQ ID NO: 7) and the antisense strand comprises the nucleotide sequence 5′-UGACAUUUUAGACUGUACAGUGG-3′ (SEQ ID NO: 8).


In one embodiment, the sense strand comprises the sense strand nucleotide sequence 5′-usasaca(Ahd)UfgUfUfGfcuuuucaasasa-3′ (SEQ ID NO: 9) and the antisense strand comprises the nucleotide sequence 5′-VPusUfsuugAfaaagcaaCfaUfuguuasgsu-3′ (SEQ ID NO: 10), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; (Ahd) is 2′-O-hexadecyl-adenosine-3′-phosphate; and VP is Vinyl-phosphonate.


In another embodiment, the sense strand comprises the nucleotides sequence 5′-ascsugu(Ahd)CfaGfUfCfuaaaauguscsa-3′ (SEQ ID NO: 11) and the antisense strand comprises the nucleotide sequence 5′-VPusGfsacaUfuuuagacUfgUfacagusgsg-3′ (SEQ ID NO: 12), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; (Ahd) is 2′-O-hexadecyl-adenosine-3′-phosphate; and VP is Vinyl-phosphonate.


The present invention further provides cells, pharmaceutical compositions for inhibiting expression of a coronavirus genome, and pharmaceutical composition comprising a lipid formulation. comprising the dsRNA agents of the invention.


In one aspect, the present invention provides a composition comprising two or more, e.g., 2, 3, or 4, double stranded RNAi agents for inhibiting expression of coronavirus genome in a cell, wherein each double stranded RNAi agent independently comprises a sense strand and an antisense strand forming a double stranded region, wherein each of the sense strands independently comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and each of the antisense strands independently comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2.


In another aspect, the present invention provides a composition comprising two or more, e.g., 2, 3, or 4, double stranded ribonucleic acid (dsRNA) agents for inhibiting expression of coronavirus genome in a cell, wherein each dsRNA agent independently comprises a sense strand and an antisense strand forming a double stranded region, wherein each of the sense strands independently comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2, and each of the antisense strands independently comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1.


In yet another aspect, the present invention provides a composition comprising two or more, e.g., 2, 3, or 4, double stranded ribonucleic acid (dsRNA) agents for inhibiting expression of coronavirus genome in a cell, wherein each dsRNA agent independently comprises a sense strand and an antisense strand forming a double stranded region, wherein each of the antisense strands independently comprises a region complementary to part of an mRNA encoding a coronavirus genome (SEQ ID NO:1), wherein each of the sense strands or each of the antisense strands are independently 14 to 30 nucleotides in length.


In one aspect, the present invention provides a composition comprising two or more, e.g., 2, 3, or 4, double stranded ribonucleic acid (dsRNA) agents for inhibiting expression of coronavirus genome in a cell, wherein each dsRNA agent independently comprises a sense strand and an antisense strand forming a double stranded region, wherein each of the antisense strands independently comprises a region complementary to part of a reverse complement of an mRNA encoding a coronavirus genome (SEQ ID NO:2), wherein each of the sense strands or each of the antisense strands are independently 14 to 30 nucleotides in length.


In another aspect, the present invention provides a composition comprising two or more, e.g., 2, 3, or 4, double stranded RNAi agents for inhibiting expression of a coronavirus genome in a cell, wherein each double stranded RNAi agent independently comprises a sense strand and an antisense strand forming a double stranded region, wherein each of the antisense strands independently comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-5, wherein each of the sense strands or each of the antisense strands are independently 14 to 30 nucleotides in length.


In one embodiment, each of the sense strands or each of the antisense strands is a sense strand or an antisense strand independently selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-5.


In another embodiment, each of the sense strands or each of the antisense strands is a sense strand or an antisense strand independently selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1184210, AD-1184270, AD-1184233, AD-1184271, AD-1184212, AD-1184228, AD-1184223, AD-1231490, AD-1231513, AD-1231485, AD-1231507, AD-1231471, AD-1231494, AD-1231496, and AD-1231497.


In yet another embodiment, each of the sense strands or each of the antisense strands is a sense strand or an antisense strand of a duplex independently selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1231490, AD-1231513, AD-1231485, AD-1231471, AD-1231496, and AD-1231497.


In one embodiment, each of the sense strands and each of the antisense strands is a sense strand and an antisense strand of a duplex independently selected from the group consisting of AD-1184137 and AD-1184150.


In one embodiment, at least one of said sense strands or at least one of said antisense strands is independently conjugated to one or more lipophilic moieties


In one embodiment, all of the sense strands or all of the antisense strand of each of the dsRNA agents are independently conjugated to one or more lipophilic moieties.


In one embodiment, each lipophilic moiety is independently conjugated to one or more positions in the double stranded region of the dsRNA agent.


In one embodiment, each lipophilic moiety is independently conjugated via a linker or a carrier.


In one embodiment, lipophilicity of each lipophilic moiety, measured by log Kow, independently exceeds 0.


In another embodiment, the hydrophobicity of each double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, independently exceeds 0.2.


In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.


In one embodiment, each of the dsRNA agents independently comprises at least one modified nucleotide.


In one embodiment, each sense strand and each antisense strand of each dsRNA agent independently comprises no more than five unmodified nucleotides.


In one embodiment, all of the nucleotides of each sense strand and all of the nucleotides of each antisense strand independently comprise a modification.


In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, a 2′-0 hexadecyl nucleotide, a nucleotide comprising a 2′-phosphate, a cytidine-2′-phosphate nucleotide, a guanosine-2′-phosphate nucleotide, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide, a a 5′-vinyl phosphonate (VP), a 2′-deoxyadenosine-3′-phosphate nucleotide, a 2′-deoxycytidine-3′-phosphate nucleotide, a 2′-deoxyguanosine-3′-phosphate nucleotide, a 2′-deoxythymidine-3′-phosphate nucleotide, a 2′-deoxyuridine nucleotide, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.


In another embodiment, the modified nucleotide is independently selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.


In yet another embodiment, the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).


In one embodiment, the modifications on the nucleotides are each independently selected from the group consisting of 2′-O-methyl modifications, 2′-deoxy-modifications, or 2′fluoro modifications.


In one embodiment, at least one of the dsRNA agents further comprises at least one phosphorothioate internucleotide linkage.


In one embodiment, at least one of the dsRNA agents comprises 6-8 phosphorothioate internucleotide linkages.


In one embodiment, each strand of each dsRNA agent is independently no more than 30 nucleotides in length.


In one embodiment, at least one strand of at least one dsRNA agent independently comprises a 3′ overhang of at least 1 nucleotide.


In another embodiment, at least one strand of at least one dsRNA agent independently comprises a 3′ overhang of at least 2 nucleotides.


In one embodiment, the double stranded region of each dsRNA agent is independently 15-30 nucleotide pairs in length.


In another embodiment, the double stranded region of each dsRNA agent is independently is 17-23 nucleotide pairs in length.


In yet another embodiment, the double stranded region of each dsRNA agent is independently is 17-25 nucleotide pairs in length.


In one embodiment, double stranded region of each dsRNA agent is independently is 23-27 nucleotide pairs in length.


In another embodiment, the double stranded region of each dsRNA agent is independently is 19-21 nucleotide pairs in length.


In one embodiment, the double stranded region of each dsRNA agent is independently is 21-23 nucleotide pairs in length.


In one embodiment, each strand of each dsRNA agent independently has 19-30 nucleotides.


In another embodiment, each strand of each dsRNA agent independently has 19-23 nucleotides.


In yet another embodiment, wherein each strand of each dsRNA agent independently has 21-23 nucleotides.


In one embodiment, each dsRNA agent comprises one or more lipophilic moieties independently conjugated to one or more internal positions on at least one strand.


In one embodiment, the one or more lipophilic moieties are each independently conjugated to one or more internal positions on at least one strand via a linker or carrier.


In one embodiment, each of the internal positions independently include all positions except the terminal two positions from each end of the at least one strand.


In one embodiment, each of the internal positions independently include all positions except the terminal three positions from each end of the at least one strand.


In one embodiment, each of the internal positions independently exclude a cleavage site region of the sense strand.


In one embodiment, each of the internal positions independently include all positions except positions 9-12, counting from the 5′-end of the sense strand.


In one embodiment, each of the internal positions independently include all positions except positions 11-13, counting from the 3′-end of the sense strand.


In one embodiment, each of the internal positions independently exclude a cleavage site region of the antisense strand.


In one embodiment, each of the the internal positions independently include all positions except positions 12-14, counting from the 5′-end of the antisense strand.


In another embodiment, each of the the internal positions independently include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end.


In one embodiment, each of the one or more lipophilic moieties are independently conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′end of each strand.


In another embodiment, the one or more lipophilic moieties are each independently conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand.


In one embodiment, each of the positions in the double stranded region independently exclude a cleavage site region of the sense strand.


In one embodiment, each of the sense strands is independently 21 nucleotides in length, each of the the antisense strands is independently 23 nucleotides in length, and each of the lipophilic moieties is independently conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.


In one embodiment, each of the lipophilic moieties is independently conjugated to position 21, position 20, position 15, position 1, or position 7 of the sense strand.


In another embodiment, each of the lipophilic moieties is independently conjugated to position 21, position 20, or position 15 of the sense strand.


In yet another embodiment, each of the lipophilic moieties is independently conjugated to position 20 or position 15 of the sense strand.


In one embodiment, each of the lipophilic moieties is independently conjugated to position 16 of the antisense strand.


In one embodiment, each of the lipophilic moieties is independently an aliphatic, alicyclic, or polyalicyclic compound.


In one embodiment, each of the lipophilic moieties is independently selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.


In one embodiment, each of the lipophilic moieties independently contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.


In one embodiment, each of the lipophilic moieties independently contains a saturated or unsaturated C6-C18 hydrocarbon chain.


In one embodiment, each of the lipophilic moieties independently contains a saturated or unsaturated C16 hydrocarbon chain.


In one embodiment, each of the saturated or unsaturated C16 hydrocarbon chain is independently conjugated to position 6, counting from the 5′-end of the strand.


In one embodiment, each of the lipophilic moieties is independently conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.


In one embodiment, each of the carriers is independently a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.


In one embodiment, each of the lipophilic moieties is independently conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.


In one embodiment, each of the lipophilic moieties is independently conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.


In one embodiment, each of the lipophilic moieties or one or more targeting ligands is independently conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.


In one embodiment, the 3′ end of at least one of the sense strands is independently protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.


In one embodiment, at least one of the dsRNA agents further comprises a targeting ligand that targets a liver tissue.


In one embodiment, each of the targeting ligands is independently a GalNAc conjugate.


In one embodiment, at least one of the dsRNA agents further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.


In another embodiment, at least one of the dsRNA agents further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In yet another embodiment, at least one of the dsRNA agents further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, at least one of the dsRNA agents further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In another embodiment, at least one of the dsRNA agents further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.


In one embodiment, at least one of the dsRNA agents further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand.


In one embodiment, each of the phosphate mimic is independently a 5′-vinyl phosphonate (VP).


In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of at least one of the duplex is independently an AU base pair.


In one embodiment, each of the sense strands independently has a total of 21 nucleotides and each of the the antisense strands independently has a total of 23 nucleotides.


In one embodiment, the composition comprises a first dsRNA agent comprising the sense strand nucleotide sequence 5′-UAACAAUGUUGCUUUUCAAAC-3′ (SEQ ID NO: 5) and an antisense strand comprising the nucleotide sequence 5′-GUUUGAAAAGCAACAUUGUUAGU-3′ (SEQ ID NO: 6); and a second dsRNA agent comprising the sense strand nucleotide sequence 5′-ACUGUACAGUCUAAAAUGUCA-3′ (SEQ ID NO: 7) and an antisense strand comprising the nucleotide sequence 5′-UGACAUUUUAGACUGUACAGUGG-3′ (SEQ ID NO: 8).


In one embodiment, the sense strand of the first dsRNA agent comprises the sense strand nucleotide sequence 5′-usasaca(Ahd)UfgUfUfGfcuuuucaasasa-3′ (SEQ ID NO: 9) and the antisense strand of the first dsRNA agent comprises the nucleotide sequence 5′-VPusUfsuugAfaaagcaaCfaUfuguuasgsu-3′ (SEQ ID NO: 10); and the sense strand of the second dsRNA agent comprises the nucleotides sequence 5′-ascsugu(Ahd)CfaGfUfCfuaaaauguscsa-3′ (SEQ ID NO:11) and the antisense strand of the second dsRNA agent comprises the nucleotide sequence 5′-VPusGfsacaUfuuuagacUfgUfacagusgsg-3′ (SEQ ID NO: 12), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; (Ahd) is 2′-O-hexadecyl-adenosine-3′-phosphate; and VP is Vinyl-phosphonate.


The present invention further provides isolated cells, comprising the compositions of the invention.


In one embodiment, the compositions of the invention are pharmaceutical compositions. In another embodiment, the compositions of the invention are pharmaceutical composition comprising a lipid formulation.


In one aspect, the present invention provides a method of inhibiting expression of a coronavirus genome in a cell. The method includes contacting the cell with the dsRNA agent of the invention, the composition of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the coronavirus genome, thereby inhibiting expression of the coronavirus genome in the cell.


In one embodiment, the cell is contacted with two or more, e.g., 2, 3, or 4, of the dsRNA agents of the invention.


In one embodiment, the cell is within a subject.


In one embodiment, the subject is a human.


In one embodiment, the expression of the coronavirus genome is inhibited by at least 50%.


In one aspect, the present invention provides a method of inhibiting replication of a coronavirus in a cell. The method includes contacting the cell with the dsRNA agent of the invention, the composition of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the RNA transcript of the coronavirus genome, thereby inhibiting replication of the coronavirus in a cell.


In one embodiment, the cell is contacted with two or more, e.g., 2, 3, or 4, of the dsRNA agents of the invention.


In one embodiment, the cell is within a subject.


In one embodiment, the subject is a human.


In one embodiment, the expression of the coronavirus genome is inhibited by at least 50%.


In one aspect, the present invention provides a method of treating a subject having a coronavirus infection. The method includes administering to the subject a therapeutically effective amount of the dsRNA agent of the invention, the composition of the invention, or the pharmaceutical composition of the invention, thereby treating the subject.


In one embodiment, the subject is administered two or more, e.g., 2, 3, or 4, dsRNA agents of the invention.


In one embodiment, the subject is a human, e.g., an immunocompromised human.


In one embodiment, the subject having the coronavirus infection is infected with a severe acute respiratory syndrome (SARS) virus, a Middle East respiratory syndrome (MERS) virus, or a severe acute respiratory syndrome 2 (SARS-2) virus.


In one embodiment, treating comprises amelioration of at least on sign or symptom of the disease.


In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.


In one embodiment, the administration of the dsRNA is pulmonary system administration, e.g., intranasal administration, or oral inhalative administration.


In one embodiment, the double-stranded RNAi agent is administered intranasally.


By pulmonary system administration, e.g., intranasal administration or oral inhalative administration, of the double-stranded RNAi agent, the method can reduce the expression of a coronavirus genome in a pulmonary system tissue, e.g., a nasopharynx tissue, an oropharynx tissue, a laryngopharynx tissue, a larynx tissue, a trachea tissue, a carina tissue, a bronchi tissue, a bronchiole tissue, or an alveoli tissue.


In one embodiment, the dsRNA agent is administered to the subject subcutaneously.


In one embodiment, the method further comprises administering to the subject an additional agent or a therapy suitable for treatment or prevention of a coronavirus-associated disorder.


In one embodiment, the additional therapeutic agent is selected from the group consisting of an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.


In one aspect the present invention provides a method of treating a subject having a coronavirus infection. The method includes administering to the subject via pulmonary system administration a therapeutically effective amount of a first dsRNA agent comprising a first sense strand and a first antisense strand forming a first double stranded region, and a therapeutically effective amount of a second dsRNA agent comprising a second sense strand and a second antisense strand forming a second double stranded region, wherein the first sense strand comprises the nucleotide sequence 5′-UAACAAUGUUGCUUUUCAAAC-3′(SEQ ID NO: 5) and the first antisense strand comprises the nucleotide sequence 5′-GUUUGAAAAGCAACAUUGUUAGU-3′ (SEQ ID NO: 6); and the second sense strand comprises the nucleotide sequence 5′-ACUGUACAGUCUAAAAUGUCA-3′ (SEQ ID NO: 7) and the second antisense strand comprises the nucleotide sequence 5′-UGACAUUUUAGACUGUACAGUGG-3′ (SEQ ID NO: 8).


In another aspect, the present invention provides a method of treating a subject having a coronavirus infection. The method includes administering to the subject via pulmonary system administration a therapeutically effective amount of a composition for inhibiting expression of a coronavirus genome in a cell, said composition comprising: a first dsRNA agent comprising a first sense strand comprising the nucleotide sequence 5′-UAACAAUGUUGCUUUUCAAAC-3′ (SEQ ID NO: 5) and a first antisense strand comprising the nucleotide sequence 5′-GUUUGAAAAGCAACAUUGUUAGU-3′ (SEQ ID NO: 6), and a second dsRNA agent comprising a second sense strand comprising the nucleotide sequence 5′-ACUGUACAGUCUAAAAUGUCA-3′ (SEQ ID NO: 7) and a second antisense strand comprising the nucleotide sequence 5′-UGACAUUUUAGACUGUACAGUGG-3′ (SEQ ID NO: 8), thereby treating the subject.


In one embodiment, the first and second dsRNA agents are present in a composition.


In one embodiment, the first and second dsRNA agents are present in separate compositions.


In another embodiment, the first and second dsRNA agents are present in the same composition.


In one embodiment, the compositions are administered to the subject at the same time.


In another embodiment, the compositions are administered to the subject at different times.


In one embodiment, the composition is a pharmaceutical composition.


In one embodiment, the first sense strand comprises the nucleotide sequence 5′-usasaca(Ahd)UfgUfUfGfcuuuucaasasa-3′ (SEQ ID NO: 9) and the first antisense strand comprises the nucleotide sequence 5′-VPusUfsuugAfaaagcaaCfaUfuguuasgsu-3′ (SEQ ID NO: 10); and the second sense strand comprises the nucleotides sequence 5′-ascsugu(Ahd)CfaGfUfCfuaaaauguscsa-3′ (SEQ ID NO: 11) and the second antisense strand comprises the nucleotide sequence 5′-VPusGfsacaUfuuuagacUfgUfacagusgsg-3′ (SEQ ID NO: 12), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; (Ahd) is 2′-O-hexadecyl-adenosine-3′-phosphate; and VP is Vinyl-phosphonate.


In one embodiment, the subject is a human.


In one embodiment, the subject having the coronavirus infection is infected with a severe acute respiratory syndrome (SARS) virus, a Middle East respiratory syndrome (MERS) virus, or a severe acute respiratory syndrome 2 (SARS-2)-CoV-2 virus.


In one embodiment, treating comprises amelioration of at least on sign or symptom of the disease.


In one embodiment, the first and second dsRNA agents are independently administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.


In one embodiment, the pulmonary system administration is via inhalation or intranasally.


In one embodiment, the methods further comprise administering to the subject an additional agent or a therapy suitable for treatment or prevention of a coronavirus-associated disorder.


In one embodiment, the additional therapeutic agent is selected from the group consisting of an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.


The present invention is further illustrated by the following detailed description and drawings.





BRIEF DESCRIPTION OF THE DRAWINGS


FIG. 1 depicts the genomes and structures of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), and the Middle East Respiratory Syndrome coronavirus (MERS-CoV).



FIG. 2 schematically depicts the concatemers and the assay used for the single dose screens of the dsRNA agents of the invention.



FIG. 3 are graphs depicting the effect of the indicated siRNAs on extracellular SARS-CoV-2 genomes determined by RT-qPCR (top graph) and the effect on of the indicated siRNAs on intracellular viral nucleocapsid protein determined by in-cell ELISA (bottom graph).



FIG. 4 depicts the effect of the indicated siRNAs on resistance selected cells infected with SARS-CoV-2 determined by RT-qPCR.



FIG. 5 depicts the effect of the indicated siRNAs on resistance selected cells infected with SARS-CoV-2 determined in a focus-forming assay.



FIG. 6 are images of an immunofluorescence assay depicting SARS-CoV-2 nucleocapsid protein staining in a focus-forming assay in the presence of 10 nM of the indicated siRNAs.



FIG. 7 is a graph depicting the effect of intranasal administration of a combination of AD-1184150 and AD-1184137 on the body weight of hamsters challenged with SARS-CoV-2.



FIG. 8 is a graph depicting the effect of intranasal administration as compared to subcutaneous administration of a combination of AD-1184150 and AD-1184137 on the body weight of hamsters challenged with SARS-CoV-2.





DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA genomes and RNA transcripts of coronavirus genes, e.g., a SARS-CoV-2 gene. The iRNAs of the invention have been designed to target a human coronavirus genome, e.g., a Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) genome, including portions of a coronavirus genome, e.g., a Severe Acute Respiratory Syndrome 2 (SARS-CoV-2) genome, that are cross-reactive with a Severe Acute Respiratory Syndrome (SARS-CoV) gene, and/or Middle East Respiratory Syndrome (MERS-CoV) gene. The coronavirus genome may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding genome (a coronavirus genome) in mammals. The present disclosure also provides methods of using the RNAi compositions of the disclosure, e.g., compositions comprising one or more, e.g., 2, 3, or 4, dsRNA agents of the invention, for inhibiting the expression of coronavirus genes or genome for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a coronavirus genome, e.g., a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV).


The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a coronavirus genome. In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 21-23 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a coronavirus genome (either of a positive-sense genomic RNA or a negative-sense genomic RNA of a coronavirus).


In certain embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA of a coronavirus genome (either of a positive-sense genomic RNA or a negative-sense genomic RNA of a coronavirus). In some embodiments, such iRNA agents having longer length antisense strands preferably may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.


The use of iRNAs of the invention enables the targeted degradation of mRNAs or RNA genomes of the corresponding viral genes (coronavirus gene) in mammals. Thus, methods and compositions including these iRNAs are useful for treating a subject having a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV).


In certain embodiments, the administration of the dsRNA to a subject results in an improvement of lung function, or a stoppage or reduction of the rate of loss of lung function, reduction of fever, reduction of cough.


The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a coronavirus genomes as well as compositions, uses, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of a coronavirus genome, e.g., subjects susceptible to or diagnosed with a coronavirus-associated disorder.


I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.


The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.


The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.


The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.


The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.


The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.


As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.


As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.


In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.


In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.


As used herein, the term “coronavirus,” (“CoV”; subfamily Coronavirinae, family Coronaviridae, order Nidovirales), refers to a group of highly diverse, enveloped, positive-sense, single-stranded RNA viruses that cause respiratory, enteric, hepatic and neurological diseases of varying severity in a broad range of animal species, including humans. Coronaviruses are subdivided into four genera: Alphacoronavirus, Betacoronavirus (13CoV), Gammacoronavirus and Deltacoronavirus.


Any coronavirus that infects humans and animals is encompassed by the term “coronavirus” as used herein. Exemplary coronaviruses encompassed by the term include the coronaviruses that cause a common cold-like respiratory illness, e.g., human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), and human coronavirus HKU1 (HCoV-HKU1); the coronavirus that causes avian infectious bronchitis virus (IBV); the coronavirus that causes murine hepatitis virus (MHV); the coronavirus that causes porcine transmissible gastroenteritis virus PRCoV; the coronavirus that causes porcine respiratory coronavirus and bovine coronavirus; the coronavirus that causes Severe Acute Respiratory Syndrome (SARS), the coronavirus that causes the Middle East respiratory syndrome (MERS), and the coronavirus that causes Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19).


The coronavirus (CoV) genome is a single-stranded, non-segmented RNA genome, which is approximately 26-32 kb. It contains 5′-methylated caps and 3′-polyadenylated tails and is arranged in the order of 5′, replicase genes, genes encoding structural proteins (spike glycoprotein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N)), polyadenylated tail and then the 3′ end. The partially overlapping 5′-terminal open reading frame 1a/b (ORF1a/b) is within the 5′ two-thirds of the CoV genome and encodes the large replicase polyprotein 1a (pp1a) and pp1ab. These polyproteins are cleaved by papain-like cysteine protease (PLpro) and 3C-like serine protease (3CLpro) to produce non-structural proteins, including RNA-dependent RNA polymerase (RdRp) and helicase (Hel), which are important enzymes involved in the transcription and replication of CoVs. The 3′ one-third of the CoV genome encodes the structural proteins (S, E, M and N), which are essential for virus-cell-receptor binding and virion assembly, and other non-structural proteins and accessory proteins that may have immunomodulatory effects. (Peiris J S., et al., 2003, Nat. Med. 10 (Suppl. 12): 88-97).


As a coronavirus is a positive-sense, single-stranded RNA virus having a 5′ methylated cap and a 3′ polyadenylated tail, once the virus enters the cell and is uncoated, the viral RNA genome attaches to the host cell's ribosome for direct translation. The host ribosome translates the initial overlapping open reading frame of the virus genome and forms a long polyprotein. The polyprotein has its own proteases which cleave the polyprotein into multiple nonstructural proteins.


A number of the nonstructural proteins coalesce to form a multi-protein replicase-transcriptase complex (RTC). The main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). It is directly involved in the replication and transcription of RNA from an RNA strand. The other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease non-structural protein for instance provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks.


One of the main functions of the complex is to replicate the viral genome. RdRp directly mediates the synthesis of negative-sense genomic RNA from the positive-sense genomic RNA. This is followed by the replication of positive-sense genomic RNA from the negative-sense genomic RNA. The other important function of the complex is to transcribe the viral genome. RdRp directly mediates the synthesis of negative-sense subgenomic RNA molecules from the positive-sense genomic RNA. This is followed by the transcription of these negative-sense subgenomic RNA molecules to their corresponding positive-sense mRNAs


The replicated positive-sense genomic RNA becomes the genome of the progeny viruses.


As use herein, the term “severe acute respiratory syndrome coronavirus” or “SARS-CoV”, refers to a coronavirus that was first discovered in 2003, which causes severe acute respiratory syndrome (SARS). SARS-CoV represents the prototype of a new lineage of coronaviruses capable of causing outbreaks of clinically significant and frequently fatal human disease. The complete genome of SARS-CoV has been identified, as well as common variants thereof. The genome of SARS-CoV is a 29,727-nucleotide polyadenylated RNA, has 11 open reading frames, and 41% of the residues are G or C (see, e.g., FIG. 1). The genomic organization is typical of coronaviruses, with the characteristic gene order (5′-replicase (rep), spike (S), envelope (E), membrane (M), nucleocapsid (N)-3′ and short untranslated regions at both termini. The SARS-CoV rep gene, which comprises about two-thirds of the genome, is predicted to encode two polyproteins that undergo co-translational proteolytic processing. There are four open reading frames (ORFs) downstream of rep that are predicted to encode the structural proteins, S, E, M and N. The hemagglutinin-esterase gene, which is present between ORF1b and S in group 2 and some group 3 coronaviruses was not found.


The amino acid and complete coding sequences of the SARS-CoV genomes are known may be found in for example, GenBank Accession Nos. AY502923.1; AP006559.1; AP006558.1; AY313906.1; AY345986.1; AY502931.1; AY282752.2; AY559097.1; AY559081.1; DQ182595.1; AY291451.1; AY568539.1; AY613947.1; and AY390556.1, the entire contents of each of which are incorporated herein by reference.


The term “SARS-CoV,” as used herein, also refers to naturally occurring RNA sequence variations of the SARS-CoV genome.


As use herein, the term “the Middle East respiratory syndrome coronavirus” or “MERS-CoV”, refers to a coronavirus that causes the Middle East respiratory syndrome (MERS), which was first identified in 2012. MERS-CoV is closely related to severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). Clinically similar to SARS, MERS-CoV infection leads to severe respiratory illness with renal failure. The genomic structure of MERS-CoV is shown in FIG. 1.


The amino acid and complete coding sequences of the MERS-CoV genomes are known and may be found in for example, GenBank Accession Nos. MK462243.1; MK462244.1; MK462245.1; MK462246.1; MK462247.1; MK462248.1; MK462249.1; MK462250.1; MK462251.1; MK462252.1; MK462253.1; MK462254.1; MK462255.1; MK462256.1; MK483839.1; and MH822886.1, the entire contents of each of which are incorporated herein by reference.


The term “MERS-CoV,” as used herein, also refers to naturally occurring RNA sequence variations of the MERS-CoV genome.


As use herein, the terms “severe acute respiratory syndrome coronavirus 2,” “SARS-CoV-2,” “2019-nCoV,” refer to the novel coronavirus that caused a pneumonia outbreak first reported in Wuhan, China in December 2019 (“COVID-19”). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that SARS-CoV-2 was most closely related (89.1% nucleotide similarity similarity) to SARS-CoV.


The amino acid and complete coding sequences of the SARS-CoV-2 genomes are known and may be found in for example, the GISAID EpiCoV™ Database (db.cngb.org/gisaid/), including Accession nos. EPI_ISL_402119; EPI_ISL_402120; EPI_ISL_402121; EPI_ISL_402123; EPI_ISL_402124; EPI_ISL_402125; EPI_ISL_402127; EPI_ISL_402128; EPI_ISL_402129; EPI_ISL_402130; EPI_ISL_402132; EPI_ISL_403928; EPI_ISL_403929; EPI_ISL_403930; EPI_ISL_403931; EPI_ISL_403932; EPI_ISL_403933; EPI_ISL_403934; EPI_ISL_403935; EPI_ISL_403936; EPI_ISL_403937; EPI_ISL_403962; EPI_ISL_404228; EPI_ISL_404253; and EPI_ISL_404895, the entire contents of watch of which are incorporated herein by reference.


The term “SARS-CoV-2,” as used herein, also refers to naturally occurring RNA sequence variations of the SARS-CoV-2 genome.


Additional examples of coronavirus genome and mRNA sequences are readily available using publicly available databases, e.g., GenBank, UniProt, and OMIM.


As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an RNA molecule, such as a coronavirus positive-sense RNA molecule or a coronavirus negative-sense RNA molecule, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an RNA molecule, such as a coronavirus positive-sense RNA molecule or a coronavirus negative-sense RNA molecule. In one embodiment, the target sequence is within the protein coding region of a coronavirus genome.


The target sequence may be from about 19-36 nucleotides in length, e.g., preferably about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.


As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.


“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.


The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulates, e.g., inhibits, the expression of a coronavirus genome in a cell, e.g., a cell within a subject, such as a mammalian subject.


In one embodiment, an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a coronavirus target mRNA sequence, either a coronavirus positive-sense RNA molecule or a coronavirus negative-sense RNA molecule, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target genome, i.e., a coronavirus genome or gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.


In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.


In another embodiment, a “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., either a coronavirus positive-sense RNA molecule or a coronavirus negative-sense RNA molecule. In some embodiments of the disclosure, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.


In general, a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.


As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.


In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide—which is acknowledged as a naturally occurring form of nucleotide—if present within a RNAi agent can be considered to constitute a modified nucleotide.


The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.


The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the target site of the dsRNA. In some embodiments, the hairpin loop can be 10 or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.


In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5′-end of first strand is linked to the 3′-end of the second strand or 3′-end of first strand is linked to 5′-end of the second strand. When the two strands are linked to each other at both ends, 5′-end of first strand is linked to 3′-end of second strand and 3′-end of first strand is linked to 5′-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleoside linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.


Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.


The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3′, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as “shRNA” herein.


Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.


In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a coronavirus target RNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).


In one embodiment, an RNAi agent of the invention is a dsRNA agent, each strand of which comprises 19-23 nucleotides that interacts with a coronavirus RNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a coronavirus RNA sequence to direct the cleavage of the target RNA.


As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.


In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.


In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.


In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.


The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.


The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a coronavirus RNA, i.e., either of a coronavirus positive-sense RNA or a coronavirus negative-sense RNA.


As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a coronavirus nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the RNAi agent.


In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand double stranded RNA agent of the invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region.


Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a coronavirus genome, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a coronavirus genome or gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a coronavirus genome is important, especially if the particular region of complementarity in a coronavirus genome is known to mutate.


The term “sense strand” or “passenger strand” as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.


As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.


As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.


As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.


Complementary sequences within a RNAi agent, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.


“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.


The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use.


As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest or target sequence (e.g., a coronavirus target sequence, either of a coronavirus positive-sense RNA or a coronavirus negative-sense RNA). For example, a polynucleotide is complementary to at least a part of a coronavirus RNA if the sequence is substantially complementary to a non-interrupted portion of a coronavirus RNA.


Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target coronavirus sequence, either of a coronavirus positive-sense RNA or a coronavirus negative-sense RNA.


In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target coronavirus sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 1, or a fragment of SEQ ID NO: 1, such as about 85%, about 90%, or about 95% complementary.


In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target coronavirus sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 2, or a fragment of SEQ ID NO: 2, such as about 85%, about 90%, or about 95% complementary.


In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target coronavirus sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-5, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-5, such as about 85%, about 90%, or about 95% complementary.


In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target coronavirus sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO: 2, or a fragment of any one of SEQ ID NO: 2, such as about 85%, about 90%, or about 95% complementary.


In another embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target coronavirus sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NO:1, or a fragment of any one of SEQ ID NO: 1, such as about 85%, about 90%, or about 95% complementary.


In some embodiments, an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target coronavirus sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of any one of Tables 2-5, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-5, such as about 85%, about 90%, or about 95% complementary.


In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.


In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.


In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.


In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.


In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.


In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.


In one embodiment, the sense strand of the iRNA agent is 21-nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3-end.


In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.


In one embodiment, at least partial suppression of the expression of a coronavirus genome, is assessed by a reduction of the amount of coronavirus genome which can be isolated from or detected in a first cell or group of cells in which a coronavirus genome is transcribed and which has or have been treated such that the expression of a coronavirus genome is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:









(

genome





in





control





cells

)

-

(

genome





in





treated





cells

)



(

genome





in





control





cells

)




100

%




In one embodiment, inhibition of expression is determined by the dual luciferase method in Example 1 wherein the RNAi agent is present at 10 nM.


The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.


Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, via inhalation, intranasal administration, or intratracheal administration, by injecting the RNAi agent into or near the tissue where the cell is located, e.g., a pulmonary system cell, or by injecting the RNAi agent into another area, or to the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT Publication No. WO 2019/217459, which is incorporated herein by reference, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the pulmonary system. In some embodiments, the RNAi agent may contain or be coupled to a ligand, e.g., one or more GalNAc derivatives as described below, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the liver. In other embodiments, the RNAi agent may contain or be coupled to a lipophilic moiety or moieties and one or more GalNAc derivatives. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.


In one embodiment, contacting a cell with an RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell.


Absorption or uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.


The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, log Kow, where Kow is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its log Kow exceeds 0. Typically, the lipophilic moiety possesses a log Kow exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the log Kow of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the log Kow of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.


The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log Kow) value of the lipophilic moiety.


Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.


In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT Publication No. WO 2019/217459. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.


Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.


The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., a rNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.


As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In a preferred embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit from reduction in coronavirus genome expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in coronavirus genome expression; a human having a disease, disorder, or condition that would benefit from reduction in coronavirus genome expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in coronavirus genome expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.


As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with coronavirus genome expression or coronavirus protein production, e.g., a coronavirus-associated disease, e.g., viral replication. Treatment also includes a reduction of one or more sign or symptoms associated with unwanted coronavirus genome expression; diminishing the extent of unwanted coronavirus genome activation or stabilization; amelioration or palliation of unwanted coronavirus genome activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.


The term “lower” in the context of the level of a coronavirus genome in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of a coronavirus genome in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., viral load, blood oxygen level, white blood cell count, kidney function, liver function. As used here, “lower” in a subject can refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production or viral replication in a subject.


The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a coronavirus-associated disease towards or to a level in a normal subject not suffering from a coronavirus-associated disease. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal.


As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder, or condition thereof, that would benefit from a reduction in expression of a coronavirus genome or production of a coronavirus protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a coronavirus-associated disease. The failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition, e.g., pneumonia (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.


As used herein, the term “coronavirus-associated disease,” is a disease or disorder that is caused by, or associated with a coronavirus infection, coronavirus genome expression or coronavirus protein production. The term “coronavirus-associated disease” includes a disease, disorder or condition that would benefit from a decrease in coronavirus genome expression, replication, or protein activity. Non-limiting examples of coronavirus-associated diseases include, for example, disease or disorders caused by infection with human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKU1), severe acute respiratory syndrome coronavirus (SARS), the Middle East respiratory syndrome coronavirus (MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 or COVID-19). The symptoms for a coronavirus-associated disease depend on the type of coronavirus and how serious the infection is. Patients with a mild to moderate upper-respiratory infection may develop symptoms such as runny nose, sneezing, headache, cough, sore throat, fever, or short of breath. In more severe cases, coronavirus infection can cause pneumonia, severe acute respiratory syndrome, kidney failure and even death. Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.


“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a coronavirus-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.


“Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having a coronavirus-associated disorder, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.


A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.


The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.


The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.


The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, bronchial fluids, sputum, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from a nasal swab. In certain embodiments, samples may be derived from a throat swab/In certain embodiments, samples may be derived from the lung, or certain types of cells in the lung. In some embodiments, the samples may be derived from the bronchioles. In some embodiments, the samples may be derived from the bronchus. In some embodiments, the samples may be derived from the alveoli. In other embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) derived from the subject. In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma or serum derived therefrom. In further embodiments, a “sample derived from a subject” refers to pulmonary system tissue (or subcomponents thereof) derived from the subject.


II. RNAi Agents of the Disclosure

Described herein are RNAi agents which inhibit the expression of a coronavirus genome. In one embodiment, the RNAi agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a coronavirus genome in a cell, such as a cell within a subject, e.g., a mammal, such as a human, e.g., a subject having a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV). The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of a target coronavirus RNA, e.g., an mRNA formed in the expression of a coronavirus genome. The region of complementarity is about 15-30 nucleotides or less in length. Upon contact with a cell expressing the coronavirus genome, the RNAi agent inhibits the expression of the coronavirus genome (e.g., a human gene, a primate gene, a non-primate gene) by at least 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flowcytometric techniques. In preferred embodiments, inhibition of expression is by at least 50% as assayed by the Dual-Glo lucifierase assay in Example a where the siRNA is at a 10 nM concentration.


A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. For example, the target sequence can be derived from the sequence of an mRNA formed during the expression of a coronavirus genome. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.


Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.


Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.


In some embodiments, the dsRNA is 15 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).


One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, a RNAi agent useful to target coronavirus expression is not generated in the target cell by cleavage of a larger dsRNA.


A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments, longer, extended overhangs are possible.


A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.


iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.


An siRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.


An siRNA can be made by separately synthesizing a single stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, after which the component strands can then be annealed.


A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given siRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the siRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.


Organic synthesis can be used to produce a discrete siRNA species. The complementary of the species to a coronavirus genome can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.


In one embodiment, RNA generated is carefully purified to remove endsiRNA is cleaved in vitro into siRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g., a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9 and Hammond Science 2001 Aug. 10; 293(5532):1146-50.


dsiRNA cleavage generally produces a plurality of siRNA species, each being a particular 21 to 23 nucleotide fragment of a source dsiRNA molecule. For example, siRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsiRNA molecule may be present.


Regardless of the method of synthesis, the siRNA preparation can be prepared in a solution (e.g., an aqueous or organic solution) that is appropriate for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process.


In one aspect, a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for coronavirus may be selected from the group of sequences provided in any one of Tables 2-5, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences of any one of Tables 2-5. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of a target coronavirus RNA, e.g., an mRNA generated in the expression of a coronavirus genome. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2-5, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2-5 for coronavirus.


In certain embodiments of the invention, the sense strand or the antisense strand of the dsRNA agent is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1184210, AD-1184270, AD-1184233, AD-1184271, AD-1184212, AD-1184228, AD-1184223, AD-1231490, AD-1231513, AD-1231485, AD-1231507, AD-1231471, AD-1231494, AD-1231496, and AD-1231497. In another embodiment, the sense strand or the antisense strand of the dsRNA agent is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1231490, AD-1231513, AD-1231485, AD-1231471, AD-1231496, and AD-1231497. In another embodiment, the sense strand or the antisense strand of the dsRNA agent is selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137 and AD-1184150. In one embodiment, the sense strand or the antisense strand of the dsRNA agent is the sense strand or antisense strand of duplex AD-1184137. In another embodiment, the sense strand or the antisense strand of the dsRNA agent is the sense strand or antisense strand of duplex AD-1184150.


In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.


It will be understood that, although the sequences provided herein are described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth in any one of Tables 2-5 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. One or more lipophilic ligands or one or more GalNAc ligands can be included in any of the positions of the RNAi agents provided in the instant application.


The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a coronavirus genome by not more than 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence using the in vitro assay with Cos7 and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure.


In addition, the RNAs described herein identify a site(s) in a coronavirus transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features RNAi agents that target within this site(s). As used herein, a RNAi agent is said to target within a particular site of an RNA transcript if the RNAi agent promotes cleavage of the transcript anywhere within that particular site. Such a RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a coronavirus genome.


An RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a coronavirus genome generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a coronavirus genome. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a coronavirus genome is important, especially if the particular region of complementarity in a coronavirus genome is known to mutate.


III. Modified RNAi Agents of the Disclosure

In one embodiment, the RNA of the RNAi agent of the disclosure e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In preferred embodiments, the RNA of an RNAi agent of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of an RNAi agent of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of an RNAi agent of the disclosure are modified. RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides.


The nucleic acids featured in the disclosure can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified RNAi agent will have a phosphorus atom in its internucleoside backbone.


Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, e.g., sodium salts, mixed salts and free acid forms are also included.


Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.


Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms 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; 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.


Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.


In other embodiments, suitable RNA mimetics are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.


Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH2—NH—CH2—, —CH2—N(CH3)—O—CH2— [known as a methylene (methylimino) or MMI backbone], —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2— and —N(CH3)—CH2—CH2— [wherein the native phosphodiester backbone is represented as —O—P—O—CH2—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.


Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: 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 can be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Exemplary suitable modifications include 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. In other embodiments, dsRNAs include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, 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, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a RNAi agent, or a group for improving the pharmacodynamic properties of a RNAi agent, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH2)2ON(CH3)2 group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH2—O—CH2—N(CH2)2. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).


Other modifications include 2′-methoxy (2′-OCH3), 2′-aminopropoxy (2′-OCH2CH2CH2NH2), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of a RNAi agent, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. RNAi agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.


An RNAi agent of the disclosure can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. 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 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 uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal 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, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.


Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.


An RNAi agent of the disclosure can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).


An RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O-N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.


Additional representative US Patents and US Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.


Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).


An RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”


An RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the-C3′ and -C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.


Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.


In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).


Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.


Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO 2011/005861.


Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the entire contents of which are incorporated herein by reference.


A. Modified RNAi agents Comprising Motifs of the Disclosure


In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic ligand, e.g., a C16 ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.


Accordingly, the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a target genome or gene (i.e., a coronavirus genome or gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15-30 nucleotides in length. For example, each strand may be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.


The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In preferred embodiments, the duplex region is 19-21 nucleotide pairs in length.


In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In preferred embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.


In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-O-methyl, thymidine (T), and any combinations thereof.


For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.


The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.


The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3-terminal end of the sense strand or, alternatively, at the 3-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.


In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.


In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.


In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.


In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand. When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16ligand).


In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.


In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1-4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.


In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.


In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.


For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1st nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1st paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.


The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.


In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adajacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.


Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.


In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.


In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.


When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.


When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two, or three nucleotides in the duplex region.


In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mistmatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.


In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.


In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.


In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense or antisense strand.


In one embodiment, the sense strand sequence may be represented by formula (I):





5′ np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Na-nq 3′  (I)


wherein:


i and j are each independently 0 or 1;


p and q are each independently 0-6;


each Na independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;


each np and nq independently represent an overhang nucleotide;


wherein Nb and Y do not have the same modification; and


XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. Preferably YYY is all 2′-F modified nucleotides.


In one embodiment, the Na or Nb comprise modifications of alternating pattern.


In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sense strand, the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end.


In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:





5′ np-Na-YYY-Nb-ZZZ—Na-nq 3′  (Ib);





5′ np-Na-XXX-Nb-YYY-Na-nq 3′  (Ic); or





5′ np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3′  (Id).


When the sense strand is represented by formula (Ib), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.


Each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the sense strand is represented as formula (Ic), Nb represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the sense strand is represented as formula (Id), each Nb independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, Nbis 0, 1, 2, 3, 4, 5 or 6. Each Na can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


Each of X, Y and Z may be the same or different from each other.


In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:





5′ np-Na-YYY-Na-nq 3′  (Ia).


When the sense strand is represented by formula (Ia), each Na independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):





5′ nq′-Na′-(Z′Z′Z′)k-Nb′-Y′Y′Y′-Nb′X′X′X′)l-N′a-np′3′  (II)


wherein:


k and l are each independently 0 or 1;


p′ and q′ are each independently 0-6;


each Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;


each np′ and nq′ independently represent an overhang nucleotide;


wherein Nb′ and Y′ do not have the same modification; and X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.


In one embodiment, the Na′ or Nb′ comprise modifications of alternating pattern.


The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1st nucleotide, from the 5′-end; or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.


In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.


In one embodiment, k is 1 and 1 is 0, or k is 0 and 1 is 1, or both k and l are 1.


The antisense strand can therefore be represented by the following formulas:





5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Na′-np′ 3′  (IIb);





5′ nq′-Na′-Y′Y′Y′-Nb′-X′X′X′-np′ 3′  (IIc); or





5′ nq′-Na′-Z′Z′Z′-Nb′-Y′Y′Y′-Nb′-X′X′X′-Na′-np′3′  (IId).


When the antisense strand is represented by formula (IIb), Nb′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the antisense strand is represented as formula (IIc), Nb′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the antisense strand is represented as formula (IId), each Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, Nb is 0, 1, 2, 3, 4, 5 or 6.


In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula:





5′ np′-Na′-Y′Y′Y′-Na′-nq′ 3′  (Ia).


When the antisense strand is represented as formula (IIa), each Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


Each of X′, Y′ and Z′ may be the same or different from each other.


Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.


In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.


In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1st nucleotide from the 5′-end, or optionally, the count starting at the 1st paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.


The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.


Accordingly, the RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):





sense: 5′ np-Na-(XXX)i-Nb-YYY-Nb-(ZZZ)j-Nb-nq 3′





antisense: 3′ np′-Na′-(X′X′X′)k-Nb′-Y′Y′Y′-Nb′-(Z′Z′Z′)lNa′-nq′ 5′  (III)


wherein:


i, j, k, and l are each independently 0 or 1;


p, p′, q, and q′ are each independently 0-6;


each Na and Na′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;


each Nb and Nb′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;


wherein


each np′, np, nq′, and nq, each of which may or may not be present, independently represents an overhang nucleotide; and


XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.


In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1; or both k and l are 0; or both k and l are 1.


Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:





5′ np-Na-YYY-Na-nq 3′





3′ np′-Na′-Y′Y′Y′-Na′nq′ 5′   (IIIa)





5′ np-Na-YYY-Nb-ZZZ-Na-nq 3′





3′ np′-Na′-Y′Y′Y′-Nb′-Z′Z′Z′-Na′nq′ 5′   (IIIb)





5′ np-Na-XXX-Nb-YYY-Na-nq 3′





3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Na′-nq′ 5′  (IIc)





5′ np-Na-XXX-Nb-YYY-Nb-ZZZ-Na-nq 3′





3′ np′-Na′-X′X′X′-Nb′-Y′Y′Y′-Nb′-Z′Z′Z′-Na-nq′ 5′   (IIId)


When the RNAi agent is represented by formula (IIIa), each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented by formula (IIIb), each Nb independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented as formula (IIIc), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.


When the RNAi agent is represented as formula (IIId), each Nb, Nb′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each Na, Na′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of Na, Na′, Nb and Nb′ independently comprises modifications of alternating pattern.


In one embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications and np′>0 and at least one np′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, optionally attached through a bivalent or trivalent branched linker.


In one embodiment, when the RNAi agent is represented by formula (IIIa), the Na modifications are 2′-O-methyl or 2′-fluoro modifications, np′>0 and at least one np′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached through a bivalent or trivalent branched linker.


In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.


In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.


In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.


Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the entire contents of each of which are hereby incorporated herein by reference.


In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:




embedded image


A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.


Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:




embedded image


E. Thermally Destabilizing Modifications


In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5′-end of the antisense strand.


The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).


Exemplified abasic modifications include, but are not limited to the following:




embedded image


Wherein R═H, Me, Et or OMe; R′ H, Me, Et or OMe; R″═H, Me, Et or OMe




embedded image


wherein B is a modified or unmodified nucleobase.


Exemplified sugar modifications include, but are not limited to the following:




embedded image


wherein B is a modified or unmodified nucleobase.


In some embodiments the thermally destabilizing modification of the duplex is selected from the group consisting of:




embedded image


wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.


The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent or at least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′, or 04′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide




embedded image


wherein B is a modified or unmodified nucleobase, R1 and R2 independently are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.


The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:




embedded image


The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.


In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W—C H-bonding to complementary base on the target mRNA, such as:




embedded image


More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.


The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.


In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:




embedded image


In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more α-nucleotide complementary to the base on the target mRNA, such as:




embedded image


wherein R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.


Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:




embedded image


R=alkyl


The alkyl for the R group can be a C1-C6alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.


As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.


In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.


In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 from the 5′-end.


In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.


In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.


In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.


In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.


Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.


In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2′-fluoro modifications on the antisense strand.


In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 14, and 16 from the 5′-end.


In some embodiments, the antisense strand comprises at least one 2′-fluoro nucleotide adjacent to the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.


In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.


In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four 2′-fluoro nucleotides.


In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.


In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5′-end of the antisense strand. Preferably, the 2 nt overhang is at the 3′-end of the antisense.


In some embodiments, the dsRNA molecule of the disclosure comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1), positions 1 to 23 of said sense strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3′ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3′ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complimentary to positions 14-17 of the 5′-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12-30 nucleotide pairs in length.


In some embodiments, the dsRNA molecule of the disclosure comprises a sense and antisense strands, wherein said dsRNA molecule comprises a sense strand having a length which is at least 25 and at most 29 nucleotides and an antisense strand having a length which is at most 30 nucleotides with the sense strand comprises a modified nucleotide that is susceptible to enzymatic degradation at position 11 from the 5′end, wherein the 3′ end of said sense strand and the 5′ end of said antisense strand form a blunt end and said antisense strand is 1-4 nucleotides longer at its 3′ end than the sense strand, wherein the duplex region which is at least 25 nucleotides in length, and said antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of said antisense strand length to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said antisense strand, thereby reducing expression of the target gene in the mammal, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.


In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.


As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. e.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.


It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.


In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.


At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.


In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc. The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.


In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.


The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.


In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.


In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.


In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.


In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).


In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).


In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).


In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.


In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.


In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.


In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.


In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.


In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.


In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.


In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at 5′-end of the antisense strand.


In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.


In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.


In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.


It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.


In some embodiments, 5′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 4′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 4′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4′-O-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4′-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.


In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.


In another embodiment, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.


Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.


As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.


The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.


The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.


In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2-5. These agents may further comprise a ligand, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both of one of more lipophilic moieties and one or more GalNAc derivatives.


IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).


In certain embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.


Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.


Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine.


Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.


Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.


The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.


In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.


Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.


The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.


In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.


When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.


A. Lipid Conjugates


In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.


A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.


In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.


In certain embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.


In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).


B. Cell Permeation Agents


In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent and can have a lipophilic and a lipophobic phase.


The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.


A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS).


An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:14)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:15)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:16)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.


An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.


An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing αvβ3 (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).


A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).


C. Carbohydrate Conjugates


In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and tri-saccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).


In certain embodiments, a carbohydrate conjugate comprises a monosaccharide.


In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).


In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments the GalNAc conjugate is conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.


In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.


In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.


In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.


In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.


In some embodiments, the GalNAc conjugate is




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In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S




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In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:




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In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:




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In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as




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Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,




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when one of X or Y is an oligonucleotide, the other is a hydrogen.


In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:




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In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands, even if such GalNAc ligands are currently projected to be of limited value for the preferred intrathecal/CNS delivery route(s) of the instant disclosure.


In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.


In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 5′end of the sense strand of a dsRNA agent, or the 5′ end of one or both sense strands of a dual targeting RNAi agent as described herein. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.


In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.


In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.


Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.


D. Linkers


In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.


The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.


A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).


Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.


A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.


A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.


Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.


In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).


i. Redox Cleavable Linking Groups


In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.


ii. Phosphate-Based Cleavable Linking Groups


In certain embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.


iii. Acid Cleavable Linking Groups


In certain embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.


iv. Ester-Based Cleavable Linking Groups


In certain embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.


v. Peptide-Based Cleavable Linking Groups


In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.


In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include but are not limited to




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when one of X or Y is an oligonucleotide, the other is a hydrogen.


In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.


In certain embodiments, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):




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wherein:


q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different;


P2A, P2B, P3A, P3B, P4A, P4B, P5A, P5B, P5C, T2A, T2B, T3A, T3B, T4A, T4B, T4A, T5B, T5C are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;


Q2A, Q2B, Q3A, Q3B, Q4A, Q4B, Q5A, Q5B, Q5C are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R′)═C(R″), C≡C or C(O);


R2A, R2B, R3A, R3B, R4A, R4B, R5A, R5B, R5C are each independently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), —C(O)CH(Ra)—NH—, CO, CH═N—O,




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or heterocyclyl;


L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5B and L5C represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and Ra is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XLIX):




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wherein L5A, L5B and L5C represent a monosaccharide, such as GalNAc derivative.


Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.


Representative U.S. Patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.


It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.


“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNA agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.


In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.


V. Delivery of an RNAi Agent of the Disclosure

The delivery of a RNAi agent of the disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV)), can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an RNAi agent of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an RNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below.


In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a RNAi agent of the disclosure (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. The non-specific effects of an RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies have shown successful knockdown of gene products when an RNAi agent is administered locally. For example, pulmonary system delivery, e.g., inhalation, of a dsRNA, e.g., SOD1, has been shown to effectively knockdown gene and protein expression in lung tissue and that there is excellent uptake of the dsRNA by the bronchioles and alveoli of the lung. Intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were also both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such seed region destabilization). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a RNAi agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making and administering cationic-RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.


Certain aspects of the instant disclosure relate to a method of reducing the expression of a coronavirus target gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In one embodiment, the cell is a hepatic cell, optionally a hepatocyte. In one embodiment, the cell is an extrahepatic cell, optionally a pulmonary system cell.


Another aspect of the disclosure relates to a method of reducing the expression of a coronavirus target gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.


Another aspect of the disclosure relates to a method of treating a subject having a coronavirus-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject. Non-limiting examples of coronavirus-associated diseases include, for example, Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV).


For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. A composition that includes a RNAi agent can be delivered to a subject by a variety of routes. Exemplary routes include pulmonary system, intravenous, intraventricular, topical, rectal, anal, vaginal, nasal, and ocular.


In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration.


In one embodiment, the double-stranded RNAi agent is administered by inhalation.


In one embodiment, the double-stranded RNAi agent is administered by intranasal administration.


In one embodiment, the double-stranded RNAi agent is administered subcutaneously.


The RNAi agents of the disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of RNAi agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.


The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be pulmonary system, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.


The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the RNAi agent in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA.


Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.


Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added.


Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.


Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.


In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.


Pulmonary System Administration


In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration. The pulmonary system includes the upper pulmonary system and the lower pulmonary system. The upper pulmonary system includes the nose and the pharynx. The pharynx includes the nasopharynx, oropharynx, and laryngopharynx. The lower pulmonary system includes the larynx, trachea, carina, bronchi, bronchioles, and alveoli.


Pulmonary system administration may be intranasal administration or oral inhalative administration. Such administration permits both systemic and local delivery of the double stranded RNAi agents of the invention.


Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization. Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays.


Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system. Suitable dosage forms for oral inhalative administration include powders and solutions. Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, N.Y.) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet nebulizers are driven by compressed air. Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode. The holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross-section size on the side from where the droplets emerge. Depending on the therapeutic application, the hole sizes and number of holes can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs.


The amount of RNAi agent for pulmonary system administration may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, preferably 50 μg to 1500 μg, more preferably 100 μg to 1000 μg.


Vector encoded RNAi agents of the Disclosure


RNAi agents targeting the coronavirus genome can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and U.S. Pat. No. 6,054,299). Expression is preferablysustained (months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).


The individual strand or strands of a RNAi agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.


RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a RNAi agent as described herein. Delivery of RNAi agent expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.


Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of a RNAi agent will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells. Other aspects to consider for vectors and constructs are known in the art.


VI. Compositions of the Invention

The present disclosure also provides compositions, including pharmaceutical compositions and formulations which include the RNAi agents of the disclosure.


For example, in one embodiment, the present invention provides compositions comprising two or more, e.g., 2, 3, or 4, dsRNA agents, e.g., dsRNA agents comprising a sense strand and an antisense strand forming a double stranded region, wherein each of the sense strands or each of the antisense strands is a sense strand or an antisense strand independently selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-5. In one embodiment, each of the sense strands or each of the antisense strands is a sense strand or an antisense strand independently selected from the sense strand or antisense strand of a duplex selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1184210, AD-1184270, AD-1184233, AD-1184271, AD-1184212, AD-1184228, AD-1184223, AD-1231490, AD-1231513, AD-1231485, AD-1231507, AD-1231471, AD-1231494, AD-1231496, and AD-1231497. In another embodiment, each of the sense strands or each of the antisense strands is a sense strand or an antisense strand of a duplex independently selected from the group consisting of AD-1184137, AD-1184147, AD-1184150, AD-1231490, AD-1231513, AD-1231485, AD-1231471, AD-1231496, and AD-1231497. In another embodiment, each of the sense strands and each of the antisense strands is a sense strand and an antisense strand of a duplex independently selected from the group consisting of AD-1184137 and AD-1184150. In on embodiment, the composition comprises a first dsRNA agent comprising the sense strand nucleotide sequence 5′-UAACAAUGUUGCUUUUCAAAC-3′(SEQ ID NO: 5) and an antisense strand comprising the nucleotide sequence 5′-GUUUGAAAAGCAACAUUGUUAGU-3′ (SEQ ID NO: 6); and a second dsRNA agent comprising the sense strand nucleotide sequence 5′-ACUGUACAGUCUAAAAUGUCA-3′ (SEQ ID NO: 7) and an antisense strand comprising the nucleotide sequence 5′-UGACAUUUUAGACUGUACAGUGG-3′ (SEQ ID NO: 8). In one embodiment, the sense strand of the first dsRNA agent comprises the sense strand nucleotide sequence 5′-usasaca(Ahd)UfgUfUfGfcuuuucaasasa-3′ (SEQ ID NO: 9) and the antisense strand of the first dsRNA agent comprises the nucleotide sequence 5′-VPusUfsuugAfaaagcaaCfaUfuguuasgsu-3′ (SEQ ID NO: 10); and the sense strand of the second dsRNA agent comprises the nucleotides sequence 5′-ascsugu(Ahd)CfaGfUfCfuaaaauguscsa-3′ (SEQ ID NO: 11) and the antisense strand of the second dsRNA agent comprises the nucleotide sequence 5′-VPusGfsacaUfuuuagacUfgUfacagusgsg-3′ (SEQ ID NO: 12), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; s is a phosphorothioate linkage; (Ahd) is 2′-O-hexadecyl-adenosine-3′-phosphate; and VP is Vinyl-phosphonate.


In another embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, or a composition, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent or the composition are useful for treating a subject who would benefit from inhibiting or reducing the expression of a coronavirus genome, e.g., a subject having a coronavirus-associated disorder, e.g., a subject having a coronavirus infection, e.g., a subject having Severe Acute Respiratory Syndrome 2 (SARS-CoV-2; COVID-19), Severe Acute Respiratory Syndrome (SARS-CoV), or Middle East Respiratory Syndrome (MERS-CoV). Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for direct delivery into the pulmonary system by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal delivery. Another example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery.


In some embodiments, the pharmaceutical compositions of the invention are pyrogen free or non-pyrogenic.


The pharmaceutical compositions of the disclosure may be administered in dosages sufficient to inhibit expression of a coronavirus genome. In general, a suitable dose of an RNAi agent of the disclosure will be a flat dose in the range of about 0.001 to about 200.0 mg about once per month to about once per year, typically about once per quarter (i.e., about once every three months) to about once per year, generally a flat dose in the range of about 1 to 50 mg about once per month to about once per year, typically about once per quarter to about once per year.


After an initial treatment regimen (e.g., loading dose), the treatments can be administered on a less frequent basis.


The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.


Advances in mouse genetics have generated a number of mouse models for the study of various coronavirus-associated diseases that would benefit from reduction in the expression of coronavirus. Such models can be used for in vivo testing of RNAi agents, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, the mouse models described elsewhere herein.


The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary system administration by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.


The RNAi agents can be delivered in a manner to target a particular tissue, such as the liver, the lung (e.g., bronchioles, alveoli, or bronchus of the lung), or both the liver and lung.


Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the RNAi agents featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). RNAi agents featured in the disclosure can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi agents can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C1-20 alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.


A. RNAi Agent Formulations Comprising Membranous Molecular Assemblies


A RNAi agent for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the RNAi agent composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the RNAi agent are delivered into the cell where the RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the RNAi agent to particular cell types.


A liposome containing an RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.


If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.


Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Nat. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Nat. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.


Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).


Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).


One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid or phosphatidylcholine or cholesterol.


Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.


Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).


Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside GM1, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).


Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside GM1, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85,:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside GM1 or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).


In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.


Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.


A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).


A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.


Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).


Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.


Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).


Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.


Liposomes that include RNAi agents can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.


Other formulations amenable to the present disclosure are described in PCT publication No. WO 2008/042973.


Transfersomes, yet another type of liposomes, are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.


Surfactants find wide application in formulations such as those described herein, particularlay in emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).


If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.


If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.


If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.


If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.


The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).


The RNAi agent for use in the methods of the disclosure can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.


A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C8 to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.


In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.


Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.


For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.


Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.


The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.


Lipid Particles


RNAi agents, e.g., dsRNAs of in the disclosure may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.


As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; United States Patent publication No. 2010/0324120 and WO 96/40964.


In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the disclosure.


Certain specific LNP formulations for delivery of RNAi agents have been described in the art, including, e.g., “LNP01” formulations as described in, e.g., WO 2008/042973, which is hereby incorporated by reference.


Additional exemplary lipid-dsRNA formulations are identified in the table below.
















cationic lipid/non-cationic




lipid/cholesterol/PEG-lipid conjugate



Ionizable/Cationic Lipid
Lipid:siRNA ratio







SNALP-1
1,2-Dilinolenyloxy-N,N-
DLinDMA/DPPC/Cholesterol/PEG-



dimethylaminopropane (DLinDMA)
cDMA




(57.1/7.1/34.4/1.4)




lipid:siRNA~7:1


2-XTC
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DPPC/Cholesterol/PEG-cDMA



dioxolane (XTC)
57.1/7.1/34.4/1.4




lipid:siRNA~7:1


LNP05
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG



dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~6:1


LNP06
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG



dioxolane (XTC)
57.5/7.5/31.5/3.5




lipid:siRNA~11:1


LNP07
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG



dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~6:1


LNP08
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG



dioxolane (XTC)
60/7.5/31/1.5,




lipid:siRNA~11:1


LNP09
2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-
XTC/DSPC/Cholesterol/PEG-DMG



dioxolane (XTC)
50/10/38.5/1.5




Lipid:siRNA 10:1


LNP10
(3aR,5s,6aS)-N,N-dimethyl-2,2-
ALN100/DSPC/Cholesterol/PEG-



di((9Z,12Z)-octadeca-9,12-
DMG



dienyl)tetrahydro-3aH-
50/10/38.5/1.5



cyclopenta[d][1,3]dioxol-5-amine
Lipid:siRNA 10:1



(ALN100)



LNP11
(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-
MC-3/DSPC/Cholesterol/PEG-DMG



tetraen-19-yl 4-(dimethylamino)butanoate
50/10/38.5/1.5



(MC3)
Lipid:siRNA 10:1


LNP12
1,1′-(2-(4-(2-((2-(bis(2-
Tech G1/DSPC/Cholesterol/PEG-



hydroxydodecyl)amino)ethyl)
DMG



(2-hydroxydodecyl)amino)ethyl)piperazin-1-
50/10/38.5/1.5



yl)ethylazanediyl)didodecan-2-ol (Tech
Lipid:siRNA 10:1



G1)



LNP13
XTC
XTC/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 33:1


LNP14
MC3
MC3/DSPC/Chol/PEG-DMG




40/15/40/5




Lipid:siRNA: 11:1


LNP15
MC3
MC3/DSPC/Chol/PEG-DSG/GalNAc-




PEG-DSG




50/10/35/4.5/0.5




Lipid:siRNA: 11:1


LNP16
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP17
MC3
MC3/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP18
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/38.5/1.5




Lipid:siRNA: 12:1


LNP19
MC3
MC3/DSPC/Chol/PEG-DMG




50/10/35/5




Lipid:siRNA: 8:1


LNP20
MC3
MC3/DSPC/Chol/PEG-DPG




50/10/38.5/1.5




Lipid:siRNA: 10:1


LNP21
C12-200
C12-200/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 7:1


LNP22
XTC
XTC/DSPC/Chol/PEG-DSG




50/10/38.5/1.5




Lipid:siRNA: 10:1





DSPC: distearoylphosphatidylcholine


DPPC: dipalmitoylphosphatidylcholine


PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000)


PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000)


PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)


SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in WO 2009/127060, which is hereby incorporated by reference.


XTC comprising formulations are described in WO 2010/088537, the entire contents of which are hereby incorporated herein by reference.


MC3 comprising formulations are described, e.g., in United States Patent Publication No. 2010/0324120, the entire contents of which are hereby incorporated by reference.


ALNY-100 comprising formulations are described in WO 2010/054406, the entire contents of which are hereby incorporated herein by reference.


C12-200 comprising formulations are described in WO 2010/129709, the entire contents of which are hereby incorporated herein by reference.






Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. 2003/0027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.


Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.


Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.


Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the brain when treating APP-associated diseases or disorders.


The pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.


The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.


Additional Formulations


i. Emulsions


The compositions of the present disclosure can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 m in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.


Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).


Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).


Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.


A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).


Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.


Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.


The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.


ii. Microemulsions


In one embodiment of the present disclosure, the compositions of RNAi agents and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used, and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).


The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.


Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (M0310), hexaglycerol monooleate (P0310), hexaglycerol pentaoleate (P0500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (M0750), decaglycerol sequioleate (S0750), decaglycerol decaoleate (DA0750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.


Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or RNAi agents. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of RNAi agents and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of RNAi agents and nucleic acids.


Microemulsions of the present disclosure can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the RNAi agents and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.


iii. Microparticles


An RNAi agent of the disclosure may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.


iv. Penetration Enhancers


In one embodiment, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly RNAi agents, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.


Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.


Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of RNAi agents through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).


Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C1-20 alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).


The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).


Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of RNAi agents through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).


As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of RNAi agents through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).


Agents that enhance uptake of RNAi agents at the cellular level can also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.


Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.


vi. Excipients


In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).


Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.


Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.


Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.


vii. Other Components


The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.


Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.


In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more RNAi agents and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a coronavirus-associated disorder. Examples of such agents include, but are not limited to SSRIs, venlafaxine, bupropion, and atypical antipsychotics.


Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.


The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the disclosure lies generally within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.


In addition to their administration, as discussed above, the RNAi agents featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by nucleotide repeat expression. In any event, the administering physician can adjust the amount and timing of RNAi agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.


VII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device. For example, the kit can include a delivery device suitable for pulmonary system administration, e.g., a device suitable for oral inhalative administration including nebulizers, metered-dose inhalers, and dry powder inhalers.


VIII. Methods for Inhibiting Coronavirus Expression

The present disclosure also provides methods of inhibiting expression of a coronavirus genome in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, a composition comprising a double stranded RNAi agent of the invention, or a pharmaceutical compostions comprising a double stranded RNAi agent of the invention in an amount effective to inhibit expression of a coronavirus genome in the cell, thereby inhibiting expression of coronavirus in the cell. In some embodiments, the methods include contacting a cell with two or more double stranded RNAi agents, as described herein, e.g., any two or more, e.g., 2, 3, or 4, of the dsRNA agents selected from the group of dsRNA agents in Tables 2-5. In certain embodiments of the methods including two or more double stranded RNAi agents, the two or more double stranded RNAi agents may be present in the same composition, in separate compositions, or any combination thereof. In some embodiments, the methods of the invention include contacting a cell with a composition comprising two or more, e.g., 2, 3, or 4, double stranded RNAi agents of the invention, e.g., any two or more of the dsRNA agents selected from the group of dsRNA agents in Tables 2-5. In certain embodiments of the disclosure, expression of a coronavirus genome is inhibited preferentially in the pulmonary system (e.g., lung, bronchial, alveoli) cells. In other embodiments of the disclosure, expression of a coronavirus genome is inhibited preferentially in the liver (e.g., hepatocytes). In certain embodiments of the disclosure, expression of a coronavirus genome is inhibited in the pulmonary system (e.g., lung, bronchial, alveoli) cells and in liver (e.g., hepatocytes) cells.


Contacting of a cell with a RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.


In some embodiments of the methods of the invention which include contacting a cell with two or more double stranded RNAi agents, as described herein, e.g., any two or more, e.g., 2, 3, or 4, of the dsRNA agents selected from the group of dsRNA agents in Tables 2-5, the cell may be contacted with a first agent (or a composition comprising a first agent) at a first time, a second agent (or a composition comprising a second agent) at a second time, a third agent (or a composition comprising a third agent) at a third time, and a fourth agent (or a composition comprising a fourth agent) at a fourth time; or the cell may be contacted with all of the agents (or a composition comprising all of the agents) at the same time, Alternatively, the cell may be contacted with a first agent (or a composition comprising a first agent) at a first time and a second, third, and/or fourth agent (or a composition comprising a second, third, and/or fourth agent) at a second time. Other combinations of contacting the cell with two or more agents (or compositions comprising two or more dsRNA agents) of the invention are also contemplated.


Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest.


The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via Lipofectamine™-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., preferably 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art.


The phrase “inhibiting expression of a coronavirus genome” or “inhibiting expression of coronavirus,” as used herein, includes inhibition of expression of any coronavirus genome as well as variants or mutants of a coronavirus genome that encode a coronavirus protein. Thus, the coronavirus genome may be a wild-type coronavirus genome, a mutant coronavirus genome, or a transgenic coronavirus genome in the context of a genetically manipulated cell, group of cells, or organism.


“Inhibiting expression of a coronavirus genome” includes any level of inhibition of a coronavirus genome, e.g., at least partial suppression of the expression of a coronavirus genome, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, preferably at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method. In a preferred method, inhibition is measured at a 10 nM concentration of the siRNA using the luciferase assay provided in Example 1.


The expression of a coronavirus genome may be assessed based on the level of any variable associated with coronavirus genome expression, e.g., coronavirus RNA level or coronavirus protein level or coronavirus replication.


Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).


In some embodiments of the methods of the disclosure, expression of a coronavirus genome is inhibited by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of coronavirus, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a coronavirus genome.


Inhibition of the expression of a coronavirus genome may be manifested by a reduction of the amount of genome expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a coronavirus genome is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of a coronavirus genome is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a RNAi agent targeted to the genome of interest). The degree of inhibition may be expressed in terms of:









(

genome





in





control





cells

)

-

(

genome





in





treated





cells

)



(

genome





in





control





cells

)




100

%




In other embodiments, inhibition of the expression of a coronavirus genome may be assessed in terms of a reduction of a parameter that is functionally linked to a coronavirus genome expression, e.g., coronavirus protein expression. Coronavirus genome silencing may be determined in any cell expressing a coronavirus genome, either endogenous or heterologous from an expression construct, and by any assay known in the art.


Inhibition of the expression of a coronavirus protein may be manifested by a reduction in the level of the coronavirus protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of genome suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.


A control cell or group of cells that may be used to assess the inhibition of the expression of a coronavirus genome includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.


The level of coronavirus genome that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of coronavirus in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., RNA of the coronavirus genome. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating coronavirus genome may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference.


In some embodiments, the level of expression of coronavirus is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific coronavirus nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.


Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to coronavirus RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known RNA detection methods for use in determining the level of coronavirus RNA.


An alternative method for determining the level of expression of coronavirus in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of coronavirus is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of coronavirus genome expression or protein level.


The expression level of coronavirus RNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of coronavirus expression level may also comprise using nucleic acid probes in solution.


In some embodiments, the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of coronavirus nucleic acids.


The level of coronavirus protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of coronavirus proteins.


In some embodiments, the efficacy of the methods of the disclosure in the treatment of a coronavirus-related disease is assessed by a decrease in coronavirus genome level (e.g, by assessment of a coronavirus level, e.g., in the lung, by biopsy, or otherwise).


In some embodiments, the efficacy of the methods of the disclosure in the treatment of a coronavirus-related disease is assessed by a decrease in coronavirus genome level (e.g, by assessment of a liver sample for coronavirus level, by biopsy, or otherwise).


In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of a coronavirus genome may be assessed using measurements of the level or change in the level of coronavirus genome or coronavirus protein in a sample derived from a specific site within the subject, e.g., lung and/or liver cells. In certain embodiments, the methods include a clinically relevant inhibition of expression of coronavirus, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of coronavirus.


As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.


IX. Methods of Treating or Preventing Coronavirus-Associated Diseases

The present disclosure also provides methods of using an RNAi agent of the disclosure, two or more, e.g., 2, 3, or 4, double stranded RNAi agents of the disclosure (e.g., each agent independently targeting a portion of a coronavirus genome), a composition (such as a pharmaceutical composition) containing a RNAi agent of the disclosure, two or more, e.g., 2, 3, or 4, compositions (such as pharmaceutical compositions), each independently comprising a double stranded RNAi agent of the invention, or a composition comprising two or more, e.g., 2, 3, or 4, double stranded RNAi agents of the disclosure to reduce or inhibit coronavirus expression in a cell. The methods include contacting the cell with a dsRNA of the disclosure, a composition of the disclosure, or a pharmaceutical composition of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the RNA transcripts of a coronavirus genome, thereby inhibiting expression of the coronavirus genome in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of coronavirus may be determined by determining the RNA expression level of a coronavirus genome using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a coronavirus protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques.


In the methods of the disclosure the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.


A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a coronavirus genome. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human lung cell. In one embodiment, the cell is a human cell, e.g., a human liver cell. In one embodiment, the cell is a human cell, e.g., a human lung cell and a human liver cell.


Coronavirus genome expression is inhibited in the cell by at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or about 100%, i.e., to below the level of detection. In preferred embodiments, coronavirus expression is inhibited by at least 50%.


The in vivo methods of the disclosure may include administering to a subject a composition containing a RNAi agent, where the RNAi agent includes a nucleotide sequence that is complementary to at least a part of target coronavirus sequence, e.g., an RNA transcript of the coronavirus genome, of the coronavirus to be treated. In some embodiments, the subject is administered two or more, e.g., 2, 3, or 4, compositions, each independently comprising an RNAi agent of the invention. The compositions may be the same or different. In other embodiments, the subject is administered a composition comprising two or more, e.g., 2, 3, or 4, dsRNA agents, each independently targeting a portion of the coronavirus genome.


When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by pulmonary system delivery, e.g., inhalation or intranasal delivery.


In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of coronavirus, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.


In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration, e.g., intranasal administration or oral inhalative administration. Pulmonary system administration may be via a syringe, a dropper, atomization, or use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI) device.


The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.


In one aspect, the present disclosure also provides methods for inhibiting the expression of a coronavirus genome in a mammal. The methods include administering to the mammal a dsRNA that targets a coronavirus genome in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the RNA transcript of the coronavirus genome, thereby inhibiting expression of the coronavirus genome in the cell. In some embodiments, the dsRNA is present in a composition, such as a pharmaceutical composition. In some embodiments, the mammal is administered two or more, e.g., 2, 3, or 4, dsRNA agents of the invention. In some embodiments, each dsRNA agent administered to the subject is independently present in a composition. In other embodiments, the mammal is administered a composition comprising two or more, e.g., 2, 3, or 4, dsRNAs of the invention.


Reduction in genome expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a lung biopsy sample serves as the tissue material for monitoring the reduction in coronavirus genome or protein expression (or of a proxy therefore).


The present disclosure further provides methods of treatment of a subject in need thereof. The treatment methods of the disclosure include administering an RNAi agent of the disclosure to a subject, e.g., a subject that would benefit from inhibition of coronavirus expression, in a therapeutically effective amount of a RNAi agent targeting a coronavirus genome or a pharmaceutical composition comprising a RNAi agent targeting a coronavirus genome. In some embodiments, the subject is administered a therapeutically effective amount of two or more, e.g., 2, 3, or 4, dsRNA agents of the invention. In some embodiments, each dsRNA agent administered to the subject is independently present in a composition. In other embodiments, the subject is administered a composition comprising two or more, e.g., 2, 3, or 4, dsRNAs of the invention.


In addition, the present disclosure provides methods of preventing, treating or inhibiting the progression of a coronavirus-associated disease or disorder, such as severe acute respiratory syndrome (SARS), the Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome-2 (SARS-2). The methods include administering to the subject a therapeutically effective amount of any of the RNAi agent, e.g., dsRNA agents, or the pharmaceutical composition provided herein, thereby preventing, treating, or inhibiting the progression of the coronavirus-associated disease or disorder in the subject. In some embodiments, the mammal is administered a therapeutically effective amount of two or more, e.g., 2, 3, or 4, dsRNA agents of the invention. In some embodiments, each dsRNA agent administered to the subject is independently present in a composition. In other embodiments, the mammal is administered a composition comprising two or more, e.g., 2, 3, or 4, dsRNAs of the invention.


In some embodiments of the methods of the invention which include administering two or more double stranded RNAi agents, as described herein, e.g., any two or more, e.g., 2, 3, or 4, of the dsRNA agents selected from the group of dsRNA agents in Tables 2-5, the the subject may be administered a first agent (or a composition comprising a first agent) at a first time, a second agent (or a composition comprising a second agent) at a second time, a third agent (or a compositions comprising a third agent) at a third time, and a fourth agent (or a composition comprising a fourth agent) at a fourth time; or the the subject may be administered all of the agents (or a composition comprising all of the agents at the same time, Alternatively, the subject may be administered a first agent (or a composition comprising a first agent) at a first time and a second, third, and/or fourth agent (or a compostion comprising a second, third and.or fourth agent) at a second time. Other combinations of contacting the cell with two or more agents of the invention are also contemplated.


An RNAi agent of the disclosure may be administered as a “free RNAi agent.” A free RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject.


Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.


Subjects that would benefit from a reduction or inhibition of coronavirus genome expression are those having a coronavirus-associated disease, e.g., subjects of an age greater than 60 years and/or subjects who are immunocompromised.


The disclosure further provides methods for the use of a RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of coronavirus expression, e.g., a subject having a coronavirus-associated disorder, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an RNAi agent targeting coronavirus is administered in combination with, e.g., an agent useful in treating a coronavirus-associated disorder as described elsewhere herein or as otherwise known in the art. For example, additional agents and treatments suitable for treating a subject that would benefit from reduction in coronavirus expression, e.g., a subject having a coronavirus-associated disorder, may include agents currently used to treat symptoms of coronavirus. The RNAi agent and additional therapeutic agents may be administered at the same time or in the same combination, e.g., via pulmonary system administration, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.


Exemplary additional therapeutics and treatments include, for example, an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.


In one embodiment, the method includes administering a composition featured herein such that expression of the target coronavirus genome is decreased, for at least one month. In preferred embodiments, expression is decreased for at least 2 months, or 6 months.


Preferably, the RNAi agents useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target coronavirus genome. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and performed as described herein.


Administration of the dsRNA according to the methods of the disclosure may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a coronavirus-associated disorder. By “reduction” in this context is meant a statistically significant or clinically significant decrease in such level. The reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.


Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a RNAi agent targeting coronavirus or pharmaceutical composition thereof, “effective against” a coronavirus-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating coronavirus-associated disorders and the related causes.


A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50%, or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.


Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using a RNAi agent or RNAi agent formulation as described herein.


Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg.


The RNAi agent can be administered via the pulmonary system over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the RNAi agent can reduce coronavirus genome levels, e.g., in a cell, tissue, blood, lung sample or other compartment of the patient by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70,% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In a preferred embodiment, administration of the RNAi agent can reduce coronavirus genome levels, e.g., in a cell, tissue, blood, pulmonary system sample or other compartment of the patient by at least 50%.


Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.


Alternatively, the RNAi agent can be administered by pulmonary system administration or subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired, e.g., monthly dose of RNAi agent to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimine may include administration of a therapeutic amount of RNAi agent on a regular basis, such as monthly or extending to once a quarter, twice per year, once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (i.e., about once every three months).


Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RNAi agents and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.


An informal Sequence Listing is filed herewith and forms part of the specification as filed.


This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are hereby incorporated herein by reference.


EXAMPLES
Example 1. iRNA Synthesis
Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.


siRNA Design


The selection of siRNA designs targeting coronavirus was driven by two primary factors: a) potency and b), the desire to employ siRNA with near-perfect matches with greater than 90% fractional coverage of the known coronaviruses sequences, e.g., SARS-CoV, MERS-CoV and SARS-CoV-2.


A detailed list of a set of the unmodified siRNA sense and antisense strand sequences targeting SARS-CoV-2 is shown in Table 2 and 4.


A detailed list of a set of the modified siRNA sense and antisense strand sequences targeting SARS-CoV-2 is shown in Tables 3 and 5.


siRNA Synthesis


siRNAs were synthesized and annealed using routine methods known in the art. Briefly, siRNA sequences were synthesized on a 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. The solid support was controlled pore glass (500-1000 Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universal solid support (AM Chemicals), or the first nucleotide of interest. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, Wis.), Hongene (China), or Chemgenes (Wilmington, Mass., USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs. Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”).


Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 μL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2′-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 μL of dimethyl sulfoxide (DMSO) and 300 μL TEA.3HF and the solution was incubated for approximately 30 mins at 60° C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively.


Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 μM in 1× PBS in 96 well plates, the plate sealed, incubated at 100° C. for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.


Example 2. In Vitro Screening of siRNA Duplexes
SARS-CoV-02 Expression Plasmid Construction

Dual-Glo® Luciferase constructs generated in the psiCHECK-2 vector which was obtained from Promega (Catalog No. C8021 (Madison, Wis.)). The SARS-CoV-2 vector sequences were synthesized and incorporated into the psiCHECK2 vector by Blue Heron Biotech (Bothell, Wash.). The final constructs are referred to CV-concat-02 and CV-concat-21 (see, FIG. 2). The nucleotide sequence of SARS-CoV-2 incorporated into the psiCHECK-2 vector to generate CV-concat-02 is provided in SEQ ID NO:3 and the nucleotide sequence of SARS-CoV-2 incorporated into the psiCHECK-2 vector to generate CV-concat-21 is provided in SEQ ID NO:4,


Cell Culture and Transfections

Cos7 cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO2 in DMEM (ATCC) supplemented with 10% FBS, before being released from the plate by trypsinization. Cos7 cells were co-transfected with psiCHECK2-CV-concat-02 or psiCHECK2-CV-concat-21 plasmids and siRNA in 384-well plates at a density of 5×104 cells per well using Lipofectamine™ 2000 transfection reagent (Catalog No. 11668019, Invitrogen (Carlsbad, Calif.)). For each well of a 384 well plate, 0.1 μl of Lipofectamine was added to 50 ng of plasmid vector in 5 μl of Opti-MEM and allowed to complex at room temperature for 15 minutes. The mixture was then added to the cells which were resuspended in 40 μl of fresh complete media. Cells were incubated for 48 hours before luciferase is measured.


Single dose experiments are performed at 1 nM or 10 nM final duplex concentration.


Dual-Glo® Luciferase Assay (Promega, Cat No. E2980)


Forty-eight hours after the siRNAs were transfected, Firefly (transfection control) and Renilla (fused to SARS-CoV-2 target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 20 μl of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture was incubated at room temperature for 30 minutes before lunimescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 20 μl of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates are incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent, quench the firefly luciferase signal and sustain luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (SARS-CoV-2) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but are not treated with siRNA or are treated with a non-targeting siRNA. All transfections are done at n=4 or greater.


The results of the single dose screens of the duplexes in Tables 2 and 3 are provided in Table 6 and the results of the single dose screens of the duplexes in Tables 4 and 5 are provided in Table 7.









TABLE 1







Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers,


when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds.








Abbreviation
Nucleotide(s)





A
Adenosine-3’-phosphate


Ab
beta-L-adenosine- custom-character -phosphate


Abs
beta-L-adenosine-3′-phosphorothioate


Af
2’-fluoroadenosine-3’-phosphate


Afs
2’-fluoroadenosine-3’-phosphorothioate


As
adenosine-3’-phosphorothioate


C
cytidine-3’-phosphate


Cb
beta-L-cytidine-custom-character -phosphate


Cbs
beta-L-cytidine-3′-phosphorothioate


Cf
2’-fluorocytidine-3’-phosphate


Cfs
2’-fluorocytidine-3’-phosphorothioate


Cs
cytidine-3’-phosphorothioate


G
guanosine-3’-phosphate


Gb
beta-L-guanosine- custom-character -phosphate


Gbs
beta-L-guanosine- custom-character -phosphorothioate


Gf
2’-fluoroguanosine-3’-phosphate


Gfs
2’-fluoroguanosine-3’-phosphorothioate


Gs
guanosine-3’-phosphorothioate


T
5’-methyluridine-3’-phosphate


Tf
2’-fluoro-5-methyluridine-3’-phosphate


Tfs
2’-fluoro-5-methyluridine-3’-phosphorothioate


Ts
5-methyluridine-3’-phosphorothioate


U
Uridine-3’-phosphate


Uf
2’-fluorouridine-3’-phosphate


Ufs
2’-fluorouridine-3’-phosphorothioate


Us
uridine-3’-phosphorothioate


N
any nucleotide, modified or unmodified


a
2′-O-methyladenosine-3’-phosphate


as
2′-O-methyladenosine-3’-phosphorothioate


c
2′-O-methylcytidine-3’-phosphate


cs
2′-O-methylcytidine-3’-phosphorothioate


g
2′-O-methylguanosine-3’-phosphate


gs
2′-O-methylguanosine-3’-phosphorothioate


t
2’-O-methyl-5-methyluridine-3’-phosphate


ts
2’-O-methyl-5-methyluridine-3’-phosphorothioate


u
2′-O-methyluridine-3’-phosphate


us
2′-O-methyluridine-3’-phosphorothioate


s
phosphorothioate linkage


L96
N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol(Hyp-(GalNAc-alkyl)3)








embedded image







Y34
2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose)


Y44
inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate)


L10
N-(cholesterylcarboxamidocaproy1)-4-hydroxyprolinol (Hyp-C6-Chol)


(Agn)
Adenosine-glycol nucleic acid (GNA)


(Cgn)
Cytidine-glycol nucleic acid (GNA)


(Ggn)
Guanosine-glycol nucleic acid (GNA)


(Tgn)
Thymidine-glycol nucleic acid (GNA) S-Isomer


P
Phosphate


VP
Vinyl-phosphonate


dA

custom-character -deoxyadenosine- custom-character -phosphate



dAs

custom-character -deoxyadenosine- custom-character -phosphorothioate



dC

custom-character -deoxycytidine- custom-character -phosphate



dCs

custom-character -deoxycytidine- custom-character -phosphorothioate



dG

custom-character -deoxyguanosine- custom-character -phosphate



dGs

custom-character -deoxyguanosine- custom-character -phosphorothioate



dT

custom-character -deoxythymidine- custom-character -phosphate



dTs

custom-character -deoxythymidine- custom-character -phosphorothioate



dU

custom-character -deoxyuridine



dUs

custom-character -deoxyuridine-custom-character -phosphorothioate



(C2p)
cytidine- custom-character -phosphate


(G2p)
guanosine- custom-character -phosphate


(U2p)
uridine- custom-character -phosphate


(A2p)
adenosine- custom-character -phosphate


(Ahd)
2′-O-hexadecyl-adenosine-3′-phosphate


(Ahds)
2′-O-hexadecyl-adenosine-3′-phosphorothioate


(Chd)
2′-O-hexadecyl-cytidine-3′-phosphate


(Chds)
2′-O-hexadecyl-cytidine-3′-phosphorothioate


(Ghd)
2′-O-hexadecyl-guanosine-3′-phosphate


(Ghds)
2′-O-hexadecyl-guanosine-3′-phosphorothioate


(Uhd)
2′-O-hexadecyl-uridine-3′-phosphate


(Uhds)
2′-O-hexadecyl-uridine-3′-phosphorothioate
















TABLE 2







Unmodified Sense and Antisense Strand Coronavirus dsRNA Sequences





























Strand













of the













Viral













RNA




SEQ

SEQ






Targeted


Duplex
Sense Strand
ID
Antisense Strand
ID
Target RNA
mRNA
mRNA
Targets
Targets
Targets
by the


Name
Sequence 5′ to 3′
NO:
Sequence 5′ to 3′
NO
Accession Version
start
end
SARS2?
SARS?
MERS?
Agent





















AD-
AUCUGUUCUCUAAACGAACU
17
AAGUUCGUUUAGAGAACAGAUC
372
BetaCoV/Wuhan-Hu-
55
77
TRUE
TRUE
FALSE
+


1183935
U

U

1/2019|EPI_ISL_402125











AD-
UCUGUUCUCUAAACGAACUU
18
AAAGUUCGUUUAGAGAACAGAU
373
BetaCoV/Wuhan-Hu-
56
78
TRUE
TRUE
FALSE
+


1183936
U

C

1/2019|EPI_ISL_402125











AD-
GUUCUCUAAACGAACUUUAA
19
UUUAAAGUUCGUUUAGAGAACA
374
BetaCoV/Wuhan-Hu-
59
81
TRUE
TRUE
FALSE
+


1183937
A

G

1/2019|EPL_ISL_402125











AD-
CUGCUUACGGUUUCGUCCGU
20
CACGGACGAAACCGUAAGCAGC
375
BetaCoV/Wuhan-Hu-
188
210
TRUE
TRUE
FALSE
+


1183938
G

C

1/2019|EPL_ISL_402125











AD-
GGUGUGACCGAAAGGUAAGA
21
AUCUUACCUUUCGGUCACACCC
376
BetaCoV/Wuhan-Hu-
245
267
TRUE
TRUE
FALSE
+


1183939
U

G

1/2019|EPL_ISL_402125











AD-
CCGAAAGGUAAGAUGGAGAG
22
GCUCUCCAUCUUACCUUUCGGU
377
BetaCoV/Wuhan-Hu-
252
274
TRUE
TRUE
FALSE
+


1183940
C

C

1/2019|EPL_ISL_402125











AD-
AAACACACGUCCAACUCAGU
23
AACUGAGUUGGACGUGUGUUUU
378
BetaCoV/Wuhan-Hu-
295
317
TRUE
TRUE
FALSE
+


1183941
U

C

1/2019|EPL_ISL_402125











AD-
ACACACGUCCAACUCAGUUU
24
CAAACUGAGUUGGACGUGUGUU
379
BetaCoV/Wuhan-Hu-
297
319
TRUE
TRUE
FALSE
+


1183942
G

U

1/2019|EPL_ISL_402125











AD-
GUCCAACUCAGUUUGCCUGU
25
AACAGGCAAACUGAGUUGGACG
380
BetaCoV/Wuhan-Hu-
303
325
TRUE
TRUE
FALSE
+


1183943
U

U

1/2019|EPL_ISL_402125











AD-
CUUCUUCGUAAGAACGGUAA
26
AUUACCGUUCUUACGAAGAAGA
381
BetaCoV/Wuhan-Hu-
627
649
TRUE
TRUE
FALSE
+


1183944
U

A

1/2019|EPL_ISL_402125











AD-
UUCUUCGUAAGAACGGUAAU
27
UAUUACCGUUCUUACGAAGAAG
382
BetaCoV/Wuhan-Hu-
628
650
TRUE
TRUE
FALSE
+


1183945
A

A

1/2019|EPL_ISL_402125











AD-
UCUUCGUAAGAACGGUAAUA
28
UUAUUACCGUUCUUACGAAGAA
383
BetaCoV/Wuhan-Hu-
629
651
TRUE
TRUE
FALSE
+


1183946
A

G

1/2019|EPL_ISL_402125











AD-
UUCCUUAAACUUCUCUUCAA
29
CUUGAAGAGAAGUUUAAGGAA
384
BetaCoV/Wuhan-Hu-
2156
2178
TRUE
FALSE
FALSE



1183947
G

GG

1/2019|EPL_ISL_402125











AD-
UCACCUAAAUUCAAGGCUUU
30
UAAAGCCUUGAAUUUAGGUGAA
385
BetaCoV/Wuhan-Hu-
2365
2387
TRUE
FALSE
FALSE



1183948
A

A

1/2019|EPL_ISL_402125











AD-
CUCUGGAUUUAACACACUUU
31
GAAAGUGUGUUAAAUCCAGAGA
386
BetaCoV/Wuhan-Hu-
2418
2440
TRUE
FALSE
FALSE



1183949
C

A

1/2019|EPL_ISL_402125











AD-
CUUCUCAUUAAGUACUUUAU
32
GAUAAAGUACUUAAUGAGAAG
387
BetaCoV/Wuhan-Hu-
2813
2835
TRUE
FALSE
FALSE



1183950
C

UG

1/2019|EPL_ISL_402125











AD-
ACUCUUCUUCUUCACAAUCA
33
GUGAUUGUGAAGAAGAAGAGU
388
BetaCoV/Wuhan-Hu-
3066
3088
TRUE
FALSE
FALSE



1183951
C

UU

1/2019|EPL_ISL_402125











AD-
UUAUUUAAAACUUACUGACA
34
UUGUCAGUAAGUUUUAAAUAAC
389
BetaCoV/Wuhan-Hu-
3353
3375
TRUE
TRUE
FALSE
+


1183952
A

C

1/2019|EPL_ISL_402125











AD-
UAUCUACACAAACUCUUAAA
35
CUUUAAGAGUUUGUGUAGAUAC
390
BetaCoV/Wuhan-Hu-
3747
3769
TRUE
FALSE
FALSE



1183953
G

U

1/2019|EPL_ISL_402125











AD-
AUCUUGUUUUCUCUGUUCAA
36
GUUGAACAGAGAAAACAAGAUG
391
BetaCoV/Wuhan-Hu-
3929
3951
TRUE
FALSE
FALSE



1183954
C

A

1/2019|EPL_ISL_402125











AD-
CACUUUUAUCACCUCUCUUA
37
UUAAGAGAGGUGAUAAAAGUG
392
BetaCoV/Wuhan-Hu-
4848
4870
TRUE
FALSE
FALSE



118395
A

UA

1/2019|EPL_ISL_402125











AD-
UCUCUGAAGAAGUAGUGGAA
38
UUUCCACUACUUCUUCAGAGAC
393
BetaCoV/Wuhan-Hu-
6403
6425
TRUE
TRUE
FALSE
+


1183956
A

U

1/2019|EPL_ISL_402125











AD-
UCUGAAGAAGUAGUGGAAAA
39
AUUUUCCACUACUUCUUCAGAG
394
BetaCoV/Wuhan-Hu-
6405
6427
TRUE
TRUE
FALSE
+


1183957
U

A

1/2019|EPL_ISL_402125











AD-
CUGAAGAAGUAGUGGAAAAU
40
GAUUUUCCACUACUUCUUCAGA
395
BetaCoV/Wuhan-Hu-
6406
6428
TRUE
TRUE
FALSE
+


1183958
C

G

1/2019|EPL_ISL_402125











AD-
AGUGGAAAAUCCUACCAUAC
41
UGUAUGGUAGGAUUUUCCACUA
396
BetaCoV/Wuhan-Hu-
6416
6438
TRUE
TRUE
FALSE
+


1183959
A

C

1/2019|EPL_ISL_402125











AD-
GUGGAAAAUCCUACCAUACA
42
CUGUAUGGUAGGAUUUUCCACU
397
BetaCoV/Wuhan-Hu-
6417
6439
TRUE
TRUE
FALSE
+


1183960
G

A

1/2019|EPL_ISL_402125











AD-
GCUCUUCUAAACCUCAUAAA
43
UUUUAUGAGGUUUAGAAGAGC
398
BetaCoV/Wuhan-Hu-
9454
9476
TRUE
FALSE
FALSE



1183961
A

UU

1/2019|EPL_ISL_402125











AD-
AUGGUUCACCAUCUGGUGUU
44
AAACACCAGAUGGUGAACCAUU
399
BetaCoV/Wuhan-Hu-
10408
10430
TRUE
TRUE
FALSE
+


1183962
U

G

1/2019|EPL_ISL_402125











AD-
CAUGUGGUAGUGUUGGUUUU
45
UAAAACCAACACUACCACAUGA
400
BetaCoV/Wuhan-Hu-
10483
10505
TRUE
TRUE
FALSE
+


1183963
A

A

1/2019|EPL_ISL_402125











AD-
CAGUCAUAAUCUAUGUUAAA
46
UUUUAACAUAGAUUAUGACUGU
401
BetaCoV/Wuhan-Hu-
10501
10523
TRUE
FALSE
FALSE



1183964
A

G

1/2019|EPL_ISL_402125











AD-
UUUUGAUGUUGUUAGACAAU
47
CAUUGUCUAACAACAUCAAAAG
402
BetaCoV/Wuhan-Hu-
10931
10953
TRUE
TRUE
FALSE
+


1183965
G

G

1/2019|EPL_ISL_402125











AD-
AUGGUAAUGCUUUAGAUCAA
48
CUUGAUCUAAAGCAUUACCAUA
403
BetaCoV/Wuhan-Hu-
11431
11453
TRUE
TRUE
FALSE
+


1183966
G

A

1/2019|EPL_ISL_402125











AD-
GCUAGAUUCCCUAAGAGUGA
49
AUCACUCUUAGGGAAUCUAGCC
404
BetaCoV/Wuhan-Hu-
12843
12865
TRUE
TRUE
FALSE
+


1183967
U

C

1/2019|EPL_ISL_402125











AD-
ACAACAUCUUAACACAAUUA
50
CUAAUUGUGUUAAGAUGUUGU
405
BetaCoV/Wuhan-Hu-
13140
13162
TRUE
FALSE
FALSE



1183968
G

GU

1/2019|EPL_ISL_402125











AD-
UAAGAUGUUGUGUACACACA
51
GUGUGUGUACACAACAUCUUAA
406
BetaCoV/Wuhan-Hu-
13148
13170
TRUE
TRUE
FALSE
+


1183969
C

C

1/2019|EPL_ISL_402125











AD-
AGAUGUUGUGUACACACACU
52
CAGUGUGUGUACACAACAUCUU
407
BetaCoV/Wuhan-Hu-
13150
13172
TRUE
TRUE
FALSE
+


1183970
G

A

1/2019|EPL_ISL_402125











AD-
GUUGUGUACACACACUGGUA
53
GUACCAGUGUGUGUACACAACA
408
BetaCoV/Wuhan-Hu-
13154
13176
TRUE
TRUE
FALSE
+


118397
C

U

1/2019|EPL_ISL_402125











AD-
CAGGCACUAGUACUGAUGUC
54
CGACAUCAGUACUAGUGCCUGU
409
BetaCoV/Wuhan-Hu-
13509
13531
TRUE
TRUE
FALSE
+


1183972
G

G

1/2019|EPL_ISL_402125











AD-
CCGUCUAUUCUAAACUUAAA
55
CUUUAAGUUUAGAAUAGACGGU
410
BetaCoV/Wuhan-Hu-
13743
13765
TRUE
FALSE
FALSE



1183973
G

G

1/2019|EPL_ISL_402125











AD-
UUUUAAAUAUUGGGAUCAGA
56
GUCUGAUCCCAAUAUUUAAAAU
411
BetaCoV/Wuhan-Hu-
14296
14318
TRUE
FALSE
TRUE
+


1183974
C

A

1/2019|EPL_ISL_402125











AD-
UUUAAAUAUUGGGAUCAGAC
57
UGUCUGAUCCCAAUAUUUAAAA
412
BetaCoV/Wuhan-Hu-
14297
14319
TRUE
FALSE
TRUE
+


1183975
A

U

1/2019|EPL_ISL_402125











AD-
CACCUACAAGUUUUGGACCA
58
GUGGUCCAAAACUUGUAGGUGG
413
BetaCoV/Wuhan-Hu-
14403
14425
TRUE
TRUE
FALSE
+


1183976
C

G

1/2019|EPL_ISL_402125











AD-
AAUCAGGAUGUAAACUUACA
59
AUGUAAGUUUACAUCCUGAUUA
414
BetaCoV/Wuhan-Hu-
14504
14526
TRUE
TRUE
FALSE
+


1183977
U

U

1/2019|EPL_ISL_402125











AD-
AGGAUGUAAACUUACAUAGC
60
AGCUAUGUAAGUUUACAUCCUG
415
BetaCoV/Wuhan-Hu-
14508
14530
TRUE
TRUE
FALSE
+


1183978
U

A

1/2019|EPL_ISL_402125











AD-
ACAAUGUUGCUUUUCAAACU
61
CAGUUUGAAAAGCAACAUUGUU
416
BetaCoV/Wuhan-Hu-
14646
14668
TRUE
TRUE
FALSE
+


1183979
G

A

1/2019|EPL_ISL_402125











AD-
UGUUGCUUUUCAAACUGUCA
62
UUGACAGUUUGAAAAGCAACAU
417
BetaCoV/Wuhan-Hu-
14650
14672
TRUE
TRUE
FALSE
+


1183980
A

U

1/2019|EPL_ISL_402125











AD-
UUGCUUUUCAAACUGUCAAA
63
GUUUGACAGUUUGAAAAGCAAC
418
BetaCoV/Wuhan-Hu-
14652
14674
TRUE
TRUE
FALSE
+


1183981
C

A

1/2019|EPL_ISL_402125











AD-
GCUUUUCAAACUGUCAAACC
64
GGGUUUGACAGUUUGAAAAGCA
419
BetaCoV/Wuhan-Hu-
14654
14676
TRUE
TRUE
FALSE
+


1183982
C

A

1/2019|EPL_ISL_402125











AD-
UAUGACUUUGCUGUGUCUAA
65
CUUAGACACAGCAAAGUCAUAG
420
BetaCoV/Wuhan-Hu-
14696
14718
TRUE
TRUE
FALSE
+


1183983
G

A

1/2019|EPL_ISL_402125











AD-
GUUUCUUUAAGGAAGGAAGU
66
AACUUCCUUCCUUAAAGAAACC
421
BetaCoV/Wuhan-Hu-
14718
14740
TRUE
TRUE
FALSE
+


1183984
U

C

1/2019|EPL_ISL_402125











AD-
UUUCUUUAAGGAAGGAAGUU
67
GAACUUCCUUCCUUAAAGAAAC
422
BetaCoV/Wuhan-Hu-
14719
14741
TRUE
TRUE
FALSE
+


1183985
C

C

1/2019|EPL_ISL_402125











AD-
AGGAAGGAAGUUCUGUUGAA
68
AUUCAACAGAACUUCCUUCCUU
423
BetaCoV/Wuhan-Hu-
14727
14749
TRUE
TRUE
FALSE
+


1183986
U

A

1/2019|EPL_ISL_402125











AD-
AACACUUCUUCUUUGCUCAG
69
CCUGAGCAAAGAAGAAGUGUUU
424
BetaCoV/Wuhan-Hu-
14751
14773
TRUE
TRUE
FALSE
+


1183987
G

U

1/2019|EPL_ISL_402125











AD-
ACUUCUUCUUUGCUCAGGAU
70
CAUCCUGAGCAAAGAAGAAGUG
425
BetaCoV/Wuhan-Hu-
14754
14776
TRUE
TRUE
FALSE
+


1183988
G

U

1/2019|EPL_ISL_402125











AD-
AAUGUGUGAUAUCAGACAAC
71
AGUUGUCUGAUAUCACACAUUG
426
BetaCoV/Wuhan-Hu-
14824
14846
TRUE
TRUE
FALSE
+


1183989
U

U

1/2019|EPL_ISL_402125











AD-
CCAUUUAAUAAAUGGGGUAA
72
CUUACCCCAUUUAUUAAAUGGA
427
BetaCoV/Wuhan-Hu-
14951
14973
TRUE
TRUE
FALSE
+


1183990
G

A

1/2019|EPL_ISL_402125











AD-
CAAUGAGUUAUGAGGAUCAA
73
CUUGAUCCUCAUAACUCAUUGA
428
BetaCoV/Wuhan-Hu-
14991
15013
TRUE
TRUE
FALSE
+


1183991
G

A

1/2019|EPL_ISL_402125











AD-
UGCAAAGAAUAGAGCUCGCA
74
GUGCGAGCUCUAUUCUUUGCAC
429
BetaCoV/Wuhan-Hu-
15085
15107
TRUE
TRUE
TRUE
+


1183992
C

U

1/2019|EPL_ISL_402125











AD-
GCAAAGAAUAGAGCUCGCAC
75
GGUGCGAGCUCUAUUCUUUGCA
430
BetaCoV/Wuhan-Hu-
15086
15108
TRUE
TRUE
TRUE
+


1183993
C

C

1/2019|EPL_ISL_402125











AD-
CAAAGAAUAGAGCUCGCACC
76
CGGUGCGAGCUCUAUUCUUUGC
431
BetaCoV/Wuhan-Hu-
15087
15109
TRUE
TRUE
TRUE
+


1183994
G

A

1/2019|EPL_ISL_402125











AD-
AAAGAAUAGAGCUCGCACCG
77
ACGGUGCGAGCUCUAUUCUUUG
432
BetaCoV/Wuhan-Hu-
15088
15110
TRUE
TRUE
TRUE
+


1183995
U

C

1/2019|EPL_ISL_402125











AD-
UGUCUCUAUCUGUAGUACUA
78
AUAGUACUACAGAUAGAGACAC
433
BetaCoV/Wuhan-Hu-
15115
15137
TRUE
TRUE
FALSE
+


1183996
U

C

1/2019|EPL_ISL_402125











AD-
CUCUAUCUGUAGUACUAUGA
79
GUCAUAGUACUACAGAUAGAGA
434
BetaCoV/Wuhan-Hu-
15118
15140
TRUE
TRUE
FALSE
+


1183997
C

C

1/2019|EPL_ISL_402125











AD-
UCACCUUAUGGGUUGGGAUU
80
UAAUCCCAACCCAUAAGGUGAG
435
BetaCoV/Wuhan-Hu-
15274
15296
TRUE
TRUE
TRUE
+


1183998
A

G

1/2019|EPL_ISL_402125











AD-
CACCUUAUGGGUUGGGAUUA
81
AUAAUCCCAACCCAUAAGGUGA
436
BetaCoV/Wuhan-Hu-
15275
15297
TRUE
TRUE
TRUE
+


1183999
U

G

1/2019|EPL_ISL_402125











AD-
ACCUUAUGGGUUGGGAUUAU
82
GAUAAUCCCAACCCAUAAGGUG
437
BetaCoV/Wuhan-Hu-
15276
15298
TRUE
TRUE
TRUE
+


1184000
C

A

1/2019|EPL_ISL_402125











AD-
CCUUAUGGGUUGGGAUUAUC
83
GGAUAAUCCCAACCCAUAAGGU
438
BetaCoV/Wuhan-Hu-
15277
15299
TRUE
TRUE
TRUE
+


1184001
C

G

1/2019|EPL_ISL_402125











AD-2
ACUUGUUCUUGCUCGCAAAC
84
UGUUUGCGAGCAAGAACAAGUG
439
BetaCoV/Wuhan-Hu-
15343
15365
TRUE
TRUE
FALSE
+


1184002
A

A

1/2019|EPL_ISL_402125











AD-
CUUGUUCUUGCUCGCAAACA
85
AUGUUUGCGAGCAAGAACAAGU
440
BetaCoV/Wuhan-Hu-
15344
15366
TRUE
TRUE
FALSE
+


1184003
U

G

1/2019|EPL_ISL_402125











AD-
CACAACUGCUUAUGCUAAUA
86
CUAUUAGCAUAAGCAGUUGUGG
441
BetaCoV/Wuhan-Hu-
15493
15515
TRUE
TRUE
FALSE
+


1184004
G

C

1/2019|EPL_ISL_402125











AD-
UGAGUGUCUCUAUAGAAAUA
87
CUAUUUCUAUAGAGACACUCAU
442
BetaCoV/Wuhan-Hu-
15622
15644
TRUE
TRUE
FALSE
+


1184005
G

A

1/2019|EPL_ISL_402125











AD-
UGGACUGAGACUGACCUUAC
88
AGUAAGGUCAGUCUCAGUCCAA
443
BetaCoV/Wuhan-Hu-
15836
15858
TRUE
TRUE
FALSE
+


1184006
U

C

1/2019|EPL_ISL_402125











AD-
UUACCCAGAUCCAUCAAGAA
89
AUUCUUGAUGGAUCUGGGUAAG
444
BetaCoV/Wuhan-Hu-
15928
15950
TRUE
TRUE
FALSE
+


1184007
U

G

1/2019|EPL_ISL_402125











AD-
GAUGGUACACUUAUGAUUGA
90
UUCAAUCAUAAGUGUACCAUCU
445
BetaCoV/Wuhan-Hu-
15989
16011
TRUE
TRUE
FALSE
+


1184008
A

G

1/2019|EPL_ISL_402125











AD-
AAACAUCCUAAUCAGGAGUA
91
AUACUCCUGAUUAGGAUGUUUA
446
BetaCoV/Wuhan-Hu-
16049
16071
TRUE
TRUE
FALSE
+


1184009
U

G

1/2019|EPL_ISL_402125











AD-
AACAUCCUAAUCAGGAGUAU
92
CAUACUCCUGAUUAGGAUGUUU
447
BetaCoV/Wuhan-Hu-
16050
16072
TRUE
TRUE
FALSE
+


1184010
G

A

1/2019|EPL_ISL_402125











AD-
AGGAGUAUGCUGAUGUCUUU
93
GAAAGACAUCAGCAUACUCCUG
448
BetaCoV/Wuhan-Hu-
16062
16084
TRUE
TRUE
FALSE
+


1184011
C

A

1/2019|EPL_ISL_402125











AD-
CCUGAGUUUUAUGAGGCUAU
94
CAUAGCCUCAUAAAACUCAGGU
449
BetaCoV/Wuhan-Hu-
16190
16212
TRUE
TRUE
FALSE
+


1184012
G

U

1/2019|EPL_ISL_402125











AD-
GCAGCAGAAACGCUCAAAGC
95
AGCUUUGAGCGUUUCUGCUGCA
450
BetaCoV/Wuhan-Hu-
16634
16656
TRUE
TRUE
FALSE
+


1184013
U

A

1/2019|EPL_ISL_402125











AD-
CCUGGUACUGGUAAGAGUCA
96
AUGACUCUUACCAGUACCAGGU
451
BetaCoV/Wuhan-Hu-
17084
17106
TRUE
TRUE
FALSE
+


1184014
U

G

1/2019|EPL_ISL_402125











AD-
UAUAGAUAAAUGUAGUAGAA
97
AUUCUACUACAUUUAUCUAUAG
452
BetaCoV/Wuhan-Hu-
17212
17234
TRUE
TRUE
FALSE
+


1184015
U

G

1/2019|EPL_ISL_402125











AD-
CUGCUGAAAUUGUUGACACU
98
CAGUGUCAACAAUUUCAGCAGG
453
BetaCoV/Wuhan-Hu-
17568
17590
TRUE
TRUE
FALSE
+


1184016
G

A

1/2019|EPL_ISL_402125











AD-
GCUGAAAUUGUUGACACUGU
99
CACAGUGUCAACAAUUUCAGCA
454
BetaCoV/Wuhan-Hu-
17570
17592
TRUE
TRUE
FALSE
+


1184017
G

G

1/2019|EPL_ISL_402125











AD-
UGAAAUUGUUGACACUGUGA
100
CUCACAGUGUCAACAAUUUCAG
455
BetaCoV/Wuhan-Hu-
17572
17594
TRUE
TRUE
FALSE
+


1184018
G

C

1/2019|EPL_ISL_402125











AD-
AAAUUGUUGACACUGUGAGU
101
CACUCACAGUGUCAACAAUUUC
456
BetaCoV/Wuhan-Hu-
17574
17596
TRUE
TRUE
FALSE
+


1184019
G

A

1/2019|EPL_ISL_402125











AD-
UGUUGACACUGUGAGUGCUU
102
AAAGCACUCACAGUGUCAACAA
457
BetaCoV/Wuhan-Hu-
17578
17600
TRUE
TRUE
FALSE
+


1184020
U

U

1/2019|EPL_ISL_402125











AD-
GCAUGAUGUUUCAUCUGCAA
103
AUUGCAGAUGAAACAUCAUGCG
458
BetaCoV/Wuhan-Hu-
17677
17699
TRUE
TRUE
FALSE
+


1184021
U

U

1/2019|EPL_ISL_402125











AD-
UUCACCUUAUAAUUCACAGA
104
UUCUGUGAAUUAUAAGGUGAA
459
BetaCoV/Wuhan-Hu-
17770
17792
TRUE
TRUE
TRUE
+


1184022
A

AU

1/2019|EPL_ISL_402125











AD-
UUCACCUUAUAAUUCACAGA
104
UUCUGUGAAUUAUAAGGUGAA
459
BetaCoV/Wuhan-Hu-
17770
17792
TRUE
TRUE
TRUE
+


1184022
A

AU

1/2019|EPL_ISL_402125











AD-
UCACCUUAUAAUUCACAGAA
105
AUUCUGUGAAUUAUAAGGUGA
460
BetaCoV/Wuhan-Hu-
17771
17793
TRUE
TRUE
TRUE
+


1184023
U

AA

1/2019|EPL_ISL_402125











AD-
ACCUUAUAAUUCACAGAAUG
106
GCAUUCUGUGAAUUAUAAGGUG
461
BetaCoV/Wuhan-Hu-
17773
17795
TRUE
TRUE
TRUE
+


1184024
C

A

1/2019|EPL_ISL_402125











AD-
CCUUAUAAUUCACAGAAUGC
107
AGCAUUCUGUGAAUUAUAAGGU
462
BetaCoV/Wuhan-Hu-
17774
17796
TRUE
FALSE
TRUE
+


1184025
U

G

1/2019|EPL_ISL_402125











AD-
CCUUAUAAUUCACAGAAUGC
107
AGCAUUCUGUGAAUUAUAAGGU
462
BetaCoV/Wuhan-Hu-
17774
17796
TRUE
FALSE
TRUE
+


1184025
U

G

1/2019|EPL_ISL_402125











AD-
UAUAAUUCACAGAAUGCUGU
108
UACAGCAUUCUGUGAAUUAUAA
463
BetaCoV/Wuhan-Hu-
17777
17799
TRUE
FALSE
TRUE
+


1184026
A

G

1/2019|EPL_ISL_402125











AD-
AACUGUUGAUUCAUCACAGG
109
CCCUGUGAUGAAUCAACAGUUU
464
BetaCoV/Wuhan-Hu-
17827
17849
TRUE
TRUE
FALSE
+


1184027
G

G

1/2019|EPL_ISL_402125











AD-
GAAUAUGACUAUGUCAUAUU
110
GAAUAUGACAUAGUCAUAUUCU
465
BetaCoV/Wuhan-Hu-
17852
17874
TRUE
TRUE
FALSE
+


1184028
C

G

1/2019|EPL_ISL_402125











AD-
CUUGUCAUAAAGGUCUCUAU
111
GAUAGAGACCUUUAUGACAAGU
466
BetaCoV/Wuhan-Hu-
17968
17990
TRUE
FALSE
FALSE



1184029
C

U

1/2019|EPL_ISL_402125











AD-
CUCAUCUCUAUGAUGGGUUU
112
AAAACCCAUCAUAGAGAUGAGU
467
BetaCoV/Wuhan-Hu-
18197
18219
TRUE
TRUE
FALSE
+


1184030
U

C

1/2019|EPL_ISL_402125











AD-
AGGUCCUAUUUUCACAAAAU
113
UAUUUUGUGAAAAUAGGACCUG
468
BetaCoV/Wuhan-Hu-
18628
18650
TRUE
FALSE
FALSE



1184031
A

A

1/2019|EPL_ISL_402125











AD-
UUUACAAACAAUUUGAUACU
114
AAGUAUCAAAUUGUUUGUAAAC
469
BetaCoV/Wuhan-Hu-
19566
19588
TRUE
TRUE
FALSE
+


1184032
U

C

1/2019|EPL_ISL_402125











AD-
AUAUACUCAACUGUGUCAAU
115
UAUUGACACAGUUGAGUAUAUU
470
BetaCoV/Wuhan-Hu-
20794
20816
TRUE
TRUE
FALSE
+


1184033
A

U

1/2019|EPL_ISL_402125











AD-
UAGUACUUUCUUUUGAACUU
116
GAAGUUCAAAAGAAAGUACUAC
471
BetaCoV/Wuhan-Hu-
23092
23114
TRUE
TRUE
FALSE
+


1184034
C

U

1/2019|EPL_ISL_402125











AD-
UGGUAACACUAAUAGUAAAA
117
AUUUUACUAUUAGUGUUACCAC
472
BetaCoV/Wuhan-Hu-
23712
23734
TRUE
FALSE
FALSE



1184035
U

A

1/2019|EPL_ISL_402125











AD-
CAAUACCAUUAAACCUAUAA
118
CUUAUAGGUUUAAUGGUAUUG
473
BetaCoV/Wuhan-Hu-
24270
24292
TRUE
FALSE
FALSE



1184036
G

GA

1/2019|EPL_ISL_402125











AD-
AAUGUUCUCUAUGAGAACCA
119
UUGGUUCUCAUAGAGAACAUUC
474
BetaCoV/Wuhan-Hu-
24300
24322
TRUE
TRUE
FALSE
+


1184037
A

U

1/2019|EPL_ISL_402125











AD-
UUCUGCUAAUCUUGCUGCUA
120
GUAGCAGCAAGAUUAGCAGAAG
475
BetaCoV/Wuhan-Hu-
24620
24642
TRUE
TRUE
FALSE
+


1184038
C

C

1/2019|EPL_ISL_402125











AD-
CUGCUAAUCUUGCUGCUACU
121
UAGUAGCAGCAAGAUUAGCAGA
476
BetaCoV/Wuhan-Hu-
24622
24644
TRUE
TRUE
FALSE
+


1184039
A

A

1/2019|EPL_ISL_402125











AD-
AUCAUACAUCACCAGAUGUU
122
CAACAUCUGGUGAUGUAUGAUU
477
BetaCoV/Wuhan-Hu-
25033
25055
TRUE
TRUE
FALSE
+


1184040
G

C

1/2019|EPL_ISL_402125











AD-
GUCAAAUUACAUUACACAUA
123
UUAUGUGUAAUGUAAUUUGAC
478
BetaCoV/Wuhan-Hu-
25362
25384
TRUE
TRUE
FALSE
+


1184041
A

UC

1/2019|EPL_ISL_402125











AD-
ACGACGACUACUAGCGUGCC
124
AGGCACGCUAGUAGUCGUCGUC
479
BetaCoV/Wuhan-Hu-
26192
26214
TRUE
TRUE
FALSE
+


1184042
U

G

1/2019|EPL_ISL_402125











AD-
CGACGACUACUAGCGUGCCU
125
AAGGCACGCUAGUAGUCGUCGU
480
BetaCoV/Wuhan-Hu-
26193
26215
TRUE
TRUE
FALSE
+


1184043
U

C

1/2019|EPL_ISL_402125











AD-
ACGACUACUAGCGUGCCUUU
126
CAAAGGCACGCUAGUAGUCGUC
481
BetaCoV/Wuhan-Hu-
26195
26217
TRUE
TRUE
FALSE
+


1184044
G

G

1/2019|EPL_ISL_402125











AD-
CGACUACUAGCGUGCCUUUG
127
ACAAAGGCACGCUAGUAGUCGU
482
BetaCoV/Wuhan-Hu-
26196
26218
TRUE
TRUE
FALSE
+


1184045
U

C

1/2019|EPL_ISL_402125











AD-
GAGUACGAACUUAUGUACUC
128
UGAGUACAUAAGUUCGUACUCA
483
BetaCoV/Wuhan-Hu-
26231
26253
TRUE
TRUE
FALSE
+


1184046
A

U

1/2019|EPL_ISL_402125











AD-
UACGAACUUAUGUACUCAUU
129
GAAUGAGUACAUAAGUUCGUAC
484
BetaCoV/Wuhan-Hu-
26234
26256
TRUE
TRUE
FALSE
+


1184047
C

U

1/2019|EPL_ISL_402125











AD-
UACUCAUUCGUUUCGGAAGA
130
CUCUUCCGAAACGAAUGAGUAC
485
BetaCoV/Wuhan-Hu-
26246
26268
TRUE
TRUE
FALSE
+


1184048
G

A

1/2019|EPL_ISL_402125











AD-
UAGUUAAUAGCGUACUUCUU
131
AAAGAAGUACGCUAUUAACUAU
486
BetaCoV/Wuhan-Hu-
26280
26302
TRUE
TRUE
FALSE
+


1184049
U

U

1/2019|EPL_ISL_402125











AD-
UUCUUGCUUUCGUGGUAUUC
132
AGAAUACCACGAAAGCAAGAAA
487
BetaCoV/Wuhan-Hu-
26301
26323
TRUE
TRUE
FALSE
+


1184050
U

A

1/2019|EPL_ISL_402125











AD-
UUCGUGGUAUUCUUGCUAGU
133
AACUAGCAAGAAUACCACGAAA
488
BetaCoV/Wuhan-Hu-
26309
26331
TRUE
TRUE
FALSE
+


1184051
U

G

1/2019|EPL_ISL_402125











AD-
UAGCCAUCCUUACUGCGCUU
134
GAAGCGCAGUAAGGAUGGCUAG
489
BetaCoV/Wuhan-Hu-
26334
26356
TRUE
TRUE
FALSE
+


1184052
C

U

1/2019|EPL_ISL_402125











AD-
AGCCAUCCUUACUGCGCUUC
135
CGAAGCGCAGUAAGGAUGGCUA
490
BetaCoV/Wuhan-Hu-
26335
26357
TRUE
TRUE
FALSE
+


1184053
G

G

1/2019|EPL_ISL_402125











AD-
GCCAUCCUUACUGCGCUUCG
136
UCGAAGCGCAGUAAGGAUGGCU
491
BetaCoV/Wuhan-Hu-
26336
26358
TRUE
TRUE
FALSE
+


1184054
A

A

1/2019|EPL_ISL_402125











AD-
CCAUCCUUACUGCGCUUCGA
137
AUCGAAGCGCAGUAAGGAUGGC
492
BetaCoV/Wuhan-Hu-
26337
26359
TRUE
TRUE
FALSE
+


1184055
U

U

1/2019|EPL_ISL_402125











AD-
ACUGCGCUUCGAUUGUGUGC
138
CGCACACAAUCGAAGCGCAGUA
493
BetaCoV/Wuhan-Hu-
26345
26367
TRUE
TRUE
FALSE
+


1184056
G

A

1/2019|EPL_ISL_402125











AD-
UGCGCUUCGAUUGUGUGCGU
139
UACGCACACAAUCGAAGCGCAG
494
BetaCoV/Wuhan-Hu-
26347
26369
TRUE
TRUE
FALSE
+


1184057
A

U

1/2019|EPL_ISL_402125











AD-
CGCUUCGAUUGUGUGCGUAC
140
AGUACGCACACAAUCGAAGCGC
495
BetaCoV/Wuhan-Hu-
26349
26371
TRUE
TRUE
FALSE
+


1184058
U

A

1/2019|EPL_ISL_402125











AD-
GCUUCGAUUGUGUGCGUACU
141
CAGUACGCACACAAUCGAAGCG
496
BetaCoV/Wuhan-Hu-
26350
26372
TRUE
TRUE
FALSE
+


1184059
G

C

1/2019|EPL_ISL_402125











AD-
GUUCCUGAUCUUCUGGUCUA
142
UUAGACCAGAAGAUCAGGAACU
497
BetaCoV/Wuhan-Hu-
26450
26472
TRUE
TRUE
FALSE
+


1184060
A

C

1/2019|EPL_ISL_402125











AD-
UUCCUGAUCUUCUGGUCUAA
143
UUUAGACCAGAAGAUCAGGAAC
498
BetaCoV/Wuhan-Hu-
26451
26473
TRUE
TRUE
FALSE
+


1184061
A

U

1/2019|EPL_ISL_402125











AD-
CCUGAUCUUCUGGUCUAAAC
144
CGUUUAGACCAGAAGAUCAGGA
499
BetaCoV/Wuhan-Hu-
26453
26475
TRUE
TRUE
FALSE
+


1184062
G

A

1/2019|EPL_ISL_402125











AD-
UCUUCUGGUCUAAACGAACU
145
UAGUUCGUUUAGACCAGAAGAU
500
BetaCoV/Wuhan-Hu-
26458
26480
TRUE
TRUE
FALSE
+


1184063
A

C

1/2019|EPL_ISL_402125











AD-
CUUCUGGUCUAAACGAACUA
146
UUAGUUCGUUUAGACCAGAAGA
501
BetaCoV/Wuhan-Hu-
26459
26481
TRUE
TRUE
FALSE
+


1184064
A

U

1/2019|EPL_ISL_402125











AD-
GGAACCUAGUAAUAGGUUUC
147
GGAAACCUAUUACUAGGUUCCA
502
BetaCoV/Wuhan-Hu-
26579
26601
TRUE
TRUE
FALSE
+


1184065
C

U

1/2019|EPL_ISL_402125











AD-
UGCUACAUCACGAACGCUUU
148
GAAAGCGUUCGUGAUGUAGCAA
503
BetaCoV/Wuhan-Hu-
27030
27052
TRUE
TRUE
FALSE
+


1184066
C

C

1/2019|EPL_ISL_402125











AD-
GCUACAUCACGAACGCUUUC
149
AGAAAGCGUUCGUGAUGUAGCA
504
BetaCoV/Wuhan-Hu-
27031
27053
TRUE
TRUE
FALSE
+


1184067
U

A

1/2019|EPL_ISL_402125











AD-
CUACAUCACGAACGCUUUCU
150
AAGAAAGCGUUCGUGAUGUAGC
505
BetaCoV/Wuhan-Hu-
27032
27054
TRUE
TRUE
FALSE
+


1184068
U

A

1/2019|EPL_ISL_402125











AD-
ACGCUUUCUUAUUACAAAUU
151
CAAUUUGUAAUAAGAAAGCGUU
506
BetaCoV/Wuhan-Hu-
27043
27065
TRUE
TRUE
FALSE
+


1184069
G

C

1/2019|EPL_ISL_402125











AD-
UGUACAGUAAGUGACAACAG
152
UCUGUUGUCACUUACUGUACAA
507
BetaCoV/Wuhan-Hu-
27180
27202
TRUE
TRUE
FALSE
+


1184070
A

G

1/2019|EPL_ISL_402125











AD-
AAGUGACAACAGAUGUUUCA
153
AUGAAACAUCUGUUGUCACUUA
508
BetaCoV/Wuhan-Hu-
27188
27210
TRUE
TRUE
FALSE
+


1184071
U

C

1/2019|EPL_ISL_402125











AD-
CUACUCUAAUAUACCAUUUA
154
CUAAAUGGUAUAUUAGAGUAG
509
BetaCoV/Wuhan-Hu-
28021
28043
TRUE
FALSE
FALSE



1184072
G

GA

1/2019|EPL_ISL_402125











AD-
CCCCAAGGUUUACCCAAUAA
155
AUUAUUGGGUAAACCUUGGGGC
510
BetaCoV/Wuhan-Hu-
28395
28417
TRUE
TRUE
FALSE
+


1184073
U

C

1/2019|EPL_ISL_402125











AD-
UACCCAAUAAUACUGCGUCU
156
AAGACGCAGUAUUAUUGGGUAA
511
BetaCoV/Wuhan-Hu-
28405
28427
TRUE
TRUE
FALSE
+


1184074
U

A

1/2019|EPL_ISL_402125











AD-
GAUGACCAAAUUGGCUACUA
157
GUAGUAGCCAAUUUGGUCAUCU
512
BetaCoV/Wuhan-Hu-
28512
28534
TRUE
TRUE
FALSE
+


1184075
C

G

1/2019|EPL_ISL_402125











AD-
GCUACUACCGAAGAGCUACC
158
UGGUAGCUCUUCGGUAGUAGCC
513
BetaCoV/Wuhan-Hu-
28525
28547
TRUE
TRUE
FALSE
+


1184076
A

A

1/2019|EPL_ISL_402125











AD-
UCGUGCUACAACUUCCUCAA
159
CUUGAGGAAGUUGUAGCACGAU
514
BetaCoV/Wuhan-Hu-
28741
28763
TRUE
TRUE
FALSE
+


1184077
G

U

1/2019|EPL_ISL_402125











AD-
CGUGCUACAACUUCCUCAAG
160
CCUUGAGGAAGUUGUAGCACGA
515
BetaCoV/Wuhan-Hu-
28742
28764
TRUE
TRUE
FALSE
+


1184078
G

U

1/2019|EPL_ISL_402125











AD-
UGCUACAACUUCCUCAAGGA
161
UUCCUUGAGGAAGUUGUAGCAC
516
BetaCoV/Wuhan-Hu-
28744
28766
TRUE
TRUE
FALSE
+


1184079
A

G

1/2019|EPL_ISL_402125











AD-
UACAACUUCCUCAAGGAACA
162
UUGUUCCUUGAGGAAGUUGUAG
517
BetaCoV/Wuhan-Hu-
28747
28769
TRUE
TRUE
FALSE
+


1184080
A

C

1/2019|EPL_ISL_402125











AD-
ACAACUUCCUCAAGGAACAA
163
GUUGUUCCUUGAGGAAGUUGUA
518
BetaCoV/Wuhan-Hu-
28748
28770
TRUE
TRUE
FALSE
+


1184081
C

G

1/2019|EPL_ISL_402125











AD-
ACUUCCUCAAGGAACAACAU
164
AAUGUUGUUCCUUGAGGAAGUU
519
BetaCoV/Wuhan-Hu-
28751
28773
TRUE
TRUE
FALSE
+


1184082
U

G

1/2019|EPL_ISL_402125











AD-
UCCUCAAGGAACAACAUUGC
165
GGCAAUGUUGUUCCUUGAGGAA
520
BetaCoV/Wuhan-Hu-
28754
28776
TRUE
TRUE
FALSE
+


1184083
C

G

1/2019|EPL_ISL_402125











AD-
CAAGGAACAACAUUGCCAAA
166
UUUUGGCAAUGUUGUUCCUUGA
521
BetaCoV/Wuhan-Hu-
28758
28780
TRUE
TRUE
FALSE
+


1184084
A

G

1/2019|EPL_ISL_402125











AD-
AAGGAACAACAUUGCCAAAA
167
CUUUUGGCAAUGUUGUUCCUUG
522
BetaCoV/Wuhan-Hu-
28759
28781
TRUE
TRUE
FALSE
+


1184085
G

A

1/2019|EPL_ISL_402125











AD-
ACAUUGCCAAAAGGCUUCUA
168
GUAGAAGCCUUUUGGCAAUGUU
523
BetaCoV/Wuhan-Hu-
28767
28789
TRUE
TRUE
FALSE
+


1184086
C

G

1/2019|EPL_ISL_402125











AD-
GACAGAUUGAACCAGCUUGA
169
CUCAAGCUGGUUCAAUCUGUCA
524
BetaCoV/Wuhan-Hu-
28944
28966
TRUE
TRUE
FALSE
+


1184087
G

A

1/2019|EPL_ISL_402125











AD-
CAAACUGUCACUAAGAAAUC
170
AGAUUUCUUAGUGACAGUUUGG
525
BetaCoV/Wuhan-Hu-
29001
29023
TRUE
TRUE
FALSE
+


1184088
U

C

1/2019|EPL_ISL_402125











AD-
GUCCAGAACAAACCCAAGGA
171
UUCCUUGGGUUUGUUCUGGACC
526
BetaCoV/Wuhan-Hu-
29104
29126
TRUE
TRUE
FALSE
+


1184089
A

A

1/2019|EPL_ISL_402125











AD-
GGCCGCAAAUUGCACAAUUU
172
CAAAUUGUGCAAUUUGCGGCCA
527
BetaCoV/Wuhan-Hu-
29173
29195
TRUE
TRUE
FALSE
+


1184090
G

A

1/2019|EPL_ISL_402125











AD-
GCAUGGAAGUCACACCUUCG
173
CCGAAGGUGUGACUUCCAUGCC
528
BetaCoV/Wuhan-Hu-
29233
29255
TRUE
TRUE
FALSE
+


1184091
G

A

1/2019|EPL_ISL_402125











AD-
UAUAGUCUACUCUUGUGCAG
174
UCUGCACAAGAGUAGACUAUAU
529
BetaCoV/Wuhan-Hu-
29595
29617
TRUE
TRUE
FALSE
+


1184092
A

A

1/2019|EPL_ISL_402125











AD-
UAGUCUACUCUUGUGCAGAA
175
AUUCUGCACAAGAGUAGACUAU
530
BetaCoV/Wuhan-Hu-
29597
29619
TRUE
TRUE
FALSE
+


1184093
U

A

1/2019|EPL_ISL_402125











AD-
AGUCUACUCUUGUGCAGAAU
176
CAUUCUGCACAAGAGUAGACUA
531
BetaCoV/Wuhan-Hu-
29598
29620
TRUE
TRUE
FALSE
+


1184094
G

U

1/2019|EPL_ISL_402125











AD-
UCUACUCUUGUGCAGAAUGA
177
UUCAUUCUGCACAAGAGUAGAC
532
BetaCoV/Wuhan-Hu-
29600
29622
TRUE
TRUE
FALSE
+


1184095
A

U

1/20191EPLISL_402125











AD-
CUCUUGUGCAGAAUGAAUUC
178
AGAAUUCAUUCUGCACAAGAGU
533
BetaCoV/Wuhan-Hu-
29604
29626
TRUE
TRUE
FALSE
+


1184096
U

A

1/2019|EPL_ISL_402125











AD-
UAGUUAACUUUAAUCUCACA
179
AUGUGAGAUUAAAGUUAACUAC
534
BetaCoV/Wuhan-Hu-
29650
29672
TRUE
TRUE
FALSE
+


1184097
U

A

1/2019|EPL_ISL_402125











AD-
UAACUUUAAUCUCACAUAGC
180
UGCUAUGUGAGAUUAAAGUUA
535
BetaCoV/Wuhan-Hu-
29654
29676
TRUE
TRUE
FALSE
+


1184098
A

AC

1/2019|EPL_ISL_402125











AD-
UUUAAUCUCACAUAGCAAUC
181
AGAUUGCUAUGUGAGAUUAAA
536
BetaCoV/Wuhan-Hu-
29658
29680
TRUE
TRUE
FALSE
+


1184099
U

GU

1/2019|EPL_ISL_402125











AD-
UUAAUCUCACAUAGCAAUCU
182
AAGAUUGCUAUGUGAGAUUAA
537
BetaCoV/Wuhan-Hu-
29659
29681
TRUE
TRUE
FALSE
+


1184100
U

AG

1/2019|EPL_ISL_402125











AD-
GGGAGGACUUGAAAGAGCCA
183
GUGGCUCUUUCAAGUCCUCCCU
538
BetaCoV/Wuhan-Hu-
29699
29721
TRUE
TRUE
FALSE
+


1184101
C

A

1/2019|EPL_ISL_402125











AD-
GUAGAUCUGUUCUCUAAACG
184
UCGUUUAGAGAACAGAUCUACA
539
BetaCoV/Wuhan-Hu-
51
73
TRUE
TRUE
FALSE
+


1184102
A

A

1/2019|EPL_ISL_402125











AD-
AGAUCUGUUCUCUAAACGAA
185
GUUCGUUUAGAGAACAGAUCUA
540
BetaCoV/Wuhan-Hu-
53
75
TRUE
TRUE
FALSE
+


1184103
C

C

1/2019|EPL_ISL_402125











AD-
UUCUCUAAACGAACUUUAAA
186
UUUUAAAGUUCGUUUAGAGAAC
541
BetaCoV/Wuhan-Hu-
60
82
TRUE
TRUE
FALSE
+


1184104
A

A

1/2019|EPL_ISL_402125











AD-
UCUCUAAACGAACUUUAAAA
187
AUUUUAAAGUUCGUUUAGAGA
542
BetaCoV/Wuhan-Hu-
61
83
TRUE
TRUE
FALSE
+


1184105
U

AC

1/2019|EPL_ISL_402125











AD-
CUAAACGAACUUUAAAAUCU
188
CAGAUUUUAAAGUUCGUUUAGA
543
BetaCoV/Wuhan-Hu-
64
86
TRUE
TRUE
FALSE
+


1184106
G

G

1/2019|EPL_ISL_402125











AD-
UAAACGAACUUUAAAAUCUG
189
ACAGAUUUUAAAGUUCGUUUAG
544
BetaCoV/Wuhan-Hu-
65
87
TRUE
TRUE
FALSE
+


1184107
U

A

1/2019|EPL_ISL_402125











AD-
AAACGAACUUUAAAAUCUGU
190
CACAGAUUUUAAAGUUCGUUUA
545
BetaCoV/Wuhan-Hu-
66
88
TRUE
TRUE
FALSE
+


1184108
G

G

1/2019|EPL_ISL_402125











AD-
ACGAACUUUAAAAUCUGUGU
191
CACACAGAUUUUAAAGUUCGUU
546
BetaCoV/Wuhan-Hu-
68
90
TRUE
TRUE
FALSE
+


1184109
G

U

1/2019|EPL_ISL_402125











AD-
GCUGCUUACGGUUUCGUCCG
192
ACGGACGAAACCGUAAGCAGCC
547
BetaCoV/Wuhan-Hu-
187
209
TRUE
TRUE
FALSE
+


1184110
U

U

1/2019|EPL_ISL_402125











AD-
UGCUUACGGUUUCGUCCGUG
193
ACACGGACGAAACCGUAAGCAG
548
BetaCoV/Wuhan-Hu-
189
211
TRUE
TRUE
FALSE
+


1184111
U

C

1/2019|EPL_ISL_402125











AD-
GCUUACGGUUUCGUCCGUGU
194
AACACGGACGAAACCGUAAGCA
549
BetaCoV/Wuhan-Hu-
190
212
TRUE
TRUE
FALSE
+


1184112
U

G

1/2019|EPL_ISL_402125











AD-
CUUACGGUUUCGUCCGUGUU
195
CAACACGGACGAAACCGUAAGC
550
BetaCoV/Wuhan-Hu-
191
213
TRUE
TRUE
FALSE
+


1184113
G

A

1/2019|EPL_ISL_402125











AD-
UUACGGUUUCGUCCGUGUUG
196
GCAACACGGACGAAACCGUAAG
551
BetaCoV/Wuhan-Hu-
192
214
TRUE
TRUE
FALSE
+


1184114
C

C

1/2019|EPL_ISL_402125











AD-
UGUGACCGAAAGGUAAGAUG
197
CCAUCUUACCUUUCGGUCACAC
552
BetaCoV/Wuhan-Hu-
247
269
TRUE
TRUE
FALSE
+


1184115
G

C

1/2019|EPL_ISL_402125











AD-
GACCGAAAGGUAAGAUGGAG
198
UCUCCAUCUUACCUUUCGGUCA
553
BetaCoV/Wuhan-Hu-
250
272
TRUE
TRUE
FALSE
+


1184116
A

C

1/2019|EPL_ISL_402125











AD-
AAAACACACGUCCAACUCAG
199
ACUGAGUUGGACGUGUGUUUUC
554
BetaCoV/Wuhan-Hu-
294
316
TRUE
TRUE
FALSE
+


1184117
U

U

1/2019|EPL_ISL_402125











AD-
CACACGUCCAACUCAGUUUG
200
GCAAACUGAGUUGGACGUGUGU
555
BetaCoV/Wuhan-Hu-
298
320
TRUE
TRUE
FALSE
+


1184118
C

U

1/2019|EPL_ISL_402125











AD-
ACACGUCCAACUCAGUUUGC
201
GGCAAACUGAGUUGGACGUGUG
556
BetaCoV/Wuhan-Hu-
299
321
TRUE
TRUE
FALSE
+


1184119
C

U

1/2019|EPL_ISL_402125











AD-
CACGUCCAACUCAGUUUGCC
202
AGGCAAACUGAGUUGGACGUGU
557
BetaCoV/Wuhan-Hu-
300
322
TRUE
TRUE
FALSE
+


1184120
U

G

1/2019|EPL_ISL_402125











AD-
ACGUCCAACUCAGUUUGCCU
203
CAGGCAAACUGAGUUGGACGUG
558
BetaCoV/Wuhan-Hu-
301
323
TRUE
TRUE
FALSE
+


1184121
G

U

1/2019|EPL_ISL_402125











AD-
CGUCCAACUCAGUUUGCCUG
204
ACAGGCAAACUGAGUUGGACGU
559
BetaCoV/Wuhan-Hu-
302
324
TRUE
TRUE
FALSE
+


1184122
U

G

1/2019|EPL_ISL_402125











AD-
GUUCUUCUUCGUAAGAACGG
205
ACCGUUCUUACGAAGAAGAACC
560
BetaCoV/Wuhan-Hu-
624
646
TRUE
TRUE
FALSE
+


1184123
U

U

1/2019|EPL_ISL_402125











AD-
UUCUUCUUCGUAAGAACGGU
206
UACCGUUCUUACGAAGAAGAAC
561
BetaCoV/Wuhan-Hu-
625
647
TRUE
TRUE
FALSE
+


1184124
A

C

1/2019|EPL_ISL_402125











AD-
UCUUCUUCGUAAGAACGGUA
207
UUACCGUUCUUACGAAGAAGAA
562
BetaCoV/Wuhan-Hu-
626
648
TRUE
TRUE
FALSE
+


1184125
A

C

1/2019|EPL_ISL_402125











AD-
AUCUUCUUCUUGCUCUUCUU
208
GAAGAAGAGCAAGAAGAAGAU
563
BetaCoV/Wuhan-Hu-
3179
3201
TRUE
FALSE
FALSE



1184126
C

UG

1/2019|EPL_ISL_402125











AD-
GGUUAUUUAAAACUUACUGA
209
GUCAGUAAGUUUUAAAUAACCA
564
BetaCoV/Wuhan-Hu-
3351
3373
TRUE
TRUE
FALSE
+


1184127
C

C

1/2019|EPL_ISL_402125











AD-
UCUACACAAACUCUUAAAGA
210
UUCUUUAAGAGUUUGUGUAGA
565
BetaCoV/Wuhan-Hu-
3745
3767
TRUE
FALSE
FALSE



1184128
A

UA

1/2019|EPL_ISL_402125











AD-
AAGAAGUAGUGGAAAAUCCU
211
UAGGAUUUUCCACUACUUCUUC
566
BetaCoV/Wuhan-Hu-
6409
6431
TRUE
TRUE
FALSE
+


1184129
A

A

1/2019|EPL_ISL_402125











AD-
ACAAUUGUGUACUUUUACUA
212
CUAGUAAAAGUACACAAUUGUA
567
BetaCoV/Wuhan-Hu-
6794
6816
TRUE
TRUE
FALSE
+


1184130
G

G

1/2019|EPL_ISL_402125











AD-
ACACUCUUAACAGUAUUCUU
213
AAAGAAUACUGUUAAGAGUGUC
568
BetaCoV/Wuhan-Hu-
6862
6884
TRUE
FALSE
FALSE



1184131
U

G

1/2019|EPL_ISL_402125











AD-
AUGUACAACUAUUGUUAAUG
214
CCAUUAACAAUAGUUGUACAUU
569
BetaCoV/Wuhan-Hu-
7529
7551
TRUE
TRUE
FALSE
+


1184132
G

C

1/2019|EPL_ISL_402125











AD-
UGUACAACUAUUGUUAAUGG
215
ACCAUUAACAAUAGUUGUACAU
570
BetaCoV/Wuhan-Hu-
7530
7552
TRUE
TRUE
FALSE
+


1184133
U

U

1/2019|EPL_ISL_402125











AD-
CCAGGAGUUUUCUGUGGUGU
216
UACACCACAGAAAACUCCUGGU
571
BetaCoV/Wuhan-Hu-
9306
9328
TRUE
TRUE
FALSE
+


1184134
A

A

1/2019|EPL_ISL_402125











AD-
UCAUGUGGUAGUGUUGGUUU
217
AAAACCAACACUACCACAUGAA
572
BetaCoV/Wuhan-Hu-
10482
10504
TRUE
TRUE
FALSE
+


1184135
U

C

1/2019|EPL_ISL_402125











AD-
CACUGUACAGUCUAAAAUGU
218
GACAUUUUAGACUGUACAGUGG
573
BetaCoV/Wuhan-Hu-
11831
11853
TRUE
TRUE
FALSE
+


1184136
C

C

1/2019|EPL_ISL_402125











AD-
ACUGUACAGUCUAAAAUGUC
7
UGACAUUUUAGACUGUACAGUG
8
BetaCoV/Wuhan-Hu-
11832
11854
TRUE
TRUE
FALSE
+


1184137
A

G

1/2019|EPL_ISL_402125











AD-
UUUGAAUGUGGCUAAAUCUG
219
UCAGAUUUAGCCACAUUCAAAG
574
BetaCoV/Wuhan-Hu-
12212
12234
TRUE
TRUE
FALSE
+


1184138
A

A

1/2019|EPL_ISL_402125











AD-
ACAACCUAAAUAGAGGUAUG
220
CCAUACCUCUAUUUAGGUUGUU
575
BetaCoV/Wuhan-Hu-
12967
12989
TRUE
TRUE
FALSE
+


1184139
G

U

1/2019|EPL_ISL_402125











AD-
AACCUAAAUAGAGGUAUGGU
221
UACCAUACCUCUAUUUAGGUUG
576
BetaCoV/Wuhan-Hu-
12969
12991
TRUE
TRUE
FALSE
+


1184140
A

U

1/2019|EPL_ISL_402125











AD-
UCAUCCAAAUCCUAAAGGAU
222
AAUCCUUUAGGAUUUGGAUGAU
577
BetaCoV/Wuhan-Hu-
13268
13290
TRUE
TRUE
FALSE
+


1184141
U

C

1/2019|EPL_ISL_402125











AD-
CACAGUCUGUACCGUCUGCG
223
CCGCAGACGGUACAGACUGUGU
578
BetaCoV/Wuhan-Hu-
13364
13386
TRUE
TRUE
FALSE
+


1184142
G

U

1/2019|EPL_ISL_402125











AD-
ACGGGUUUGCGGUGUAAGUG
224
GCACUUACACCGCAAACCCGUU
579
BetaCoV/Wuhan-Hu-
13465
13487
TRUE
TRUE
FALSE
+


1184143
C

U

1/2019|EPL_ISL_402125











AD-
AGGCACUAGUACUGAUGUCG
225
ACGACAUCAGUACUAGUGCCUG
580
BetaCoV/Wuhan-Hu-
13510
13532
TRUE
TRUE
FALSE
+


1184144
U

U

1/2019|EPL_ISL_402125











AD-
UUCUUACUUUGUAGUUAAGA
226
CUCUUAACUACAAAGUAAGAAU
581
BetaCoV/Wuhan-Hu-
13639
13661
TRUE
TRUE
FALSE
+


1184145
G

C

1/2019|EPL_ISL_402125











AD-
CUCUAACUACCAACAUGAAG
227
UCUUCAUGUUGGUAGUUAGAGA
582
BetaCoV/Wuhan-Hu-
13669
13691
TRUE
TRUE
FALSE
+


1184146
A

A

1/2019|EPL_ISL_402125











AD-
UUGUGAUGAUGAUUAUUUCA
228
UUGAAAUAAUCAUCAUCACAAC
583
BetaCoV/Wuhan-Hu-
13891
13913
TRUE
TRUE
FALSE
+


1184147
A

A

1/2019|EPL_ISL_402125











AD-
UGUGAUGAUGAUUAUUUCAA
229
AUUGAAAUAAUCAUCAUCACAA
584
BetaCoV/Wuhan-Hu-
13892
13914
TRUE
TRUE
FALSE
+


1184148
U

C

1/2019|EPL_ISL_402125











AD-
AUCAGGAUGUAAACUUACAU
230
UAUGUAAGUUUACAUCCUGAUU
585
BetaCoV/Wuhan-Hu-
14505
14527
TRUE
TRUE
FALSE
+


1184149
A

A

1/2019|EPL_ISL_402125











AD-
UAACAAUGUUGCUUUUCAAA
5
GUUUGAAAAGCAACAUUGUUAG
6
BetaCoV/Wuhan-Hu-
14644
14666
TRUE
TRUE
FALSE
+


1184150
C

U

1/2019|EPL_ISL_402125











AD-
UUUUCAAACUGUCAAACCCG
231
CCGGGUUUGACAGUUUGAAAAG
586
BetaCoV/Wuhan-Hu-
14656
14678
TRUE
TRUE
FALSE
+


1184151
G

C

1/2019|EPL_ISL_402125











AD-
ACUGUCAAACCCGGUAAUUU
232
AAAAUUACCGGGUUUGACAGUU
587
BetaCoV/Wuhan-Hu-
14663
14685
TRUE
TRUE
FALSE
+


1184152
U

U

1/2019|EPL_ISL_402125











AD-
CUUUAAGGAAGGAAGUUCUG
233
ACAGAACUUCCUUCCUUAAAGA
588
BetaCoV/Wuhan-Hu-
14722
14744
TRUE
TRUE
FALSE
+


1184153
U

A

1/2019|EPL_ISL_402125











AD-
UUUAAGGAAGGAAGUUCUGU
234
AACAGAACUUCCUUCCUUAAAG
589
BetaCoV/Wuhan-Hu-
14723
14745
TRUE
TRUE
FALSE
+


1184154
U

A

1/2019|EPL_ISL_402125











AD-
UUAAGGAAGGAAGUUCUGUU
235
CAACAGAACUUCCUUCCUUAAA
590
BetaCoV/Wuhan-Hu-
14724
14746
TRUE
TRUE
FALSE
+


1184155
G

G

1/2019|EPL_ISL_402125











AD-
UAAGGAAGGAAGUUCUGUUG
236
UCAACAGAACUUCCUUCCUUAA
591
BetaCoV/Wuhan-Hu-
14725
14747
TRUE
TRUE
FALSE
+


1184156
A

A

1/2019|EPL_ISL_402125











AD-
AAAACACUUCUUCUUUGCUC
237
UGAGCAAAGAAGAAGUGUUUU
592
BetaCoV/Wuhan-Hu-
14749
14771
TRUE
TRUE
FALSE
+


1184157
A

AA

1/2019|EPL_ISL_402125











AD-
GGUAAGGCUAGACUUUAUUA
238
AUAAUAAAGUCUAGCCUUACCC
593
BetaCoV/Wuhan-Hu-
14966
14988
TRUE
TRUE
FALSE
+


1184158
U

C

1/2019|EPL_ISL_402125











AD-
GUAAGGCUAGACUUUAUUAU
239
CAUAAUAAAGUCUAGCCUUACC
594
BetaCoV/Wuhan-Hu-
14967
14989
TRUE
TRUE
FALSE
+


1184159
G

C

1/2019|EPL_ISL_402125











AD-
GAUCAAGAUGCACUUUUCGC
240
UGCGAAAAGUGCAUCUUGAUCC
595
BetaCoV/Wuhan-Hu-
15005
15027
TRUE
TRUE
FALSE
+


1184160
A

U

1/2019|EPL_ISL_402125











AD-
UAGCUGGUGUCUCUAUCUGU
241
UACAGAUAGAGACACCAGCUAC
596
BetaCoV/Wuhan-Hu-
15108
15130
TRUE
TRUE
FALSE
+


1184161
A

G

1/2019|EPL_ISL_402125











AD-
GGUGUCUCUAUCUGUAGUAC
242
AGUACUACAGAUAGAGACACCA
597
BetaCoV/Wuhan-Hu-
15113
15135
TRUE
TRUE
FALSE
+


1184162
U

G

1/2019|EPL_ISL_402125











AD-
UCUCUAUCUGUAGUACUAUG
243
UCAUAGUACUACAGAUAGAGAC
598
BetaCoV/Wuhan-Hu-
15117
15139
TRUE
TRUE
FALSE
+


1184163
A

A

1/2019|EPL_ISL_402125











AD-
UUGUUCUUGCUCGCAAACAU
244
UAUGUUUGCGAGCAAGAACAAG
599
BetaCoV/Wuhan-Hu-
15345
15367
TRUE
TRUE
FALSE
+


1184164
A

U

1/2019|EPL_ISL_402125











AD-
UUCACUAUAUGUUAAACCAG
245
CCUGGUUUAACAUAUAGUGAAC
600
BetaCoV/Wuhan-Hu-
15451
15473
TRUE
TRUE
FALSE
+


1184165
G

C

1/2019|EPL_ISL_402125











AD-
ACAACUGCUUAUGCUAAUAG
246
ACUAUUAGCAUAAGCAGUUGUG
601
BetaCoV/Wuhan-Hu-
15494
15516
TRUE
TRUE
FALSE
+


1184166
U

G

1/2019|EPL_ISL_402125











AD-
CAACUGCUUAUGCUAAUAGU
247
CACUAUUAGCAUAAGCAGUUGU
602
BetaCoV/Wuhan-Hu-
15495
15517
TRUE
TRUE
FALSE
+


1184167
G

G

1/2019|EPL_ISL_402125











AD-
AACUGCUUAUGCUAAUAGUG
248
ACACUAUUAGCAUAAGCAGUUG
603
BetaCoV/Wuhan-Hu-
15496
15518
TRUE
TRUE
FALSE
+


1184168
U

U

1/2019|EPL_ISL_402125











AD-
ACUGCUUAUGCUAAUAGUGU
249
AACACUAUUAGCAUAAGCAGUU
604
BetaCoV/Wuhan-Hu-
15497
15519
TRUE
TRUE
FALSE
+


1184169
U

G

1/2019|EPL_ISL_402125











AD-
CACUCAUAAAGUCUGUGUUG
250
ACAACACAGACUUUAUGAGUGU
605
BetaCoV/Wuhan-Hu-
15609
15631
TRUE
FALSE
FALSE



1184170
U

C

1/2019|EPL_ISL_402125











AD-
GAGUGUCUCUAUAGAAAUAG
251
UCUAUUUCUAUAGAGACACUCA
606
BetaCoV/Wuhan-Hu-
15623
15645
TRUE
TRUE
FALSE
+


1184171
A

U

1/2019|EPL_ISL_402125











AD-
ACUGAGACUGACCUUACUAA
252
UUUAGUAAGGUCAGUCUCAGUC
607
BetaCoV/Wuhan-Hu-
15839
15861
TRUE
TRUE
FALSE
+


1184172
A

C

1/2019|EPL_ISL_402125











AD-
CAUACAAUGCUAGUUAAACA
253
CUGUUUAACUAGCAUUGUAUGU
608
BetaCoV/Wuhan-Hu-
15884
15906
TRUE
TRUE
FALSE
+


1184173
G

U

1/2019|EPL_ISL_402125











AD-
ACAGAUGGUACACUUAUGAU
254
AAUCAUAAGUGUACCAUCUGUU
609
BetaCoV/Wuhan-Hu-
15986
16008
TRUE
TRUE
FALSE
+


1184174
U

U

1/2019|EPL_ISL_402125











AD-
CAGAUGGUACACUUAUGAUU
255
CAAUCAUAAGUGUACCAUCUGU
610
BetaCoV/Wuhan-Hu-
15987
16009
TRUE
TRUE
FALSE
+


1184175
G

U

1/2019|EPL_ISL_402125











AD-
AGGAGAGUACACCUUUGAAA
256
UUUUCAAAGGUGUACUCUCCUA
611
BetaCoV/Wuhan-Hu-
16819
16841
TRUE
TRUE
FALSE
+


1184176
A

U

1/2019|EPL_ISL_402125











AD-
UAUCUCAGAUGAGUUUUCUA
257
CUAGAAAACUCAUCUGAGAUAU
612
BetaCoV/Wuhan-Hu-
17005
17027
TRUE
TRUE
FALSE
+


1184177
G

U

1/2019|EPL_ISL_402125











AD-
AUCUCAGAUGAGUUUUCUAG
258
GCUAGAAAACUCAUCUGAGAUA
613
BetaCoV/Wuhan-Hu-
17006
17028
TRUE
TRUE
FALSE
+


1184178
C

U

1/2019|EPL_ISL_402125











AD-
AUGUUGCAAAUUAUCAAAAG
259
CCUUUUGAUAAUUUGCAACAUU
614
BetaCoV/Wuhan-Hu-
17028
17050
TRUE
TRUE
FALSE
+


1184179
G

G

1/2019|EPL_ISL_402125











AD-
GUUGCAAAUUAUCAAAAGGU
260
AACCUUUUGAUAAUUUGCAACA
615
BetaCoV/Wuhan-Hu-
17030
17052
TRUE
TRUE
FALSE
+


1184180
U

U

1/2019|EPL_ISL_402125











AD-
GUACUGGUAAGAGUCAUUUU
261
CAAAAUGACUCUUACCAGUACC
616
BetaCoV/Wuhan-Hu-
17088
17110
TRUE
TRUE
FALSE
+


1184181
G

A

1/2019|EPL_ISL_402125











AD-
UACUGGUAAGAGUCAUUUUG
262
GCAAAAUGACUCUUACCAGUAC
617
BetaCoV/Wuhan-Hu-
17089
17111
TRUE
TRUE
FALSE
+


1184182
C

C

1/2019|EPL_ISL_402125











AD-
GUAGAGUGUUUUGAUAAAUU
263
GAAUUUAUCAAAACACUCUACA
618
BetaCoV/Wuhan-Hu-
17252
17274
TRUE
TRUE
FALSE
+


1184183
C

C

1/2019|EPL_ISL_402125











AD-
AGAGUGUUUUGAUAAAUUCA
264
UUGAAUUUAUCAAAACACUCUA
619
BetaCoV/Wuhan-Hu-
17254
17276
TRUE
TRUE
FALSE
+


1184184
A

C

1/2019|EPL_ISL_402125











AD-
GUGUUUUGAUAAAUUCAAAG
265
ACUUUGAAUUUAUCAAAACACU
620
BetaCoV/Wuhan-Hu-
17257
17279
TRUE
TRUE
FALSE
+


1184185
U

C

1/2019|EPL_ISL_402125











AD-
GUUUUGAUAAAUUCAAAGUG
266
UCACUUUGAAUUUAUCAAAACA
621
BetaCoV/Wuhan-Hu-
17259
17281
TRUE
TRUE
FALSE
+


1184186
A

C

1/2019|EPL_ISL_402125











AD-
CACACUAGAACCAGAAUAUU
267
AAAUAUUCUGGUUCUAGUGUGC
622
BetaCoV/Wuhan-Hu-
17479
17501
TRUE
TRUE
FALSE
+


1184187
U

C

1/2019|EPL_ISL_402125











AD-
ACACUAGAACCAGAAUAUUU
268
GAAAUAUUCUGGUUCUAGUGUG
623
BetaCoV/Wuhan-Hu-
17480
17502
TRUE
TRUE
FALSE
+


1184188
C

C

1/2019|EPL_ISL_402125











AD-
GAAAUUGUUGACACUGUGAG
269
ACUCACAGUGUCAACAAUUUCA
624
BetaCoV/Wuhan-Hu-
17573
17595
TRUE
TRUE
FALSE
+


1184189
U

G

1/2019|EPL_ISL_402125











AD-
CCCUGCUUGGAGAAAAGCUG
270
ACAGCUUUUCUCCAAGCAGGGU
625
BetaCoV/Wuhan-Hu-
17743
17765
TRUE
TRUE
FALSE
+


1184190
U

U

1/2019|EPL_ISL_402125











AD-
CUUAUAAUUCACAGAAUGCU
271
CAGCAUUCUGUGAAUUAUAAGG
626
BetaCoV/Wuhan-Hu-
17775
17797
TRUE
FALSE
TRUE
+


1184191
G

U

1/2019|EPL_ISL_402125











AD-
CUUAUAAUUCACAGAAUGCU
271
CAGCAUUCUGUGAAUUAUAAGG
626
BetaCoV/Wuhan-Hu-
17775
17797
TRUE
FALSE
TRUE
+


1184191
G

U

1/2019|EPL_ISL_402125











AD-
UUAUAAUUCACAGAAUGCUG
272
ACAGCAUUCUGUGAAUUAUAAG
627
BetaCoV/Wuhan-Hu-
17776
17798
TRUE
FALSE
TRUE
+


1184192
U

G

1/2019|EPL_ISL_402125











AD-
AAUAUGACUAUGUCAUAUUC
273
UGAAUAUGACAUAGUCAUAUUC
628
BetaCoV/Wuhan-Hu-
17853
17875
TRUE
TRUE
FALSE
+


1184193
A

U

1/2019|EPL_ISL_402125











AD-
AUAUGACUAUGUCAUAUUCA
274
GUGAAUAUGACAUAGUCAUAUU
629
BetaCoV/Wuhan-Hu-
17854
17876
TRUE
TRUE
FALSE
+


1184194
C

C

1/2019|EPL_ISL_402125











AD-
UAUGACUAUGUCAUAUUCAC
275
AGUGAAUAUGACAUAGUCAUAU
630
BetaCoV/Wuhan-Hu-
17855
17877
TRUE
TRUE
FALSE
+


1184195
U

U

1/2019|EPL_ISL_402125











AD-
AAGACUCAUCUCUAUGAUGG
276
CCCAUCAUAGAGAUGAGUCUUC
631
BetaCoV/Wuhan-Hu-
18193
18215
TRUE
TRUE
FALSE
+


1184196
G

U

1/2019|EPL_ISL_402125











AD-
AGACUCAUCUCUAUGAUGGG
277
ACCCAUCAUAGAGAUGAGUCUU
632
BetaCoV/Wuhan-Hu-
18194
18216
TRUE
TRUE
FALSE
+


1184197
U

C

1/2019|EPL_ISL_402125











AD-
UCAGAGUUUAGAAAAUGUGG
278
GCCACAUUUUCUAAACUCUGAA
633
BetaCoV/Wuhan-Hu-
19615
19637
TRUE
TRUE
FALSE
+


1184198
C

G

1/2019|EPL_ISL_402125











AD-
ACCAUCAACUUUCUUAUAAU
279
UAUUAUAAGAAAGUUGAUGGU
634
BetaCoV/Wuhan-Hu-
20152
20174
TRUE
FALSE
FALSE



1184199
A

GU

1/2019|EPL_ISL_402125











AD-
CUCCAUAAACGAUAUGUUCG
280
UCGAACAUAUCGUUUAUGGAGA
635
BetaCoV/Wuhan-Hu-
20315
20337
TRUE
FALSE
FALSE



1184200
A

U

1/2019|EPL_ISL_402125











AD-
CAUAGGAAUAAAAUCUUCUA
281
UUAGAAGAUUUUAUUCCUAUGG
636
BetaCoV/Wuhan-Hu-
20413
20435
TRUE
FALSE
FALSE



1184201
A

A

1/2019|EPL_ISL_402125











AD-
CCUAAUAUUACAAACUUGUG
282
GCACAAGUUUGUAAUAUUAGGA
637
BetaCoV/Wuhan-Hu-
22548
22570
TRUE
TRUE
FALSE
+


1184202
C

A

1/2019|EPL_ISL_402125











AD-
GCUUGUUAAACAACUUAGCU
283
GAGCUAAGUUGUUUAACAAGCG
638
BetaCoV/Wuhan-Hu-
24443
24465
TRUE
TRUE
FALSE
+


1184203
C

U

1/2019|EPL_ISL_402125











AD-
CUUGUUAAACAACUUAGCUC
284
GGAGCUAAGUUGUUUAACAAGC
639
BetaCoV/Wuhan-Hu-
24444
24466
TRUE
TRUE
FALSE
+


1184204
C

G

1/2019|EPL_ISL_402125











AD-
CUUGCUGCUACUAAAAUGUC
285
UGACAUUUUAGUAGCAGCAAGA
640
BetaCoV/Wuhan-Hu-
24630
24652
TRUE
TRUE
FALSE
+


1184205
A

U

1/2019|EPL_ISL_402125











AD-
ACUUAUGGAUUUGUUUAUGA
286
CUCAUAAACAAAUCCAUAAGUU
641
BetaCoV/Wuhan-Hu-
25387
25409
TRUE
TRUE
FALSE
+


1184206
G

C

1/2019|EPL_ISL_402125











AD-
CGACGACGACUACUAGCGUG
287
GCACGCUAGUAGUCGUCGUCGG
642
BetaCoV/Wuhan-Hu-
26190
26212
TRUE
TRUE
FALSE
+


1184207
C

U

1/2019|EPL_ISL_402125











AD-
GACGACGACUACUAGCGUGC
288
GGCACGCUAGUAGUCGUCGUCG
643
BetaCoV/Wuhan-Hu-
26191
26213
TRUE
TRUE
FALSE
+


1184208
C

G

1/2019|EPL_ISL_402125











AD-
AUGAGUACGAACUUAUGUAC
289
AGUACAUAAGUUCGUACUCAUC
644
BetaCoV/Wuhan-Hu-
26229
26251
TRUE
TRUE
FALSE
+


1184209
U

A

1/2019|EPL_ISL_402125











AD-
CGAACUUAUGUACUCAUUCG
290
ACGAAUGAGUACAUAAGUUCGU
645
BetaCoV/Wuhan-Hu-
26236
26258
TRUE
TRUE
FALSE
+


1184210
U

A

1/2019|EPL_ISL_402125











AD-
GAACUUAUGUACUCAUUCGU
291
AACGAAUGAGUACAUAAGUUCG
646
BetaCoV/Wuhan-Hu-
26237
26259
TRUE
TRUE
FALSE
+


1184211
U

U

1/2019|EPL_ISL_402125











AD-
AACUUAUGUACUCAUUCGUU
292
AAACGAAUGAGUACAUAAGUUC
647
BetaCoV/Wuhan-Hu-
26238
26260
TRUE
TRUE
FALSE
+


1184212
U

G

1/2019|EPL_ISL_402125











AD-
ACUUAUGUACUCAUUCGUUU
293
GAAACGAAUGAGUACAUAAGUU
648
BetaCoV/Wuhan-Hu-
26239
26261
TRUE
TRUE
FALSE
+


1184213
C

C

1/2019|EPL_ISL_402125











AD-
CUUAUGUACUCAUUCGUUUC
294
CGAAACGAAUGAGUACAUAAGU
649
BetaCoV/Wuhan-Hu-
26240
26262
TRUE
TRUE
FALSE
+


1184214
G

U

1/2019|EPL_ISL_402125











AD-
UUAUGUACUCAUUCGUUUCG
295
CCGAAACGAAUGAGUACAUAAG
650
BetaCoV/Wuhan-Hu-
26241
26263
TRUE
TRUE
FALSE
+


1184215
G

U

1/2019|EPL_ISL_402125











AD-
UAUGUACUCAUUCGUUUCGG
296
UCCGAAACGAAUGAGUACAUAA
651
BetaCoV/Wuhan-Hu-
26242
26264
TRUE
TRUE
FALSE
+


1184216
A

G

1/2019|EPL_ISL_402125











AD-
AUGUACUCAUUCGUUUCGGA
297
UUCCGAAACGAAUGAGUACAUA
652
BetaCoV/Wuhan-Hu-
26243
26265
TRUE
TRUE
FALSE
+


1184217
A

A

1/2019|EPL_ISL_402125











AD-
UGUACUCAUUCGUUUCGGAA
298
CUUCCGAAACGAAUGAGUACAU
653
BetaCoV/Wuhan-Hu-
26244
26266
TRUE
TRUE
FALSE
+


1184218
G

A

1/2019|EPL_ISL_402125











AD-
GUACUCAUUCGUUUCGGAAG
299
UCUUCCGAAACGAAUGAGUACA
654
BetaCoV/Wuhan-Hu-
26245
26267
TRUE
TRUE
FALSE
+


1184219
A

U

1/2019|EPL_ISL_402125











AD-
CAGGUACGUUAAUAGUUAAU
300
UAUUAACUAUUAACGUACCUGU
655
BetaCoV/Wuhan-Hu-
26268
26290
TRUE
TRUE
FALSE
+


1184220
A

C

1/2019|EPL_ISL_402125











AD-
AGGUACGUUAAUAGUUAAUA
301
CUAUUAACUAUUAACGUACCUG
656
BetaCoV/Wuhan-Hu-
26269
26291
TRUE
TRUE
FALSE
+


1184221
G

U

1/2019|EPL_ISL_402125











AD-
UAAUAGUUAAUAGCGUACUU
302
GAAGUACGCUAUUAACUAUUAA
657
BetaCoV/Wuhan-Hu-
26277
26299
TRUE
TRUE
FALSE
+


1184222
C

C

1/2019|EPL_ISL_402125











AD-
UCUUGCUUUCGUGGUAUUCU
303
AAGAAUACCACGAAAGCAAGAA
658
BetaCoV/Wuhan-Hu-
26302
26324
TRUE
TRUE
FALSE
+


1184223
U

A

1/2019|EPL_ISL_402125











AD-
CUUGCUUUCGUGGUAUUCUU
304
CAAGAAUACCACGAAAGCAAGA
659
BetaCoV/Wuhan-Hu-
26303
26325
TRUE
TRUE
FALSE
+


1184224
G

A

1/2019|EPL_ISL_402125











AD-
UUGCUUUCGUGGUAUUCUUG
305
GCAAGAAUACCACGAAAGCAAG
660
BetaCoV/Wuhan-Hu-
26304
26326
TRUE
TRUE
FALSE
+


1184225
C

A

1/2019|EPL_ISL_402125











AD-
UGCUUUCGUGGUAUUCUUGC
306
AGCAAGAAUACCACGAAAGCAA
661
BetaCoV/Wuhan-Hu-
26305
26327
TRUE
TRUE
FALSE
+


1184226
U

G

1/2019|EPL_ISL_402125











AD-
GCUUUCGUGGUAUUCUUGCU
307
UAGCAAGAAUACCACGAAAGCA
662
BetaCoV/Wuhan-Hu-
26306
26328
TRUE
TRUE
FALSE
+


1184227
A

A

1/2019|EPL_ISL_402125











AD-
UUUCGUGGUAUUCUUGCUAG
308
ACUAGCAAGAAUACCACGAAAG
663
BetaCoV/Wuhan-Hu-
26308
26330
TRUE
TRUE
FALSE
+


1184228
U

C

1/2019|EPL_ISL_402125











AD-
CUGCGCUUCGAUUGUGUGCG
309
ACGCACACAAUCGAAGCGCAGU
664
BetaCoV/Wuhan-Hu-
26346
26368
TRUE
TRUE
FALSE
+


1184229
U

A

1/2019|EPL_ISL_402125











AD-
GUACUGCUGCAAUAUUGUUA
310
UUAACAAUAUUGCAGCAGUACG
665
BetaCoV/Wuhan-Hu-
26365
26387
TRUE
TRUE
FALSE
+


1184230
A

C

1/2019|EPL_ISL_402125











AD-
CUGCUGCAAUAUUGUUAACG
311
ACGUUAACAAUAUUGCAGCAGU
666
BetaCoV/Wuhan-Hu-
26368
26390
TRUE
TRUE
FALSE
+


1184231
U

A

1/2019|EPL_ISL_402125











AD-
UGCUGCAAUAUUGUUAACGU
312
CACGUUAACAAUAUUGCAGCAG
667
BetaCoV/Wuhan-Hu-
26369
26391
TRUE
TRUE
FALSE
+


1184232
G

U

1/2019|EPL_ISL_402125











AD-
GCUGCAAUAUUGUUAACGUG
313
UCACGUUAACAAUAUUGCAGCA
668
BetaCoV/Wuhan-Hu-
26370
26392
TRUE
TRUE
FALSE
+


1184233
A

G

1/2019|EPL_ISL_402125











AD-
CUGCAAUAUUGUUAACGUGA
314
CUCACGUUAACAAUAUUGCAGC
669
BetaCoV/Wuhan-Hu-
26371
26393
TRUE
TRUE
FALSE
+


1184234
G

A

1/2019|EPL_ISL_402125











AD-
AGAGUUCCUGAUCUUCUGGU
315
GACCAGAAGAUCAGGAACUCUA
670
BetaCoV/Wuhan-Hu-
26447
26469
TRUE
TRUE
FALSE
+


1184235
C

G

1/2019|EPL_ISL_402125











AD-
CUGAUCUUCUGGUCUAAACG
316
UCGUUUAGACCAGAAGAUCAGG
671
BetaCoV/Wuhan-Hu-
26454
26476
TRUE
TRUE
FALSE
+


1184236
A

A

1/2019|EPL_ISL_402125











AD-
GAUCUUCUGGUCUAAACGAA
317
GUUCGUUUAGACCAGAAGAUCA
672
BetaCoV/Wuhan-Hu-
26456
26478
TRUE
TRUE
FALSE
+


1184237
C

G

1/2019|EPL_ISL_402125











AD-
ACAAUGGAACCUAGUAAUAG
318
CCUAUUACUAGGUUCCAUUGUU
673
BetaCoV/Wuhan-Hu-
26574
26596
TRUE
TRUE
FALSE
+


1184238
G

C

1/2019|EPL_ISL_402125











AD-
CAAUGGAACCUAGUAAUAGG
319
ACCUAUUACUAGGUUCCAUUGU
674
BetaCoV/Wuhan-Hu-
26575
26597
TRUE
TRUE
FALSE
+


1184239
U

U

1/2019|EPL_ISL_402125











AD-
AAUGGAACCUAGUAAUAGGU
320
AACCUAUUACUAGGUUCCAUUG
675
BetaCoV/Wuhan-Hu-
26576
26598
TRUE
TRUE
FALSE
+


1184240
U

U

1/2019|EPL_ISL_402125











AD-
AUGGAACCUAGUAAUAGGUU
321
AAACCUAUUACUAGGUUCCAUU
676
BetaCoV/Wuhan-Hu-
26577
26599
TRUE
TRUE
FALSE
+


1184241
U

G

1/2019|EPL_ISL_402125











AD-
AACCUAGUAAUAGGUUUCCU
322
UAGGAAACCUAUUACUAGGUUC
677
BetaCoV/Wuhan-Hu-
26581
26603
TRUE
TRUE
FALSE
+


1184242
A

C

1/2019|EPL_ISL_402125











AD-
ACCUAGUAAUAGGUUUCCUA
323
AUAGGAAACCUAUUACUAGGUU
678
BetaCoV/Wuhan-Hu-
26582
26604
TRUE
TRUE
FALSE
+


1184243
U

C

1/2019|EPL_ISL_402125











AD-
UACAUCACGAACGCUUUCUU
324
UAAGAAAGCGUUCGUGAUGUAG
679
BetaCoV/Wuhan-Hu-
27033
27055
TRUE
TRUE
FALSE
+


1184244
A

C

1/2019|EPL_ISL_402125











AD-
ACAUCACGAACGCUUUCUUA
325
AUAAGAAAGCGUUCGUGAUGUA
680
BetaCoV/Wuhan-Hu-
27034
27056
TRUE
TRUE
FALSE
+


1184245
U

G

1/2019|EPL_ISL_402125











AD-
CAUCACGAACGCUUUCUUAU
326
AAUAAGAAAGCGUUCGUGAUGU
681
BetaCoV/Wuhan-Hu-
27035
27057
TRUE
TRUE
FALSE
+


1184246
U

A

1/2019|EPL_ISL_402125











AD-
AUCACGAACGCUUUCUUAUU
327
UAAUAAGAAAGCGUUCGUGAUG
682
BetaCoV/Wuhan-Hu-
27036
27058
TRUE
TRUE
FALSE
+


1184247
A

U

1/2019|EPL_ISL_402125











AD-
UCACGAACGCUUUCUUAUUA
328
GUAAUAAGAAAGCGUUCGUGAU
683
BetaCoV/Wuhan-Hu-
27037
27059
TRUE
TRUE
FALSE
+


1184248
C

G

1/2019|EPL_ISL_402125











AD-
CACGAACGCUUUCUUAUUAC
329
UGUAAUAAGAAAGCGUUCGUGA
684
BetaCoV/Wuhan-Hu-
27038
27060
TRUE
TRUE
FALSE
+


1184249
A

U

1/2019|EPL_ISL_402125











AD-
ACGAACGCUUUCUUAUUACA
330
UUGUAAUAAGAAAGCGUUCGUG
685
BetaCoV/Wuhan-Hu-
27039
27061
TRUE
TRUE
FALSE
+


1184250
A

A

1/2019|EPL_ISL_402125











AD-
CGAACGCUUUCUUAUUACAA
331
UUUGUAAUAAGAAAGCGUUCGU
686
BetaCoV/Wuhan-Hu-
27040
27062
TRUE
TRUE
FALSE
+


1184251
A

G

1/2019|EPL_ISL_402125











AD-
GUAAGUGACAACAGAUGUUU
332
GAAACAUCUGUUGUCACUUACU
687
BetaCoV/Wuhan-Hu-
27186
27208
TRUE
TRUE
FALSE
+


1184252
C

G

1/2019|EPL_ISL_402125











AD-
UUAGCCUUUCUGCUAUUCCU
333
AAGGAAUAGCAGAAAGGCUAAA
688
BetaCoV/Wuhan-Hu-
27793
27815
TRUE
TRUE
FALSE
+


1184253
U

A

1/2019|EPL_ISL_402125











AD-
UAGCCUUUCUGCUAUUCCUU
334
CAAGGAAUAGCAGAAAGGCUAA
689
BetaCoV/Wuhan-Hu-
27794
27816
TRUE
TRUE
FALSE
+


1184254
G

A

1/2019|EPL_ISL_402125











AD-
AGCCUUUCUGCUAUUCCUUG
335
ACAAGGAAUAGCAGAAAGGCUA
690
BetaCoV/Wuhan-Hu-
27795
27817
TRUE
TRUE
FALSE
+


1184255
U

A

1/2019|EPL_ISL_402125











AD-
CCUUUCUGCUAUUCCUUGUU
336
AAACAAGGAAUAGCAGAAAGGC
691
BetaCoV/Wuhan-Hu-
27797
27819
TRUE
TRUE
FALSE
+


1184256
U

U

1/2019|EPL_ISL_402125











AD-
CUUUCUGCUAUUCCUUGUUU
337
AAAACAAGGAAUAGCAGAAAGG
692
BetaCoV/Wuhan-Hu-
27798
27820
TRUE
TRUE
FALSE
+


1184257
U

C

1/2019|EPL_ISL_402125











AD-
UUUCUGCUAUUCCUUGUUUU
338
UAAAACAAGGAAUAGCAGAAAG
693
BetaCoV/Wuhan-Hu-
27799
27821
TRUE
TRUE
FALSE
+


1184258
A

G

1/2019|EPL_ISL_402125











AD-
UUCUGCUAUUCCUUGUUUUA
339
UUAAAACAAGGAAUAGCAGAAA
694
BetaCoV/Wuhan-Hu-
27800
27822
TRUE
TRUE
FALSE
+


1184259
A

G

1/2019|EPL_ISL_402125











AD-
UCUGCUAUUCCUUGUUUUAA
340
AUUAAAACAAGGAAUAGCAGAA
695
BetaCoV/Wuhan-Hu-
27801
27823
TRUE
TRUE
FALSE
+


1184260
U

A

1/2019|EPL_ISL_402125











AD-
CUGCUAUUCCUUGUUUUAAU
341
AAUUAAAACAAGGAAUAGCAGA
696
BetaCoV/Wuhan-Hu-
27802
27824
TRUE
TRUE
FALSE
+


1184261
U

A

1/2019|EPL_ISL_402125











AD-
CCAAGGUUUACCCAAUAAUA
342
GUAUUAUUGGGUAAACCUUGGG
697
BetaCoV/Wuhan-Hu-
28397
28419
TRUE
TRUE
FALSE
+


1184262
C

G

1/2019|EPL_ISL_402125











AD-
AAGGUUUACCCAAUAAUACU
343
CAGUAUUAUUGGGUAAACCUUG
698
BetaCoV/Wuhan-Hu-
28399
28421
TRUE
TRUE
FALSE
+


1184263
G

G

1/2019|EPL_ISL_402125











AD-
AGGUUUACCCAAUAAUACUG
344
GCAGUAUUAUUGGGUAAACCUU
699
BetaCoV/Wuhan-Hu-
28400
28422
TRUE
TRUE
FALSE
+


1184264
C

G

1/2019|EPL_ISL_402125











AD-
ACCCAAUAAUACUGCGUCUU
345
CAAGACGCAGUAUUAUUGGGUA
700
BetaCoV/Wuhan-Hu-
28406
28428
TRUE
TRUE
FALSE
+


1184265
G

A

1/2019|EPL_ISL_402125











AD-
AGUCCAGAUGACCAAAUUGG
346
GCCAAUUUGGUCAUCUGGACUG
701
BetaCoV/Wuhan-Hu-
28506
28528
TRUE
TRUE
FALSE
+


1184266
C

C

1/2019|EPL_ISL_402125











AD-
GUCCAGAUGACCAAAUUGGC
347
AGCCAAUUUGGUCAUCUGGACU
702
BetaCoV/Wuhan-Hu-
28507
28529
TRUE
TRUE
FALSE
+


1184267
U

G

1/2019|EPL_ISL_402125











AD-
AUUGCCAAAAGGCUUCUACG
348
GCGUAGAAGCCUUUUGGCAAUG
703
BetaCoV/Wuhan-Hu-
28769
28791
TRUE
TRUE
FALSE
+


1184268
C

U

1/2019|EPL_ISL_402125











AD-
UUGCCAAAAGGCUUCUACGC
349
UGCGUAGAAGCCUUUUGGCAAU
704
BetaCoV/Wuhan-Hu-
28770
28792
TRUE
TRUE
FALSE
+


1184269
A

G

1/20191EPLISL_402125











AD-
GGCAGUCAAGCCUCUUCUCG
350
ACGAGAAGAGGCUUGACUGCCG
705
BetaCoV/Wuhan-Hu-
28806
28828
TRUE
TRUE
FALSE
+


1184270
U

C

1/2019|EPL_ISL_402125











AD-
AACUGUCACUAAGAAAUCUG
351
GCAGAUUUCUUAGUGACAGUUU
706
BetaCoV/Wuhan-Hu-
29003
29025
TRUE
TRUE
FALSE
+


1184271
C

G

1/2019|EPL_ISL_402125











AD-
UGUCACUAAGAAAUCUGCUG
352
GCAGCAGAUUUCUUAGUGACAG
707
BetaCoV/Wuhan-Hu-
29006
29028
TRUE
TRUE
FALSE
+


1184272
C

U

1/2019|EPL_ISL_402125











AD-
AACUGUGACUCUUCUUCCUG
353
GCAGGAAGAAGAGUCACAGUUU
708
BetaCoV/Wuhan-Hu-
29441
29463
TRUE
TRUE
FALSE
+


1184273
C

G

1/2019|EPL_ISL_402125











AD-
UCUUGUGCAGAAUGAAUUCU
354
GAGAAUUCAUUCUGCACAAGAG
709
BetaCoV/Wuhan-Hu-
29605
29627
TRUE
TRUE
FALSE
+


1184274
C

U

1/2019|EPL_ISL_402125











AD-
CUUGUGCAGAAUGAAUUCUC
355
CGAGAAUUCAUUCUGCACAAGA
710
BetaCoV/Wuhan-Hu-
29606
29628
TRUE
TRUE
FALSE
+


1184275
G

G

1/2019|EPL_ISL_402125











AD-
UUGUGCAGAAUGAAUUCUCG
356
ACGAGAAUUCAUUCUGCACAAG
711
BetaCoV/Wuhan-Hu-
29607
29629
TRUE
TRUE
FALSE
+


1184276
U

A

1/2019|EPL_ISL_402125











AD-
UGUGCAGAAUGAAUUCUCGU
357
UACGAGAAUUCAUUCUGCACAA
712
BetaCoV/Wuhan-Hu-
29608
29630
TRUE
TRUE
FALSE
+


1184277
A

G

1/2019|EPL_ISL_402125











AD-
GUGCAGAAUGAAUUCUCGUA
358
UUACGAGAAUUCAUUCUGCACA
713
BetaCoV/Wuhan-Hu-
29609
29631
TRUE
TRUE
FALSE
+


1184278
A

A

1/2019|EPL_ISL_402125











AD-
UGCAGAAUGAAUUCUCGUAA
359
GUUACGAGAAUUCAUUCUGCAC
714
BetaCoV/Wuhan-Hu-
29610
29632
TRUE
TRUE
FALSE
+


1184279
C

A

1/2019|EPL_ISL_402125











AD-
GCAGAAUGAAUUCUCGUAAC
360
AGUUACGAGAAUUCAUUCUGCA
715
BetaCoV/Wuhan-Hu-
29611
29633
TRUE
TRUE
FALSE
+


1184280
U

C

1/2019|EPL_ISL_402125











AD-
CAGAAUGAAUUCUCGUAACU
361
UAGUUACGAGAAUUCAUUCUGC
716
BetaCoV/Wuhan-Hu-
29612
29634
TRUE
TRUE
FALSE
+


1184281
A

A

1/2019|EPL_ISL_402125











AD-
UAAUCUCACAUAGCAAUCUU
362
AAAGAUUGCUAUGUGAGAUUA
717
BetaCoV/Wuhan-Hu-
29660
29682
TRUE
TRUE
FALSE
+


1184282
U

AA

1/2019|EPL_ISL_402125











AD-
UCUCACAUAGCAAUCUUUAA
363
AUUAAAGAUUGCUAUGUGAGA
718
BetaCoV/Wuhan-Hu-
29663
29685
TRUE
TRUE
FALSE
+


1184283
U

UU

1/2019|EPL_ISL_402125











AD-
CUCACAUAGCAAUCUUUAAU
364
GAUUAAAGAUUGCUAUGUGAG
719
BetaCoV/Wuhan-Hu-
29664
29686
TRUE
TRUE
FALSE
+


1184284
C

AU

1/2019|EPL_ISL_402125











AD-
UCACAUAGCAAUCUUUAAUC
365
UGAUUAAAGAUUGCUAUGUGA
720
BetaCoV/Wuhan-Hu-
29665
29687
TRUE
TRUE
FALSE
+


1184285
A

GA

1/2019|EPL_ISL_402125











AD-
CACAUAGCAAUCUUUAAUCA
366
CUGAUUAAAGAUUGCUAUGUGA
721
BetaCoV/Wuhan-Hu-
29666
29688
TRUE
TRUE
FALSE
+


1184286
G

G

1/2019|EPL_ISL_402125











AD-
GAGCCCUAAUGUGUAAAAUU
367
UAAUUUUACACAUUAGGGCUCU
722
BetaCoV/Wuhan-Hu-
29796
29818
TRUE
TRUE
FALSE
+


1184287
A

U

1/2019|EPL_ISL_402125











AD-
GCCCUAAUGUGUAAAAUUAA
368
AUUAAUUUUACACAUUAGGGCU
723
BetaCoV/Wuhan-Hu-
29798
29820
TRUE
TRUE
FALSE
+


1184288
U

C

1/2019|EPL_ISL_402125











AD-
CCCUAAUGUGUAAAAUUAAU
369
AAUUAAUUUUACACAUUAGGGC
724
BetaCoV/Wuhan-Hu-
29799
29821
TRUE
TRUE
FALSE
+


1184289
U

U

1/2019|EPL_ISL_402125











AD-
CCUAAUGUGUAAAAUUAAUU
370
AAAUUAAUUUUACACAUUAGGG
725
BetaCoV/Wuhan-Hu-
29800
29822
TRUE
TRUE
FALSE
+


1184290
U

C

1/2019|EPL_ISL_402125











AD-
CCCAUGUGAUUUUAAUAGCU
371
AAGCUAUUAAAAUCACAUGGGG
726
BetaCoV/Wuhan-Hu-
29834
29856
TRUE
TRUE
FALSE
+


1184291
U

A

1/2019|EPL_ISL_402125
















TABLE 3







Modified Sense and Antisense Strand Coronavirus dsRNA Sequences





















Strand of the









Viral RNA


Duplex
Sense Strand Sequence

Antisense Strand Sequence

mRNA Target Sequence

Targeted by


Name
5′ to 3′
SEQ ID NO:
5′ to 3′
SEQ ID NO:
5′ to 3′
SEQ ID NO:
the Agent

















AD-
asuscug(Uhd)UfcUfCfUfaaacgaa
727
VPusAfsgudTc(G2p)uuuagaGfaAfcag
1082
AGATCTGTTCTCTAAACGAA
1437
+


1183935
csusa

auscsu

CTT







AD-
uscsugu(Uhd)CfuCfUfAfaacgaac
728
VPusAfsagdTu(C2p)guuuagAfgAfaca
1083
GATCTGTTCTCTAAACGAAC
1438
+


1183936
ususa

gasusc

TTT







AD-
gsusucu(Chd)UfaAfAfCfgaacuuu
729
VPusUfsuadAa(G2p)uucguuUfaGfaga
1084
CTGTTCTCTAAACGAACTTT
1439
+


1183937
asasa

acsasg

AAA







AD-
csusgcu(Uhd)AfcGfGfUfuucgucc
730
VPusAfscgdGa(C2p)gaaaccGfuAfagca
1085
GGCTGCTTACGGTTTCGTCC
1440
+


1183938
gsusa

gscsc

GTG







AD-
gsgsugu(Ghd)AfcCfGfAfaagguaa
731
VPusUfscudTa(C2p)cuuucgGfuCfacac
1086
CGGGTGTGACCGAAAGGTA
1441
+


1183939
gsasa

cscsg

AGAT







AD-
cscsgaa(Ahd)GfgUfAfAfgauggag
732
VPusCfsucdTc(C2p)aucuuaCfcUfuucg
1087
GACCGAAAGGTAAGATGGA
1442
+


1183940
asgsa

gsusc

GAGC







AD-
asasaca(Chd)AfcGfUfCfcaacucag
733
VPusAfscudGa(G2p)uuggacGfuGfugu
1088
GAAAACACACGTCCAACTCA
1443
+


1183941
susa

uususc

GTT







AD-
ascsaca(Chd)GfuCfCfAfacucaguu
734
VPusAfsaadCu(G2p)aguuggAfcGfugu
1089
AAACACACGTCCAACTCAGT
1444
+


1183942
susa

gususu

TTG







AD-
gsuscca(Ahd)CfuCfAfGfuuugccu
735
VPusAfscadGg(C2p)aaacugAfgUfugg
1090
ACGTCCAACTCAGTTTGCCT
1445
+


1183943
gsusa

acsgsu

GTT







AD-
csusucu(Uhd)CfgUfAfAfgaacggu
736
VPusUfsuadCc(G2p)uucuuaCfgAfaga
1091
TTCTTCTTCGTAAGAACGGT
1446
+


1183944
asasa

agsasa

AAT







AD-
ususcuu(Chd)GfuAfAfGfaacggua
737
VPusAfsuudAc(C2p)guucuuAfcGfaag
1092
TCTTCTTCGTAAGAACGGTA
1447
+


1183945
asusa

aasgsa

ATA







AD-
uscsuuc(Ghd)UfaAfGfAfacgguaa
738
VPusUfsaudTa(C2p)cguucuUfaCfgaag
1093
CTTCTTCGTAAGAACGGTAA
1448
+


1183946
usasa

asasg

TAA







AD-
ususccu(Uhd)AfaAfCfUfucucuuc
739
VPusUfsugdAa(G2p)agaaguUfuAfagg
1094
CCTTCCTTAAACTTCTCTTCA
1449



1183947
asasa

aasgsg

AG







AD-
uscsacc(Uhd)AfaAfUfUfcaaggcu
740
VPusAfsaadGc(C2p)uugaauUfuAfggu
1095
TTTCACCTAAATTCAAGGCT
1450



1183948
ususa

gasasa

TTA







AD-
csuscug(Ghd)AfuUfUfAfacacacu
741
VPusAfsaadGu(G2p)uguuaaAfuCfcag
1096
TTCTCTGGATTTAACACACTT
1451



1183949
ususa

agsasa

TC







AD-
csusucu(Chd)AfuUfAfAfguacuuu
742
VPusAfsuadAa(G2p)uacuuaAfuGfaga
1097
CACTTCTCATTAAGTACTTTA
1452



1183950
asusa

agsusg

TC







AD-
ascsucu(Uhd)CfuUfCfUfucacaau
743
VPusUfsgadTu(G2p)ugaagaAfgAfaga
1098
AAACTCTTCTTCTTCACAATC
1453



1183951
csasa

gususu

AC







AD-
ususauu(Uhd)AfaAfAfCfuuacuga
744
VPusUfsgudCa(G2p)uaaguuUfuAfaau
1099
GGTTATTTAAAACTTACTGA
1454
+


1183952
csasa

aascsc

CAA







AD-
usasucu(Ahd)CfaCfAfAfacucuua
745
VPusUfsuudAa(G2p)aguuugUfgUfaga
1100
AGTATCTACACAAACTCTTA
1455



1183953
asasa

uascsu

AAG







AD-
asuscuu(Ghd)UfuUfUfCfucuguuc
746
VPusUfsugdAa(C2p)agagaaAfaCfaaga
1101
TCATCTTGTTTTCTCTGTTCA
1456



1183954
asasa

usgsa

AC







AD-
csascuu(Uhd)UfaUfCfAfccucucu
747
VPusUfsaadGa(G2p)aggugaUfaAfaag
1102
TACACTTTTATCACCTCTCTT
1457



1183955
usasa

ugsusa

AA







AD-
uscsucu(Ghd)AfaGfAfAfguagugg
748
VPusUfsucdCa(C2p)uacuucUfuCfaga
1103
AGTCTCTGAAGAAGTAGTGG
1458
+


1183956
asasa

gascsu

AAA







AD-
uscsuga(Ahd)GfaAfGfUfaguggaa
749
VPusUfsuudTc(C2p)acuacuUfcUfucag
1104
TCTCTGAAGAAGTAGTGGAA
1459
+


1183957
asasa

asgsa

AAT







AD-
csusgaa(Ghd)AfaGfUfAfguggaaa
750
VPusAfsuudTu(C2p)cacuacUfuCfuuca
1105
CTCTGAAGAAGTAGTGGAAA
1460
+


1183958
asusa

gsasg

ATC







AD-
asgsugg(Ahd)AfaAfUfCfcuaccau
751
VPusGfsuadTg(G2p)uaggauUfuUfcca
1106
GTAGTGGAAAATCCTACCAT
1461
+


1183959
ascsa

cusasc

ACA







AD-
gsusgga(Ahd)AfaUfCfCfuaccaua
752
VPusUfsgudAu(G2p)guaggaUfuUfucc
1107
TAGTGGAAAATCCTACCATA
1462
+


1183960
csasa

acsusa

CAG







AD-
gscsucu(Uhd)CfuAfAfAfccucaua
753
VPusUfsuudAu(G2p)agguuuAfgAfaga
1108
AAGCTCTTCTAAACCTCATA
1463



1183961
asasa

gcsusu

AAA







AD-
asusggu(Uhd)CfaCfCfAfucuggug
754
VPusAfsacdAc(C2p)agauggUfgAfacc
1109
CAATGGTTCACCATCTGGTG
1464
+


1183962
ususa

aususg

TTT







AD-
csasugu(Ghd)GfuAfGfUfguuggu
755
VPusAfsaadAc(C2p)aacacuAfcCfacau
1110
TTCATGTGGTAGTGTTGGTTT
1465
+


1183963
uususa

gsasa

TA







AD-
csasguc(Ahd)UfaAfUfCfuauguua
756
VPusUfsuudAa(C2p)auagauUfaUfgac
1111
CACAGTCATAATCTATGTTA
1466



1183964
asasa

ugsusg

AAA







AD-
ususuug(Ahd)UfgUfUfGfuuagaca
757
VPusAfsuudGu(C2p)uaacaaCfaUfcaaa
1112
CCTTTTGATGTTGTTAGACA
1467
+


1183965
asusa

asgsg

ATG







AD-
asusggu(Ahd)AfuGfCfUfuuagauc
758
VPusUfsugdAu(C2p)uaaagcAfuUfacc
1113
TTATGGTAATGCTTTAGATC
1468
+


1183966
asasa

ausasa

AAG







AD-
gscsuag(Ahd)UfuCfCfCfuaagagu
759
VPusUfscadCu(C2p)uuagggAfaUfcua
1114
GGGCTAGATTCCCTAAGAGT
1469
+


1183967
gsasa

gcscsc

GAT







AD-
ascsaac(Ahd)UfcUfUfAfacacaauu
760
VPusUfsaadTu(G2p)uguuaaGfaUfguu
1115
ACACAACATCTTAACACAAT
1470



1183968
sasa

gusgsu

TAG







AD-
usasaga(Uhd)GfuUfGfUfguacaca
761
VPusUfsgudGu(G2p)uacacaAfcAfucu
1116
GTTAAGATGTTGTGTACACA
1471
+


1183969
csasa

uasasc

CAC







AD-
asgsaug(Uhd)UfgUfGfUfacacaca
762
VPusAfsgudGu(G2p)uguacaCfaAfcau
1117
TAAGATGTTGTGTACACACA
1472
+


1183970
csusa

cususa

CTG







AD-
gsusugu(Ghd)UfaCfAfCfacacugg
763
VPusUfsacdCa(G2p)ugugugUfaCfaca
1118
ATGTTGTGTACACACACTGG
1473
+


1183971
usasa

acsasu

TAC







AD-
csasggc(Ahd)CfuAfGfUfacugaug
764
VPusGfsacdAu(C2p)aguacuAfgUfgcc
1119
CACAGGCACTAGTACTGATG
1474
+


1183972
uscsa

ugsusg

TCG







AD-
cscsguc(Uhd)AfuUfCfUfaaacuua
765
VPusUfsuudAa(G2p)uuuagaAfuAfgac
1120
CACCGTCTATTCTAAACTTA
1475



1183973
asasa

ggsusg

AAG







AD-
ususuua(Ahd)AfuAfUfUfgggauca
766
VPusUfscudGa(Tgn)cccaauAfuUfuaaa
1121
TATTTTAAATATTGGGATCA
1476
+


1183974
gsasa

asusa

GAC







AD-
ususuaa(Ahd)UfaUfUfGfggaucag
767
VPusGfsucdTg(Agn)ucccaaUfaUfuuaa
1122
ATTTTAAATATTGGGATCAG
1477
+


1183975
ascsa

asasu

ACA







AD-
csasccu(Ahd)CfaAfGfUfuuuggac
768
VPusUfsggdTc(C2p)aaaacuUfgUfagg
1123
CCCACCTACAAGTTTTGGAC
1478
+


1183976
csasa

ugsgsg

CAC







AD-
asasuca(Ghd)GfaUfGfUfaaacuua
769
VPusUfsgudAa(G2p)uuuacaUfcCfuga
1124
ATAATCAGGATGTAAACTTA
1479
+


1183977
csasa

uusasu

CAT







AD-
asgsgau(Ghd)UfaAfAfCfuuacaua
770
VPusGfscudAu(G2p)uaaguuUfaCfauc
1125
TCAGGATGTAAACTTACATA
1480
+


1183978
gscsa

cusgsa

GCT







AD-
ascsaau(Ghd)UfuGfCfUfuuucaaa
771
VPusAfsgudTu(G2p)aaaagcAfaCfauu
1126
TAACAATGTTGCTTTTCAAA
1481
+


1183979
csusa

gususa

CTG







AD-
usgsuug(Chd)UfuUfUfCfaaacugu
772
VPusUfsgadCa(G2p)uuugaaAfaGfcaa
1127
AATGTTGCTTTTCAAACTGT
1482
+


1183980
csasa

casusu

CAA







AD-
ususgcu(Uhd)UfuCfAfAfacuguca
773
VPusUfsuudGa(C2p)aguuugAfaAfagc
1128
TGTTGCTTTTCAAACTGTCA
1483
+


1183981
asasa

aascsa

AAC







AD-
gscsuuu(Uhd)CfaAfAfCfugucaaa
774
VPusGfsgudTu(G2p)acaguuUfgAfaaa
1129
TTGCTTTTCAAACTGTCAAA
1484
+


1183982
cscsa

gcsasa

CCC







AD-
usasuga(Chd)UfuUfGfCfugugucu
775
VPusUfsuadGa(C2p)acagcaAfaGfucau
1130
TCTATGACTTTGCTGTGTCTA
1485
+


1183983
asasa

asgsa

AG







AD-
gsusuuc(Uhd)UfuAfAfGfgaaggaa
776
VPusAfscudTc(C2p)uuccuuAfaAfgaaa
1131
GGGTTTCTTTAAGGAAGGAA
1486
+


1183984
gsusa

cscsc

GTT







AD-
ususucu(Uhd)UfaAfGfGfaaggaag
777
VPusAfsacdTu(C2p)cuuccuUfaAfagaa
1132
GGTTTCTTTAAGGAAGGAAG
1487
+


1183985
ususa

ascsc

TTC







AD-
asgsgaa(Ghd)GfaAfGfUfucuguug
778
VPusUfsucdAa(C2p)agaacuUfcCfuucc
1133
TAAGGAAGGAAGTTCTGTTG
1488
+


1183986
asasa

ususa

AAT







AD-
asascac(Uhd)UfcUfUfCfuuugcuc
779
VPusCfsugdAg(C2p)aaagaaGfaAfgug
1134
AAAACACTTCTTCTTTGCTC
1489
+


1183987
asgsa

uususu

AGG







AD-
ascsuuc(Uhd)UfcUfUfUfgcucagg
780
VPusAfsucdCu(G2p)agcaaaGfaAfgaa
1135
ACACTTCTTCTTTGCTCAGG
1490
+


1183988
asusa

gusgsu

ATG







AD-
asasugu(Ghd)UfgAfUfAfucagaca
781
VPusGfsuudGu(C2p)ugauauCfaCfaca
1136
ACAATGTGTGATATCAGACA
1491
+


1183989
ascsa

uusgsu

ACT







AD-
cscsauu(Uhd)AfaUfAfAfauggggu
782
VPusUfsuadCc(C2p)cauuuaUfuAfaau
1137
TTCCATTTAATAAATGGGGT
1492
+


1183990
asasa

ggsasa

AAG







AD-
csasaug(Ahd)GfuUfAfUfgaggauc
783
VPusUfsugdAu(C2p)cucauaAfcUfcau
1138
TTCAATGAGTTATGAGGATC
1493
+


1183991
asasa

ugsasa

AAG







AD-
usgscaa(Ahd)GfaAfUfAfgagcucg
784
VPusUfsgcdGa(G2p)cucuauUfcUfuug
1139
AGTGCAAAGAATAGAGCTC
1494
+


1183992
csasa

cascsu

GCAC







AD-
gscsaaa(Ghd)AfaUfAfGfagcucgc
785
VPusGfsugdCg(Agn)gcucuaUfuCfuuu
1140
GTGCAAAGAATAGAGCTCGC
1495
+


1183993
ascsa

gcsasc

ACC







AD-
csasaag(Ahd)AfuAfGfAfgcucgca
786
VPusGfsgudGc(G2p)agcucuAfuUfcuu
1141
TGCAAAGAATAGAGCTCGCA
1496
+


1183994
cscsa

ugscsa

CCG







AD-
asasaga(Ahd)UfaGfAfGfcucgcac
787
VPusCfsggdTg(C2p)gagcucUfaUfucu
1142
GCAAAGAATAGAGCTCGCA
1497
+


1183995
csgsa

uusgsc

CCGT







AD-
usgsucu(Chd)UfaUfCfUfguaguac
788
VPusUfsagdTa(C2p)uacagaUfaGfagac
1143
GGTGTCTCTATCTGTAGTAC
1498
+


1183996
usasa

ascsc

TAT







AD-
csuscua(Uhd)CfuGfUfAfguacuau
789
VPusUfscadTa(G2p)uacuacAfgAfuaga
1144
GTCTCTATCTGTAGTACTAT
1499
+


1183997
gsasa

gsasc

GAC







AD-
uscsacc(Uhd)UfaUfGfGfguuggga
790
VPusAfsaudCc(C2p)aacccaUfaAfggug
1145
CCTCACCTTATGGGTTGGGA
1500
+


1183998
ususa

asgsg

TTA







AD-
csasccu(Uhd)AfuGfGfGfuugggau
791
VPusUfsaadTc(C2p)caacccAfuAfaggu
1146
CTCACCTTATGGGTTGGGAT
1501
+


1183999
usasa

gsasg

TAT







AD-
ascscuu(Ahd)UfgGfGfUfugggauu
792
VPusAfsuadAu(C2p)ccaaccCfaUfaagg
1147
TCACCTTATGGGTTGGGATT
1502
+


1184000
asusa

usgsa

ATC







AD-
cscsuua(Uhd)GfgGfUfUfgggauua
793
VPusGfsaudAa(Tgn)cccaacCfcAfuaag
1148
CACCTTATGGGTTGGGATTA
1503
+


1184001
uscsa

gsusg

TCC







AD-
ascsuug(Uhd)UfcUfUfGfcucgcaa
794
VPusGfsuudTg(C2p)gagcaaGfaAfcaag
1149
TCACTTGTTCTTGCTCGCAA
1504
+


1184002
ascsa

usgsa

ACA







AD-
csusugu(Uhd)CfuUfGfCfucgcaaa
795
VPusUfsgudTu(G2p)cgagcaAfgAfaca
1150
CACTTGTTCTTGCTCGCAAA
1505
+


1184003
csasa

agsusg

CAT







AD-
csascaa(Chd)UfgCfUfUfaugcuaa
796
VPusUfsaudTa(G2p)cauaagCfaGfuug
1151
GCCACAACTGCTTATGCTAA
1506
+


1184004
usasa

ugsgsc

TAG







AD-
usgsagu(Ghd)UfcUfCfUfauagaaa
797
VPusUfsaudTu(C2p)uauagaGfaCfacuc
1152
TATGAGTGTCTCTATAGAAA
1507
+


1184005
usasa

asusa

TAG







AD-
usgsgac(Uhd)GfaGfAfCfugaccuu
798
VPusGfsuadAg(G2p)ucagucUfcAfguc
1153
GTTGGACTGAGACTGACCTT
1508
+


1184006
ascsa

casasc

ACT







AD-
ususacc(Chd)AfgAfUfCfcaucaag
799
VPusUfsucdTu(G2p)auggauCfuGfggu
1154
CCTTACCCAGATCCATCAAG
1509
+


1184007
asasa

aasgsg

AAT







AD-
gsasugg(Uhd)AfcAfCfUfuaugauu
800
VPusUfscadAu(C2p)auaaguGfuAfcca
1155
CAGATGGTACACTTATGATT
1510
+


1184008
gsasa

ucsusg

GAA







AD-
asasaca(Uhd)CfcUfAfAfucaggag
801
VPusUfsacdTc(C2p)ugauuaGfgAfugu
1156
CTAAACATCCTAATCAGGAG
1511
+


1184009
usasa

uusasg

TAT







AD-
asascau(Chd)CfuAfAfUfcaggagu
802
VPusAfsuadCu(C2p)cugauuAfgGfaug
1157
TAAACATCCTAATCAGGAGT
1512
+


1184010
asusa

uususa

ATG







AD-
asgsgag(Uhd)AfuGfCfUfgaugucu
803
VPusAfsaadGa(C2p)aucagcAfuAfcucc
1158
TCAGGAGTATGCTGATGTCT
1513
+


1184011
ususa

usgsa

TTC







AD-
cscsuga(Ghd)UfuUfUfAfugaggcu
804
VPusAfsuadGc(C2p)ucauaaAfaCfucag
1159
AACCTGAGTTTTATGAGGCT
1514
+


1184012
asusa

gsusu

ATG







AD-
gscsagc(Ahd)GfaAfAfCfgcucaaa
805
VPusGfscudTu(G2p)agcguuUfcUfgcu
1160
TTGCAGCAGAAACGCTCAAA
1515
+


1184013
gscsa

gcsasa

GCT







AD-
cscsugg(Uhd)AfcUfGfGfuaagagu
806
VPusUfsgadCu(C2p)uuaccaGfuAfcca
1161
CACCTGGTACTGGTAAGAGT
1516
+


1184014
csasa

ggsusg

CAT







AD-
usasuag(Ahd)UfaAfAfUfguaguag
807
VPusUfsucdTa(C2p)uacauuUfaUfcuau
1162
CCTATAGATAAATGTAGTAG
1517
+


1184015
asasa

asgsg

AAT







AD-
csusgcu(Ghd)AfaAfUfUfguugaca
808
VPusAfsgudGu(C2p)aacaauUfuCfagc
1163
TCCTGCTGAAATTGTTGACA
1518
+


1184016
csusa

agsgsa

CTG







AD-
gscsuga(Ahd)AfuUfGfUfugacacu
809
VPusAfscadGu(G2p)ucaacaAfuUfuca
1164
CTGCTGAAATTGTTGACACT
1519
+


1184017
gsusa

gcsasg

GTG







AD-
usgsaaa(Uhd)UfgUfUfGfacacugu
810
VPusUfscadCa(G2p)ugucaaCfaAfuuu
1165
GCTGAAATTGTTGACACTGT
1520
+


1184018
gsasa

casgsc

GAG







AD-
asasauu(Ghd)UfuGfAfCfacuguga
811
VPusAfscudCa(C2p)agugucAfaCfaau
1166
TGAAATTGTTGACACTGTGA
1521
+


1184019
gsusa

uuscsa

GTG







AD-
usgsuug(Ahd)CfaCfUfGfugagugc
812
VPusAfsagdCa(C2p)ucacagUfgUfcaac
1167
ATTGTTGACACTGTGAGTGC
1522
+


1184020
ususa

asasu

TTT







AD-
gscsaug(Ahd)UfgUfUfUfcaucugc
813
VPusUfsugdCa(G2p)augaaaCfaUfcau
1168
ACGCATGATGTTTCATCTGC
1523
+


1184021
asasa

gcsgsu

AAT







AD-
ususcac(Chd)UfuAfUfAfauucaca
814
VPusUfscudGu(G2p)aauuauAfaGfgug
1169
ATTTCACCTTATAATTCACA
1524
+


1184022
gsasa

aasasu

GAA







AD-
ususcac(Chd)UfuAfUfAfauucaca
814
VPusUfscudGu(G2p)aauuauAfaGfgug
1169
ATTTCACCTTATAATTCACA
1524
+


1184022
gsasa

aasasu

GAA







AD-
uscsacc(Uhd)UfaUfAfAfuucacag
815
VPusUfsucdTg(Tgn)gaauuaUfaAfggu
1170
TTTCACCTTATAATTCACAG
1525
+


1184023
asasa

gasasa

AAT







AD-
ascscuu(Ahd)UfaAfUfUfcacagaa
816
VPusCfsaudTc(Tgn)gugaauUfaUfaagg
1171
TCACCTTATAATTCACAGAA
1526
+


1184024
usgsa

usgsa

TGC







AD-
cscsuua(Uhd)AfaUfUfCfacagaau
817
VPusGfscadTu(C2p)ugugaaUfuAfuaa
1172
CACCTTATAATTCACAGAAT
1527
+


1184025
gscsa

ggsusg

GCT







AD-
cscsuua(Uhd)AfaUfUfCfacagaau
817
VPusGfscadTu(C2p)ugugaaUfuAfuaa
1172
CACCTTATAATTCACAGAAT
1527
+


1184025
gscsa

ggsusg

GCT







AD-
usasuaa(Uhd)UfcAfCfAfgaaugcu
818
VPusAfscadGc(Agn)uucuguGfaAfuua
1173
CTTATAATTCACAGAATGCT
1528
+


1184026
gsusa

uasasg

GTA







AD-
asascug(Uhd)UfgAfUfUfcaucaca
819
VPusCfscudGu(G2p)augaauCfaAfcag
1174
CAAACTGTTGATTCATCACA
1529
+


1184027
gsgsa

uususg

GGG







AD-
gsasaua(Uhd)GfaCfUfAfugucaua
820
VPusAfsaudAu(G2p)acauagUfcAfuau
1175
CAGAATATGACTATGTCATA
1530
+


1184028
ususa

ucsusg

TTC







AD-
csusugu(Chd)AfuAfAfAfggucucu
821
VPusAfsuadGa(G2p)accuuuAfuGfaca
1176
AACTTGTCATAAAGGTCTCT
1531



1184029
asusa

agsusu

ATC







AD-
csuscau(Chd)UfcUfAfUfgaugggu
822
VPusAfsaadCc(C2p)aucauaGfaGfauga
1177
GACTCATCTCTATGATGGGT
1532
+


1184030
ususa

gsusc

TTT







AD-
asgsguc(Chd)UfaUfUfUfucacaaa
823
VPusAfsuudTu(G2p)ugaaaaUfaGfgac
1178
TCAGGTCCTATTTTCACAAA
1533



1184031
asusa

cusgsa

ATA







AD-
ususuac(Ahd)AfaCfAfAfuuugaua
824
VPusAfsgudAu(C2p)aaauugUfuUfgua
1179
GGTTTACAAACAATTTGATA
1534
+


1184032
csusa

aascsc

CTT







AD-
asusaua(Chd)UfcAfAfCfuguguca
825
VPusAfsuudGa(C2p)acaguuGfaGfuau
1180
AAATATACTCAACTGTGTCA
1535
+


1184033
asusa

aususu

ATA







AD-
usasgua(Chd)UfuUfCfUfuuugaac
826
VPusAfsagdTu(C2p)aaaagaAfaGfuacu
1181
AGTAGTACTTTCTTTTGAACT
1536
+


1184034
ususa

ascsu

TC







AD-
usgsgua(Ahd)CfaCfUfAfauaguaa
827
VPusUfsuudTa(C2p)uauuagUfgUfuac
1182
TGTGGTAACACTAATAGTAA
1537



1184035
asasa

cascsa

AAT







AD-
csasaua(Chd)CfaUfUfAfaaccuaua
828
VPusUfsuadTa(G2p)guuuaaUfgGfuau
1183
TCCAATACCATTAAACCTAT
1538



1184036
sasa

ugsgsa

AAG







AD-
asasugu(Uhd)CfuCfUfAfugagaac
829
VPusUfsggdTu(C2p)ucauagAfgAfaca
1184
AGAATGTTCTCTATGAGAAC
1539
+


1184037
csasa

uuscsu

CAA







AD-
ususcug(Chd)UfaAfUfCfuugcugc
830
VPusUfsagdCa(G2p)caagauUfaGfcaga
1185
GCTTCTGCTAATCTTGCTGCT
1540
+


1184038
usasa

asgsc

AC







AD-
csusgcu(Ahd)AfuCfUfUfgcugcua
831
VPusAfsgudAg(C2p)agcaagAfuUfagc
1186
TTCTGCTAATCTTGCTGCTAC
1541
+


1184039
csusa

agsasa

TA







AD-
asuscau(Ahd)CfaUfCfAfccagaug
832
VPusAfsacdAu(C2p)uggugaUfgUfaug
1187
GAATCATACATCACCAGATG
1542
+


1184040
ususa

aususc

TTG







AD-
gsuscaa(Ahd)UfuAfCfAfuuacaca
833
VPusUfsaudGu(G2p)uaauguAfaUfuug
1188
GAGTCAAATTACATTACACA
1543
+


1184041
usasa

acsusc

TAA







AD-
ascsgac(Ghd)AfcUfAfCfuagcgug
834
VPusGfsgcdAc(G2p)cuaguaGfuCfguc
1189
CGACGACGACTACTAGCGTG
1544
+


1184042
cscsa

guscsg

CCT







AD-
csgsacg(Ahd)CfuAfCfUfagcgugc
835
VPusAfsggdCa(C2p)gcuaguAfgUfcgu
1190
GACGACGACTACTAGCGTGC
1545
+


1184043
csusa

cgsusc

CTT







AD-
ascsgac(Uhd)AfcUfAfGfcgugccu
836
VPusAfsaadGg(C2p)acgcuaGfuAfguc
1191
CGACGACTACTAGCGTGCCT
1546
+


1184044
ususa

guscsg

TTG







AD-
csgsacu(Ahd)CfuAfGfCfgugccuu
837
VPusCfsaadAg(G2p)cacgcuAfgUfagu
1192
GACGACTACTAGCGTGCCTT
1547
+


1184045
usgsa

cgsusc

TGT







AD-
gsasgua(Chd)GfaAfCfUfuauguac
838
VPusGfsagdTa(C2p)auaaguUfcGfuacu
1193
ATGAGTACGAACTTATGTAC
1548
+


1184046
uscsa

csasu

TCA







AD-
usascga(Ahd)CfuUfAfUfguacuca
839
VPusAfsaudGa(G2p)uacauaAfgUfucg
1194
AGTACGAACTTATGTACTCA
1549
+


1184047
ususa

uascsu

TTC







AD-
usascuc(Ahd)UfuCfGfUfuucggaa
840
VPusUfscudTc(C2p)gaaacgAfaUfgagu
1195
TGTACTCATTCGTTTCGGAA
1550
+


1184048
gsasa

ascsa

GAG







AD-
usasguu(Ahd)AfuAfGfCfguacuuc
841
VPusAfsagdAa(G2p)uacgcuAfuUfaac
1196
AATAGTTAATAGCGTACTTC
1551
+


1184049
ususa

uasusu

TTT







AD-
ususcuu(Ghd)CfuUfUfCfgugguau
842
VPusGfsaadTa(C2p)cacgaaAfgCfaaga
1197
TTTTCTTGCTTTCGTGGTATT
1552
+


1184050
uscsa

asasa

CT







AD-
ususcgu(Ghd)GfuAfUfUfcuugcua
843
VPusAfscudAg(C2p)aagaauAfcCfacga
1198
CTTTCGTGGTATTCTTGCTAG
1553
+


1184051
gsusa

asasg

TT







AD-
usasgcc(Ahd)UfcCfUfUfacugcgc
844
VPusAfsagdCg(C2p)aguaagGfaUfggc
1199
ACTAGCCATCCTTACTGCGC
1554
+


1184052
ususa

uasgsu

TTC







AD-
asgscca(Uhd)CfcUfUfAfcugcgcu
845
VPusGfsaadGc(G2p)caguaaGfgAfugg
1200
CTAGCCATCCTTACTGCGCT
1555
+


1184053
uscsa

cusasg

TCG







AD-
gscscau(Chd)CfuUfAfCfugcgcuu
846
VPusCfsgadAg(C2p)gcaguaAfgGfaug
1201
TAGCCATCCTTACTGCGCTT
1556
+


1184054
csgsa

gcsusa

CGA







AD-
cscsauc(Chd)UfuAfCfUfgcgcuuc
847
VPusUfscgdAa(G2p)cgcaguAfaGfgau
1202
AGCCATCCTTACTGCGCTTC
1557
+


1184055
gsasa

ggscsu

GAT







AD-
ascsugc(Ghd)CfuUfCfGfauugugu
848
VPusGfscadCa(C2p)aaucgaAfgCfgcag
1203
TTACTGCGCTTCGATTGTGT
1558
+


1184056
gscsa

usasa

GCG







AD-
usgscgc(Uhd)UfcGfAfUfugugugc
849
VPusAfscgdCa(C2p)acaaucGfaAfgcgc
1204
ACTGCGCTTCGATTGTGTGC
1559
+


1184057
gsusa

asgsu

GTA







AD-
csgscuu(Chd)GfaUfUfGfugugcgu
850
VPusGfsuadCg(C2p)acacaaUfcGfaagc
1205
TGCGCTTCGATTGTGTGCGT
1560
+


1184058
ascsa

gscsa

ACT







AD-
gscsuuc(Ghd)AfuUfGfUfgugcgua
851
VPusAfsgudAc(G2p)cacacaAfuCfgaa
1206
GCGCTTCGATTGTGTGCGTA
1561
+


1184059
csusa

gcsgsc

CTG







AD-
gsusucc(Uhd)GfaUfCfUfucugguc
852
VPusUfsagdAc(C2p)agaagaUfcAfgga
1207
GAGTTCCTGATCTTCTGGTCT
1562
+


1184060
usasa

acsusc

AA







AD-
ususccu(Ghd)AfuCfUfUfcuggucu
853
VPusUfsuadGa(C2p)cagaagAfuCfagg
1208
AGTTCCTGATCTTCTGGTCTA
1563
+


1184061
asasa

aascsu

AA







AD-
cscsuga(Uhd)CfuUfCfUfggucuaa
854
VPusGfsuudTa(G2p)accagaAfgAfuca
1209
TTCCTGATCTTCTGGTCTAAA
1564
+


1184062
ascsa

ggsasa

CG







AD-
uscsuuc(Uhd)GfgUfCfUfaaacgaa
855
VPusAfsgudTc(G2p)uuuagaCfcAfgaa
1210
GATCTTCTGGTCTAAACGAA
1565
+


1184063
csusa

gasusc

CTA







AD-
csusucu(Ghd)GfuCfUfAfaacgaac
856
VPusUfsagdTu(C2p)guuuagAfcCfaga
1211
ATCTTCTGGTCTAAACGAAC
1566
+


1184064
usasa

agsasu

TAA







AD-
gsgsaac(Chd)UfaGfUfAfauagguu
857
VPusGfsaadAc(C2p)uauuacUfaGfguu
1212
ATGGAACCTAGTAATAGGTT
1567
+


1184065
uscsa

ccsasu

TCC







AD-
usgscua(Chd)AfuCfAfCfgaacgcu
858
VPusAfsaadGc(G2p)uucgugAfuGfuag
1213
GTTGCTACATCACGAACGCT
1568
+


1184066
ususa

casasc

TTC







AD-
gscsuac(Ahd)UfcAfCfGfaacgcuu
859
VPusGfsaadAg(C2p)guucguGfaUfgua
1214
TTGCTACATCACGAACGCTT
1569
+


1184067
uscsa

gcsasa

TCT







AD-
csusaca(Uhd)CfaCfGfAfacgcuuu
860
VPusAfsgadAa(G2p)cguucgUfgAfugu
1215
TGCTACATCACGAACGCTTT
1570
+


1184068
csusa

agscsa

CTT







AD-
ascsgcu(Uhd)UfcUfUfAfuuacaaa
861
VPusAfsaudTu(G2p)uaauaaGfaAfagc
1216
GAACGCTTTCTTATTACAAA
1571
+


1184069
ususa

gususc

TTG







AD-
usgsuac(Ahd)GfuAfAfGfugacaac
862
VPusCfsugdTu(G2p)ucacuuAfcUfgua
1217
CTTGTACAGTAAGTGACAAC
1572
+


1184070
asgsa

casasg

AGA







AD-
asasgug(Ahd)CfaAfCfAfgauguuu
863
VPusUfsgadAa(C2p)aucuguUfgUfcac
1218
GTAAGTGACAACAGATGTTT
1573
+


1184071
csasa

uusasc

CAT







AD-
csusacu(Chd)UfaAfUfAfuaccauu
864
VPusUfsaadAu(G2p)guauauUfaGfagu
1219
TCCTACTCTAATATACCATTT
1574



1184072
usasa

agsgsa

AG







AD-
cscscca(Ahd)GfgUfUfUfacccaaua
865
VPusUfsuadTu(G2p)gguaaaCfcUfugg
1220
GGCCCCAAGGTTTACCCAAT
1575
+


1184073
sasa

ggscsc

AAT







AD-
usasccc(Ahd)AfuAfAfUfacugcgu
866
VPusAfsgadCg(C2p)aguauuAfuUfggg
1221
TTTACCCAATAATACTGCGT
1576
+


1184074
csusa

uasasa

CTT







AD-
gsasuga(Chd)CfaAfAfUfuggcuac
867
VPusUfsagdTa(G2p)ccaauuUfgGfuca
1222
CAGATGACCAAATTGGCTAC
1577
+


1184075
usasa

ucsusg

TAC







AD-
gscsuac(Uhd)AfcCfGfAfagagcua
868
VPusGfsgudAg(C2p)ucuucgGfuAfgua
1223
TGGCTACTACCGAAGAGCTA
1578
+


1184076
cscsa

gcscsa

CCA







AD-
uscsgug(Chd)UfaCfAfAfcuuccuc
869
VPusUfsugdAg(G2p)aaguugUfaGfcac
1224
AATCGTGCTACAACTTCCTC
1579
+


1184077
asasa

gasusu

AAG







AD-
csgsugc(Uhd)AfcAfAfCfuuccuca
870
VPusCfsuudGa(G2p)gaaguuGfuAfgca
1225
ATCGTGCTACAACTTCCTCA
1580
+


1184078
asgsa

cgsasu

AGG







AD-
usgscua(Chd)AfaCfUfUfccucaag
871
VPusUfsccdTu(G2p)aggaagUfuGfuag
1226
CGTGCTACAACTTCCTCAAG
1581
+


1184079
gsasa

cascsg

GAA







AD-
usascaa(Chd)UfuCfCfUfcaaggaac
872
VPusUfsgudTc(C2p)uugaggAfaGfuug
1227
GCTACAACTTCCTCAAGGAA
1582
+


1184080
sasa

uasgsc

CAA







AD-
ascsaac(Uhd)UfcCfUfCfaaggaaca
873
VPusUfsugdTu(C2p)cuugagGfaAfguu
1228
CTACAACTTCCTCAAGGAAC
1583
+


1184081
sasa

gusasg

AAC







AD-
ascsuuc(Chd)UfcAfAfGfgaacaaca
874
VPusAfsugdTu(G2p)uuccuuGfaGfgaa
1229
CAACTTCCTCAAGGAACAAC
1584
+


1184082
susa

gususg

ATT







AD-
uscscuc(Ahd)AfgGfAfAfcaacauu
875
VPusGfscadAu(G2p)uuguucCfuUfgag
1230
CTTCCTCAAGGAACAACATT
1585
+


1184083
gscsa

gasasg

GCC







AD-
csasagg(Ahd)AfcAfAfCfauugcca
876
VPusUfsuudGg(C2p)aauguuGfuUfccu
1231
CTCAAGGAACAACATTGCCA
1586
+


1184084
asasa

ugsasg

AAA







AD-
asasgga(Ahd)CfaAfCfAfuugccaa
877
VPusUfsuudTg(G2p)caauguUfgUfucc
1232
TCAAGGAACAACATTGCCAA
1587
+


1184085
asasa

uusgsa

AAG







AD-
ascsauu(Ghd)CfcAfAfAfaggcuuc
878
VPusUfsagdAa(G2p)ccuuuuGfgCfaau
1233
CAACATTGCCAAAAGGCTTC
1588
+


1184086
usasa

gususg

TAC







AD-
gsascag(Ahd)UfuGfAfAfccagcuu
879
VPusUfscadAg(C2p)ugguucAfaUfcug
1234
TTGACAGATTGAACCAGCTT
1589
+


1184087
gsasa

ucsasa

GAG







AD-
csasaac(Uhd)GfuCfAfCfuaagaaau
880
VPusGfsaudTu(C2p)uuagugAfcAfguu
1235
GCCAAACTGTCACTAAGAAA
1590
+


1184088
scsa

ugsgsc

TCT







AD-
gsuscca(Ghd)AfaCfAfAfacccaag
881
VPusUfsccdTu(G2p)gguuugUfuCfugg
1236
TGGTCCAGAACAAACCCAAG
1591
+


1184089
gsasa

acscsa

GAA







AD-
gsgsccg(Chd)AfaAfUfUfgcacaau
882
VPusAfsaadTu(G2p)ugcaauUfuGfcgg
1237
TTGGCCGCAAATTGCACAAT
1592
+


1184090
ususa

ccsasa

TTG







AD-
gscsaug(Ghd)AfaGfUfCfacaccuu
883
VPusCfsgadAg(G2p)ugugacUfuCfcau
1238
TGGCATGGAAGTCACACCTT
1593
+


1184091
csgsa

gcscsa

CGG







AD-
usasuag(Uhd)CfuAfCfUfcuugugc
884
VPusCfsugdCa(C2p)aagaguAfgAfcua
1239
TATATAGTCTACTCTTGTGC
1594
+


1184092
asgsa

uasusa

AGA







AD-
usasguc(Uhd)AfcUfCfUfugugcag
885
VPusUfsucdTg(C2p)acaagaGfuAfgacu
1240
TATAGTCTACTCTTGTGCAG
1595
+


1184093
asasa

asusa

AAT







AD-
asgsucu(Ahd)CfuCfUfUfgugcaga
886
VPusAfsuudCu(G2p)cacaagAfgUfaga
1241
ATAGTCTACTCTTGTGCAGA
1596
+


1184094
asusa

cusasu

ATG







AD-
uscsuac(Uhd)CfuUfGfUfgcagaau
887
VPusUfscadTu(C2p)ugcacaAfgAfgua
1242
AGTCTACTCTTGTGCAGAAT
1597
+


1184095
gsasa

gascsu

GAA







AD-
csuscuu(Ghd)UfgCfAfGfaaugaau
888
VPusGfsaadTu(C2p)auucugCfaCfaaga
1243
TACTCTTGTGCAGAATGAAT
1598
+


1184096
uscsa

gsusa

TCT







AD-
usasguu(Ahd)AfcUfUfUfaaucuca
889
VPusUfsgudGa(G2p)auuaaaGfuUfaac
1244
TGTAGTTAACTTTAATCTCA
1599
+


1184097
csasa

uascsa

CAT







AD-
usasacu(Uhd)UfaAfUfCfucacaua
890
VPusGfscudAu(G2p)ugagauUfaAfagu
1245
GTTAACTTTAATCTCACATA
1600
+


1184098
gscsa

uasasc

GCA







AD-
ususuaa(Uhd)CfuCfAfCfauagcaa
891
VPusGfsaudTg(C2p)uaugugAfgAfuua
1246
ACTTTAATCTCACATAGCAA
1601
+


1184099
uscsa

aasgsu

TCT







AD-
ususaau(Chd)UfcAfCfAfuagcaau
892
VPusAfsgadTu(G2p)cuauguGfaGfauu
1247
CTTTAATCTCACATAGCAAT
1602
+


1184100
csusa

aasasg

CTT







AD-
gsgsgag(Ghd)AfcUfUfGfaaagagc
893
VPusUfsggdCu(C2p)uuucaaGfuCfcuc
1248
TAGGGAGGACTTGAAAGAG
1603
+


1184101
csasa

ccsusa

CCAC







AD-
gsusaga(Uhd)CfuGfUfUfcucuaaa
894
VPusCfsguuUfagagaacAfgAfucuacsas
1249
TTGTAGATCTGTTCTCTAAA
1604
+


1184102
csgsa

a

CGA







AD-
asgsauc(Uhd)GfuUfCfUfcuaaacg
895
VPusUfsucgUfuuagagaAfcAfgaucusas
1250
GTAGATCTGTTCTCTAAACG
1605
+


1184103
asasa

c

AAC







AD-
ususcuc(Uhd)AfaAfCfGfaacuuua
896
VPusUfsuuaAfaguucguUfuAfgagaascs
1251
TGTTCTCTAAACGAACTTTA
1606
+


1184104
asasa

a

AAA







AD-
uscsucu(Ahd)AfaCfGfAfacuuuaa
897
VPusUfsuuuAfaaguucgUfuUfagagasas
1252
GTTCTCTAAACGAACTTTAA
1607
+


1184105
asasa

c

AAT







AD-
csusaaa(Chd)GfaAfCfUfuuaaaauc
898
VPusAfsgauUfuuaaaguUfcGfuuuagsas
1253
CTCTAAACGAACTTTAAAAT
1608
+


1184106
susa

g

CTG







AD-
usasaac(Ghd)AfaCfUfUfuaaaauc
899
VPusCfsagaUfuuuaaagUfuCfguuuasgs
1254
TCTAAACGAACTTTAAAATC
1609
+


1184107
usgsa

a

TGT







AD-
asasacg(Ahd)AfcUfUfUfaaaaucu
900
VPusAfscagAfuuuuaaaGfuUfcguuusas
1255
CTAAACGAACTTTAAAATCT
1610
+


1184108
gsusa

g

GTG







AD-
ascsgaa(Chd)UfuUfAfAfaaucugu
901
VPusAfscacAfgauuuuaAfaGfuucgusus
1256
AAACGAACTTTAAAATCTGT
1611
+


1184109
gsusa

u

GTG







AD-
gscsugc(Uhd)UfaCfGfGfuuucguc
902
VPusCfsggaCfgaaaccgUfaAfgcagcscs
1257
AGGCTGCTTACGGTTTCGTC
1612
+


1184110
csgsa

u

CGT







AD-
usgscuu(Ahd)CfgGfUfUfucguccg
903
VPusCfsacgGfacgaaacCfgUfaagcasgsc
1258
GCTGCTTACGGTTTCGTCCG
1613
+


1184111
usgsa



TGT







AD-
gscsuua(Chd)GfgUfUfUfcguccgu
904
VPusAfscacGfgacgaaaCfcGfuaagcsas
1259
CTGCTTACGGTTTCGTCCGT
1614
+


1184112
gsusa

g

GTT







AD-
csusuac(Ghd)GfuUfUfCfguccgug
905
VPusAfsacaCfggacgaaAfcCfguaagscs
1260
TGCTTACGGTTTCGTCCGTGT
1615
+


1184113
ususa

a

TG







AD-
ususacg(Ghd)UfuUfCfGfuccgugu
906
VPusCfsaacAfcggacgaAfaCfcguaasgs
1261
GCTTACGGTTTCGTCCGTGTT
1616
+


1184114
usgsa

c

GC







AD-
usgsuga(Chd)CfgAfAfAfgguaaga
907
VPusCfsaucUfuaccuuuCfgGfucacascs
1262
GGTGTGACCGAAAGGTAAG
1617
+


1184115
usgsa

c

ATGG







AD-
gsasccg(Ahd)AfaGfGfUfaagaugg
908
VPusCfsuccAfucuuaccUfuUfcggucsas
1263
GTGACCGAAAGGTAAGATG
1618
+


1184116
asgsa

c

GAGA







AD-
asasaac(Ahd)CfaCfGfUfccaacuca
909
VPusCfsugaGfuuggacgUfgUfguuuusc
1264
AGAAAACACACGTCCAACTC
1619
+


1184117
sgsa

su

AGT







AD-
csascac(Ghd)UfcCfAfAfcucaguu
910
VPusCfsaaaCfugaguugGfaCfgugugsus
1265
AACACACGTCCAACTCAGTT
1620
+


1184118
usgsa

u

TGC







AD-
ascsacg(Uhd)CfcAfAfCfucaguuu
911
VPusGfscaaAfcugaguuGfgAfcgugusgs
1266
ACACACGTCCAACTCAGTTT
1621
+


1184119
gscsa

u

GCC







AD-
csascgu(Chd)CfaAfCfUfcaguuug
912
VPusGfsgcaAfacugaguUfgGfacgugsus
1267
CACACGTCCAACTCAGTTTG
1622
+


1184120
cscsa

g

CCT







AD-
ascsguc(Chd)AfaCfUfCfaguuugc
913
VPusAfsggcAfaacugagUfuGfgacgusgs
1268
ACACGTCCAACTCAGTTTGC
1623
+


1184121
csusa

u

CTG







AD-
csgsucc(Ahd)AfcUfCfAfguuugcc
914
VPusCfsaggCfaaacugaGfuUfggacgsus
1269
CACGTCCAACTCAGTTTGCC
1624
+


1184122
usgsa

g

TGT







AD-
gsusucu(Uhd)CfuUfCfGfuaagaac
915
VPusCfscguUfcuuacgaAfgAfagaacscs
1270
AGGTTCTTCTTCGTAAGAAC
1625
+


1184123
gsgsa

u

GGT







AD-
ususcuu(Chd)UfuCfGfUfaagaacg
916
VPusAfsccgUfucuuacgAfaGfaagaascs
1271
GGTTCTTCTTCGTAAGAACG
1626
+


1184124
gsusa

c

GTA







AD-
uscsuuc(Uhd)UfcGfUfAfagaacgg
917
VPusUfsaccGfuucuuacGfaAfgaagasas
1272
GTTCTTCTTCGTAAGAACGG
1627
+


1184125
usasa

c

TAA







AD-
asuscuu(Chd)UfuCfUfUfgcucuuc
918
VPusAfsagaAfgagcaagAfaGfaagausus
1273
CAATCTTCTTCTTGCTCTTCT
1628



1184126
ususa

g

TC







AD-
gsgsuua(Uhd)UfuAfAfAfacuuacu
919
VPusUfscagUfaaguuuuAfaAfuaaccsas
1274
GTGGTTATTTAAAACTTACT
1629
+


1184127
gsasa

c

GAC







AD-
uscsuac(Ahd)CfaAfAfCfucuuaaa
920
VPusUfscuuUfaagaguuUfgUfguagasus
1275
TATCTACACAAACTCTTAAA
1630



1184128
gsasa

a

GAA







AD-
asasgaa(Ghd)UfaGfUfGfgaaaauc
921
VPusAfsggaUfuuuccacUfaCfuucuuscs
1276
TGAAGAAGTAGTGGAAAAT
1631
+


1184129
csusa

a

CCTA







AD-
ascsaau(Uhd)GfuGfUfAfcuuuuac
922
VPusUfsaguAfaaaguacAfcAfauugusas
1277
CTACAATTGTGTACTTTTACT
1632
+


1184130
usasa

g

AG







AD-
ascsacu(Chd)UfuAfAfCfaguauuc
923
VPusAfsagaAfuacuguuAfaGfaguguscs
1278
CGACACTCTTAACAGTATTC
1633



1184131
ususa

g

TTT







AD-
asusgua(Chd)AfaCfUfAfuuguuaa
924
VPusCfsauuAfacaauagUfuGfuacausus
1279
GAATGTACAACTATTGTTAA
1634
+


1184132
usgsa

c

TGG







AD-
usgsuac(Ahd)AfcUfAfUfuguuaau
925
VPusCfscauUfaacaauaGfuUfguacasus
1280
AATGTACAACTATTGTTAAT
1635
+


1184133
gsgsa

u

GGT







AD-
cscsagg(Ahd)GfuUfUfUfcuguggu
926
VPusAfscacCfacagaaaAfcUfccuggsus
1281
TACCAGGAGTTTTCTGTGGT
1636
+


1184134
gsusa

a

GTA







AD-
uscsaug(Uhd)GfgUfAfGfuguugg
927
VPusAfsaacCfaacacuaCfcAfcaugasasc
1282
GTTCATGTGGTAGTGTTGGT
1637
+


1184135
uususa



TTT







AD-
csascug(Uhd)AfcAfGfUfcuaaaau
928
VPusAfscauUfuuagacuGfuAfcagugsgs
1283
GCCACTGTACAGTCTAAAAT
1638
+


1184136
gsusa

c

GTC







AD-
ascsugu(Ahd)CfaGfUfCfuaaaaug
11
VPusGfsacaUfuuuagacUfgUfacagusgs
12
CCACTGTACAGTCTAAAATG
1639
+


1184137
uscsa

g

TCA







AD-
ususuga(Ahd)UfgUfGfGfcuaaauc
929
VPusCfsagaUfuuagccaCfaUfucaaasgs
1284
TCTTTGAATGTGGCTAAATC
1640
+


1184138
usgsa

a

TGA







AD-
ascsaac(Chd)UfaAfAfUfagaggua
930
VPusCfsauaCfcucuauuUfaGfguugusus
1285
AAACAACCTAAATAGAGGT
1641
+


1184139
usgsa

u

ATGG







AD-
asasccu(Ahd)AfaUfAfGfagguaug
931
VPusAfsccaUfaccucuaUfuUfagguusgs
1286
ACAACCTAAATAGAGGTATG
1642
+


1184140
gsusa

u

GTA







AD-
uscsauc(Chd)AfaAfUfCfcuaaagg
932
VPusAfsuccUfuuaggauUfuGfgaugasus
1287
GATCATCCAAATCCTAAAGG
1643
+


1184141
asusa

c

ATT







AD-
csascag(Uhd)CfuGfUfAfccgucug
933
VPusCfsgcaGfacgguacAfgAfcugugsus
1288
AACACAGTCTGTACCGTCTG
1644
+


1184142
csgsa

u

CGG







AD-
ascsggg(Uhd)UfuGfCfGfguguaag
934
VPusCfsacuUfacaccgcAfaAfcccgusus
1289
AAACGGGTTTGCGGTGTAAG
1645
+


1184143
usgsa

u

TGC







AD-
asgsgca(Chd)UfaGfUfAfcugaugu
935
VPusCfsgacAfucaguacUfaGfugccusgs
1290
ACAGGCACTAGTACTGATGT
1646
+


1184144
csgsa

u

CGT







AD-
ususcuu(Ahd)CfuUfUfGfuaguuaa
936
VPusUfscuuAfacuacaaAfgUfaagaasus
1291
GATTCTTACTTTGTAGTTAA
1647
+


1184145
gsasa

c

GAG







AD-
csuscua(Ahd)CfuAfCfCfaacaugaa
937
VPusCfsuucAfuguugguAfgUfuagagsa
1292
TTCTCTAACTACCAACATGA
1648
+


1184146
sgsa

sa

AGA







AD-
ususgug(Ahd)UfgAfUfGfauuauu
938
VPusUfsgaaAfuaaucauCfaUfcacaascsa
1293
TGTTGTGATGATGATTATTTC
1649
+


1184147
ucsasa



AA







AD-
usgsuga(Uhd)GfaUfGfAfuuauuuc
939
VPusUfsugaAfauaaucaUfcAfucacasas
1294
GTTGTGATGATGATTATTTC
1650
+


1184148
asasa

c

AAT







AD-
asuscag(Ghd)AfuGfUfAfaacuuac
940
VPusAfsuguAfaguuuacAfuCfcugausus
1295
TAATCAGGATGTAAACTTAC
1651
+


1184149
asusa

a

ATA







AD-
usasaca(Ahd)UfgUfUfGfcuuuuca
9
VPusUfsuugAfaaagcaaCfaUfuguuasgs
10
ACTAACAATGTTGCTTTTCA
1652
+


1184150
asasa

u

AAC







AD-
ususuuc(Ahd)AfaCfUfGfucaaacc
941
VPusCfsgggUfuugacagUfuUfgaaaasgs
1296
GCTTTTCAAACTGTCAAACC
1653
+


1184151
csgsa

c

CGG







AD-
ascsugu(Chd)AfaAfCfCfcgguaau
942
VPusAfsaauUfaccggguUfuGfacagusus
1297
AAACTGTCAAACCCGGTAAT
1654
+


1184152
ususa

u

TTT







AD-
csusuua(Ahd)GfgAfAfGfgaaguuc
943
VPusCfsagaAfcuuccuuCfcUfuaaagsas
1298
TTCTTTAAGGAAGGAAGTTC
1655
+


1184153
usgsa

a

TGT







AD-
ususuaa(Ghd)GfaAfGfGfaaguucu
944
VPusAfscagAfacuuccuUfcCfuuaaasgs
1299
TCTTTAAGGAAGGAAGTTCT
1656
+


1184154
gsusa

a

GTT







AD-
ususaag(Ghd)AfaGfGfAfaguucug
945
VPusAfsacaGfaacuuccUfuCfcuuaasas
1300
CTTTAAGGAAGGAAGTTCTG
1657
+


1184155
ususa

g

TTG







AD-
usasagg(Ahd)AfgGfAfAfguucug
946
VPusCfsaacAfgaacuucCfuUfccuuasas
1301
TTTAAGGAAGGAAGTTCTGT
1658
+


1184156
uusgsa

a

TGA







AD-
asasaac(Ahd)CfuUfCfUfucuuugc
947
VPusGfsagcAfaagaagaAfgUfguuuusas
1302
TTAAAACACTTCTTCTTTGCT
1659
+


1184157
uscsa

a

CA







AD-
gsgsuaa(Ghd)GfcUfAfGfacuuuau
948
VPusUfsaauAfaagucuaGfcCfuuaccscs
1303
GGGGTAAGGCTAGACTTTAT
1660
+


1184158
usasa

c

TAT







AD-
gsusaag(Ghd)CfuAfGfAfcuuuauu
949
VPusAfsuaaUfaaagucuAfgCfcuuacscs
1304
GGGTAAGGCTAGACTTTATT
1661
+


1184159
asusa

c

ATG







AD-
gsasuca(Ahd)GfaUfGfCfacuuuuc
950
VPusGfscgaAfaagugcaUfcUfugaucscs
1305
AGGATCAAGATGCACTTTTC
1662
+


1184160
gscsa

u

GCA







AD-
usasgcu(Ghd)GfuGfUfCfucuaucu
951
VPusAfscagAfuagagacAfcCfagcuascs
1306
CGTAGCTGGTGTCTCTATCT
1663
+


1184161
gsusa

g

GTA







AD-
gsgsugu(Chd)UfcUfAfUfcuguagu
952
VPusGfsuacUfacagauaGfaGfacaccsas
1307
CTGGTGTCTCTATCTGTAGT
1664
+


1184162
ascsa

g

ACT







AD-
uscsucu(Ahd)UfcUfGfUfaguacua
953
VPusCfsauaGfuacuacaGfaUfagagascs
1308
TGTCTCTATCTGTAGTACTAT
1665
+


1184163
usgsa

a

GA







AD-
ususguu(Chd)UfuGfCfUfcgcaaac
954
VPusAfsuguUfugcgagcAfaGfaacaasgs
1309
ACTTGTTCTTGCTCGCAAAC
1666
+


1184164
asusa

u

ATA







AD-
ususcac(Uhd)AfuAfUfGfuuaaacc
955
VPusCfsuggUfuuaacauAfuAfgugaascs
1310
GGTTCACTATATGTTAAACC
1667
+


1184165
asgsa

c

AGG







AD-
ascsaac(Uhd)GfcUfUfAfugcuaau
956
VPusCfsuauUfagcauaaGfcAfguugusgs
1311
CCACAACTGCTTATGCTAAT
1668
+


1184166
asgsa

g

AGT







AD-
csasacu(Ghd)CfuUfAfUfgcuaaua
957
VPusAfscuaUfuagcauaAfgCfaguugsus
1312
CACAACTGCTTATGCTAATA
1669
+


1184167
gsusa

g

GTG







AD-
asascug(Chd)UfuAfUfGfcuaauag
958
VPusCfsacuAfuuagcauAfaGfcaguusgs
1313
ACAACTGCTTATGCTAATAG
1670
+


1184168
usgsa

u

TGT







AD-
ascsugc(Uhd)UfaUfGfCfuaauagu
959
VPusAfscacUfauuagcaUfaAfgcagusus
1314
CAACTGCTTATGCTAATAGT
1671
+


1184169
gsusa

g

GTT







AD-
csascuc(Ahd)UfaAfAfGfucugugu
960
VPusCfsaacAfcagacuuUfaUfgagugsus
1315
GACACTCATAAAGTCTGTGT
1672



1184170
usgsa

c

TGT







AD-
gsasgug(Uhd)CfuCfUfAfuagaaau
961
VPusCfsuauUfucuauagAfgAfcacucsas
1316
ATGAGTGTCTCTATAGAAAT
1673
+


1184171
asgsa

u

AGA







AD-
ascsuga(Ghd)AfcUfGfAfccuuacu
962
VPusUfsuagUfaaggucaGfuCfucaguscs
1317
GGACTGAGACTGACCTTACT
1674
+


1184172
asasa

c

AAA







AD-
csasuac(Ahd)AfuGfCfUfaguuaaa
963
VPusUfsguuUfaacuagcAfuUfguaugsus
1318
AACATACAATGCTAGTTAAA
1675
+


1184173
csasa

u

CAG







AD-
ascsaga(Uhd)GfgUfAfCfacuuaug
964
VPusAfsucaUfaaguguaCfcAfucugusus
1319
AAACAGATGGTACACTTATG
1676
+


1184174
asusa

u

ATT







AD-
csasgau(Ghd)GfuAfCfAfcuuauga
965
VPusAfsaucAfuaaguguAfcCfaucugsus
1320
AACAGATGGTACACTTATGA
1677
+


1184175
ususa

u

TTG







AD-
asgsgag(Ahd)GfuAfCfAfccuuuga
966
VPusUfsuucAfaagguguAfcUfcuccusas
1321
ATAGGAGAGTACACCTTTGA
1678
+


1184176
asasa

u

AAA







AD-
usasucu(Chd)AfgAfUfGfaguuuuc
967
VPusUfsagaAfaacucauCfuGfagauasus
1322
AATATCTCAGATGAGTTTTC
1679
+


1184177
usasa

u

TAG







AD-
asuscuc(Ahd)GfaUfGfAfguuuucu
968
VPusCfsuagAfaaacucaUfcUfgagausas
1323
ATATCTCAGATGAGTTTTCT
1680
+


1184178
asgsa

u

AGC







AD-
asusguu(Ghd)CfaAfAfUfuaucaaa
969
VPusCfsuuuUfgauaauuUfgCfaacausus
1324
CAATGTTGCAAATTATCAAA
1681
+


1184179
asgsa

g

AGG







AD-
gsusugc(Ahd)AfaUfUfAfucaaaag
970
VPusAfsccuUfuugauaaUfuUfgcaacsas
1325
ATGTTGCAAATTATCAAAAG
1682
+


1184180
gsusa

u

GTT







AD-
gsusacu(Ghd)GfuAfAfGfagucauu
971
VPusAfsaaaUfgacucuuAfcCfaguacscs
1326
TGGTACTGGTAAGAGTCATT
1683
+


1184181
ususa

a

TTG







AD-
usascug(Ghd)UfaAfGfAfgucauuu
972
VPusCfsaaaAfugacucuUfaCfcaguascs
1327
GGTACTGGTAAGAGTCATTT
1684
+


1184182
usgsa

c

TGC







AD-
gsusaga(Ghd)UfgUfUfUfugauaaa
973
VPusAfsauuUfaucaaaaCfaCfucuacsasc
1328
GTGTAGAGTGTTTTGATAAA
1685
+


1184183
ususa



TTC







AD-
asgsagu(Ghd)UfuUfUfGfauaaauu
974
VPusUfsgaaUfuuaucaaAfaCfacucusas
1329
GTAGAGTGTTTTGATAAATT
1686
+


1184184
csasa

c

CAA







AD-
gsusguu(Uhd)UfgAfUfAfaauucaa
975
VPusCfsuuuGfaauuuauCfaAfaacacsus
1330
GAGTGTTTTGATAAATTCAA
1687
+


1184185
asgsa

c

AGT







AD-
gsusuuu(Ghd)AfuAfAfAfuucaaag
976
VPusCfsacuUfugaauuuAfuCfaaaacsas
1331
GTGTTTTGATAAATTCAAAG
1688
+


1184186
usgsa

c

TGA







AD-
csascac(Uhd)AfgAfAfCfcagaaua
977
VPusAfsauaUfucugguuCfuAfgugugsc
1332
GGCACACTAGAACCAGAAT
1689
+


1184187
ususa

sc

ATTT







AD-
ascsacu(Ahd)GfaAfCfCfagaauau
978
VPusAfsaauAfuucugguUfcUfagugusg
1333
GCACACTAGAACCAGAATAT
1690
+


1184188
ususa

Sc

TTC







AD-
gsasaau(Uhd)GfuUfGfAfcacugug
979
VPusCfsucaCfagugucaAfcAfauuucsas
1334
CTGAAATTGTTGACACTGTG
1691
+


1184189
asgsa

g

AGT







AD-
cscscug(Chd)UfuGfGfAfgaaaagc
980
VPusCfsagcUfuuucuccAfaGfcagggsus
1335
AACCCTGCTTGGAGAAAAGC
1692
+


1184190
usgsa

u

TGT







AD-
csusuau(Ahd)AfuUfCfAfcagaaug
981
VPusAfsgcaUfucugugaAfuUfauaagsgs
1336
ACCTTATAATTCACAGAATG
1693
+


1184191
csusa

u

CTG







AD-
csusuau(Ahd)AfuUfCfAfcagaaug
981
VPusAfsgcaUfucugugaAfuUfauaagsgs
1336
ACCTTATAATTCACAGAATG
1693
+


1184191
csusa

u

CTG







AD-
ususaua(Ahd)UfuCfAfCfagaaugc
982
VPusCfsagcAfuucugugAfaUfuauaasgs
1337
CCTTATAATTCACAGAATGC
1694
+


1184192
usgsa

g

TGT







AD-
asasuau(Ghd)AfcUfAfUfgucauau
983
VPusGfsaauAfugacauaGfuCfauauuscs
1338
AGAATATGACTATGTCATAT
1695
+


1184193
uscsa

u

TCA







AD-
asusaug(Ahd)CfuAfUfGfucauauu
984
VPusUfsgaaUfaugacauAfgUfcauausus
1339
GAATATGACTATGTCATATT
1696
+


1184194
csasa

c

CAC







AD-
usasuga(Chd)UfaUfGfUfcauauuc
985
VPusGfsugaAfuaugacaUfaGfucauasus
1340
AATATGACTATGTCATATTC
1697
+


1184195
ascsa

u

ACT







AD-
asasgac(Uhd)CfaUfCfUfcuaugau
986
VPusCfscauCfauagagaUfgAfgucuuscs
1341
AGAAGACTCATCTCTATGAT
1698
+


1184196
gsgsa

u

GGG







AD-
asgsacu(Chd)AfuCfUfCfuaugaug
987
VPusCfsccaUfcauagagAfuGfagucusus
1342
GAAGACTCATCTCTATGATG
1699
+


1184197
gsgsa

c

GGT







AD-
uscsaga(Ghd)UfuUfAfGfaaaaugu
988
VPusCfscacAfuuuucuaAfaCfucugasas
1343
CTTCAGAGTTTAGAAAATGT
1700
+


1184198
gsgsa

g

GGC







AD-
ascscau(Chd)AfaCfUfUfucuuaua
989
VPusAfsuuaUfaagaaagUfuGfauggusgs
1344
ACACCATCAACTTTCTTATA
1701



1184199
asusa

u

ATA







AD-
csuscca(Uhd)AfaAfCfGfauauguu
990
VPusCfsgaaCfauaucguUfuAfuggagsas
1345
ATCTCCATAAACGATATGTT
1702



1184200
csgsa

u

CGA







AD-
csasuag(Ghd)AfaUfAfAfaaucuuc
991
VPusUfsagaAfgauuuuaUfuCfcuaugsgs
1346
TCCATAGGAATAAAATCTTC
1703



1184201
usasa

a

TAA







AD-
cscsuaa(Uhd)AfuUfAfCfaaacuug
992
VPusCfsacaAfguuuguaAfuAfuuaggsas
1347
TTCCTAATATTACAAACTTG
1704
+


1184202
usgsa

a

TGC







AD-
gscsuug(Uhd)UfaAfAfCfaacuuag
993
VPusAfsgcuAfaguuguuUfaAfcaagcsgs
1348
ACGCTTGTTAAACAACTTAG
1705
+


1184203
csusa

u

CTC







AD-
csusugu(Uhd)AfaAfCfAfacuuagc
994
VPusGfsagcUfaaguuguUfuAfacaagscs
1349
CGCTTGTTAAACAACTTAGC
1706
+


1184204
uscsa

g

TCC







AD-
csusugc(Uhd)GfcUfAfCfuaaaaug
995
VPusGfsacaUfuuuaguaGfcAfgcaagsas
1350
ATCTTGCTGCTACTAAAATG
1707
+


1184205
uscsa

u

TCA







AD-
ascsuua(Uhd)GfgAfUfUfuguuuau
996
VPusUfscauAfaacaaauCfcAfuaagusus
1351
GAACTTATGGATTTGTTTAT
1708
+


1184206
gsasa

c

GAG







AD-
csgsacg(Ahd)CfgAfCfUfacuagcg
997
VPusCfsacgCfuaguaguCfgUfcgucgsgs
1352
ACCGACGACGACTACTAGCG
1709
+


1184207
usgsa

u

TGC

+





AD-
gsascga(Chd)GfaCfUfAfcuagcgu
998
VPusGfscacGfcuaguagUfcGfucgucsgs
1353
CCGACGACGACTACTAGCGT
1710



1184208
gscsa

g

GCC







AD-
asusgag(Uhd)AfcGfAfAfcuuaugu
999
VPusGfsuacAfuaaguucGfuAfcucauscs
1354
TGATGAGTACGAACTTATGT
1711
+


1184209
ascsa

a

ACT







AD-
csgsaac(Uhd)UfaUfGfUfacucauu
1000
VPusCfsgaaUfgaguacaUfaAfguucgsus
1355
TACGAACTTATGTACTCATT
1712
+


1184210
csgsa

a

CGT







AD-
gsasacu(Uhd)AfuGfUfAfcucauuc
1001
VPusAfscgaAfugaguacAfuAfaguucsgs
1356
ACGAACTTATGTACTCATTC
1713
+


1184211
gsusa

u

GTT







AD-
asascuu(Ahd)UfgUfAfCfucauucg
1002
VPusAfsacgAfaugaguaCfaUfaaguuscs
1357
CGAACTTATGTACTCATTCG
1714
+


1184212
ususa

g

TTT







AD-
ascsuua(Uhd)GfuAfCfUfcauucgu
1003
VPusAfsaacGfaaugaguAfcAfuaagusus
1358
GAACTTATGTACTCATTCGT
1715
+


1184213
ususa

c

TTC







AD-
csusuau(Ghd)UfaCfUfCfauucguu
1004
VPusGfsaaaCfgaaugagUfaCfauaagsus
1359
AACTTATGTACTCATTCGTTT
1716
+


1184214
uscsa

u

CG







AD-
ususaug(Uhd)AfcUfCfAfuucguuu
1005
VPusCfsgaaAfcgaaugaGfuAfcauaasgs
1360
ACTTATGTACTCATTCGTTTC
1717
+


1184215
csgsa

u

GG







AD-
usasugu(Ahd)CfuCfAfUfucguuuc
1006
VPusCfscgaAfacgaaugAfgUfacauasas
1361
CTTATGTACTCATTCGTTTCG
1718
+


1184216
gsgsa

g

GA







AD-
asusgua(Chd)UfcAfUfUfcguuucg
1007
VPusUfsccgAfaacgaauGfaGfuacausas
1362
TTATGTACTCATTCGTTTCGG
1719
+


1184217
gsasa

a

AA







AD-
usgsuac(Uhd)CfaUfUfCfguuucgg
1008
VPusUfsuccGfaaacgaaUfgAfguacasus
1363
TATGTACTCATTCGTTTCGG
1720
+


1184218
asasa

a

AAG







AD-
gsusacu(Chd)AfuUfCfGfuuucgga
1009
VPusCfsuucCfgaaacgaAfuGfaguacsas
1364
ATGTACTCATTCGTTTCGGA
1721
+


1184219
asgsa

u

AGA







AD-
csasggu(Ahd)CfgUfUfAfauaguua
1010
VPusAfsuuaAfcuauuaaCfgUfaccugsus
1365
GACAGGTACGTTAATAGTTA
1722
+


1184220
asusa

c

ATA







AD-
asgsgua(Chd)GfuUfAfAfuaguuaa
1011
VPusUfsauuAfacuauuaAfcGfuaccusgs
1366
ACAGGTACGTTAATAGTTAA
1723
+


1184221
usasa

u

TAG







AD-
usasaua(Ghd)UfuAfAfUfagcguac
1012
VPusAfsaguAfcgcuauuAfaCfuauuasas
1367
GTTAATAGTTAATAGCGTAC
1724
+


1184222
ususa

c

TTC







AD-
uscsuug(Chd)UfuUfCfGfugguauu
1013
VPusAfsgaaUfaccacgaAfaGfcaagasasa
1368
TTTCTTGCTTTCGTGGTATTC
1725
+


1184223
csusa



TT







AD-
csusugc(Uhd)UfuCfGfUfgguauuc
1014
VPusAfsagaAfuaccacgAfaAfgcaagsas
1369
TTCTTGCTTTCGTGGTATTCT
1726
+


1184224
ususa

a

TG







AD-
ususgcu(Uhd)UfcGfUfGfguauucu
1015
VPusCfsaagAfauaccacGfaAfagcaasgsa
1370
TCTTGCTTTCGTGGTATTCTT
1727
+


1184225
usgsa



GC







AD-
usgscuu(Uhd)CfgUfGfGfuauucuu
1016
VPusGfscaaGfaauaccaCfgAfaagcasasg
1371
CTTGCTTTCGTGGTATTCTTG
1728
+


1184226
gscsa



CT







AD-
gscsuuu(Chd)GfuGfGfUfauucuug
1017
VPusAfsgcaAfgaauaccAfcGfaaagcsas
1372
TTGCTTTCGTGGTATTCTTGC
1729
+


1184227
csusa

a

TA







AD-
ususucg(Uhd)GfgUfAfUfucuugc
1018
VPusCfsuagCfaagaauaCfcAfcgaaasgsc
1373
GCTTTCGTGGTATTCTTGCTA
1730
+


1184228
uasgsa



GT







AD-
csusgcg(Chd)UfuCfGfAfuugugug
1019
VPusCfsgcaCfacaaucgAfaGfcgcagsus
1374
TACTGCGCTTCGATTGTGTG
1731
+


1184229
csgsa

a

CGT







AD-
gsusacu(Ghd)CfuGfCfAfauauugu
1020
VPusUfsaacAfauauugcAfgCfaguacsgs
1375
GCGTACTGCTGCAATATTGT
1732
+


1184230
usasa

c

TAA







AD-
csusgcu(Ghd)CfaAfUfAfuuguuaa
1021
VPusCfsguuAfacaauauUfgCfagcagsus
1376
TACTGCTGCAATATTGTTAA
1733
+


1184231
csgsa

a

CGT







AD-
usgscug(Chd)AfaUfAfUfuguuaac
1022
VPusAfscguUfaacaauaUfuGfcagcasgs
1377
ACTGCTGCAATATTGTTAAC
1734
+


1184232
gsusa

u

GTG







AD-
gscsugc(Ahd)AfuAfUfUfguuaacg
1023
VPusCfsacgUfuaacaauAfuUfgcagcsas
1378
CTGCTGCAATATTGTTAACG
1735
+


1184233
usgsa

g

TGA







AD-
csusgca(Ahd)UfaUfUfGfuuaacgu
1024
VPusUfscacGfuuaacaaUfaUfugcagscs
1379
TGCTGCAATATTGTTAACGT
1736
+


1184234
gsasa

a

GAG







AD-
asgsagu(Uhd)CfcUfGfAfucuucug
1025
VPusAfsccaGfaagaucaGfgAfacucusas
1380
CTAGAGTTCCTGATCTTCTG
1737
+


1184235
gsusa

g

GTC







AD-
csusgau(Chd)UfuCfUfGfgucuaaa
1026
VPusCfsguuUfagaccagAfaGfaucagsgs
1381
TCCTGATCTTCTGGTCTAAA
1738
+


1184236
csgsa

a

CGA







AD-
gsasucu(Uhd)CfuGfGfUfcuaaacg
1027
VPusUfsucgUfuuagaccAfgAfagaucsas
1382
CTGATCTTCTGGTCTAAACG
1739
+


1184237
asasa

g

AAC







AD-
ascsaau(Ghd)GfaAfCfCfuaguaau
1028
VPusCfsuauUfacuagguUfcCfauugusus
1383
GAACAATGGAACCTAGTAAT
1740
+


1184238
asgsa

c

AGG







AD-
csasaug(Ghd)AfaCfCfUfaguaaua
1029
VPusCfscuaUfuacuaggUfuCfcauugsus
1384
AACAATGGAACCTAGTAATA
1741
+


1184239
gsgsa

u

GGT







AD-
asasugg(Ahd)AfcCfUfAfguaauag
1030
VPusAfsccuAfuuacuagGfuUfccauusgs
1385
ACAATGGAACCTAGTAATAG
1742
+


1184240
gsusa

u

GTT







AD-
asusgga(Ahd)CfcUfAfGfuaauagg
1031
VPusAfsaccUfauuacuaGfgUfuccausus
1386
CAATGGAACCTAGTAATAGG
1743
+


1184241
ususa

g

TTT







AD-
asasccu(Ahd)GfuAfAfUfagguuuc
1032
VPusAfsggaAfaccuauuAfcUfagguuscs
1387
GGAACCTAGTAATAGGTTTC
1744
+


1184242
csusa

c

CTA







AD-
ascscua(Ghd)UfaAfUfAfgguuucc
1033
VPusUfsaggAfaaccuauUfaCfuaggusus
1388
GAACCTAGTAATAGGTTTCC
1745
+


1184243
usasa

c

TAT







AD-
usascau(Chd)AfcGfAfAfcgcuuuc
1034
VPusAfsagaAfagcguucGfuGfauguasgs
1389
GCTACATCACGAACGCTTTC
1746
+


1184244
ususa

c

TTA







AD-
ascsauc(Ahd)CfgAfAfCfgcuuucu
1035
VPusUfsaagAfaagcguuCfgUfgaugusas
1390
CTACATCACGAACGCTTTCT
1747
+


1184245
usasa

g

TAT







AD-
csasuca(Chd)GfaAfCfGfcuuucuu
1036
VPusAfsuaaGfaaagcguUfcGfugaugsus
1391
TACATCACGAACGCTTTCTT
1748
+


1184246
asusa

a

ATT







AD-
asuscac(Ghd)AfaCfGfCfuuucuua
1037
VPusAfsauaAfgaaagcgUfuCfgugausgs
1392
ACATCACGAACGCTTTCTTA
1749
+


1184247
ususa

u

TTA







AD-
uscsacg(Ahd)AfcGfCfUfuucuuau
1038
VPusUfsaauAfagaaagcGfuUfcgugasus
1393
CATCACGAACGCTTTCTTAT
1750
+


1184248
usasa

g

TAC







AD-
csascga(Ahd)CfgCfUfUfucuuauu
1039
VPusGfsuaaUfaagaaagCfgUfucgugsas
1394
ATCACGAACGCTTTCTTATT
1751
+


1184249
ascsa

u

ACA







AD-
ascsgaa(Chd)GfcUfUfUfcuuauua
1040
VPusUfsguaAfuaagaaaGfcGfuucgusgs
1395
TCACGAACGCTTTCTTATTA
1752
+


1184250
csasa

a

CAA







AD-
csgsaac(Ghd)CfuUfUfCfuuauuac
1041
VPusUfsuguAfauaagaaAfgCfguucgsus
1396
CACGAACGCTTTCTTATTAC
1753
+


1184251
asasa

g

AAA







AD-
gsusaag(Uhd)GfaCfAfAfcagaugu
1042
VPusAfsaacAfucuguugUfcAfcuuacsus
1397
CAGTAAGTGACAACAGATGT
1754
+


1184252
ususa

g

TTC







AD-
ususagc(Chd)UfuUfCfUfgcuauuc
1043
VPusAfsggaAfuagcagaAfaGfgcuaasas
1398
TTTTAGCCTTTCTGCTATTCC
1755
+


1184253
csusa

a

TT







AD-
usasgcc(Uhd)UfuCfUfGfcuauucc
1044
VPusAfsaggAfauagcagAfaAfggcuasas
1399
TTTAGCCTTTCTGCTATTCCT
1756
+


1184254
ususa

a

TG







AD-
asgsccu(Uhd)UfcUfGfCfuauuccu
1045
VPusCfsaagGfaauagcaGfaAfaggcusas
1400
TTAGCCTTTCTGCTATTCCTT
1757
+


1184255
usgsa

a

GT







AD-
cscsuuu(Chd)UfgCfUfAfuuccuug
1046
VPusAfsacaAfggaauagCfaGfaaaggscs
1401
AGCCTTTCTGCTATTCCTTGT
1758
+


1184256
ususa

u

TT







AD-
csusuuc(Uhd)GfcUfAfUfuccuugu
1047
VPusAfsaacAfaggaauaGfcAfgaaagsgs
1402
GCCTTTCTGCTATTCCTTGTT
1759
+


1184257
ususa

c

TT







AD-
ususucu(Ghd)CfuAfUfUfccuuguu
1048
VPusAfsaaaCfaaggaauAfgCfagaaasgs
1403
CCTTTCTGCTATTCCTTGTTT
1760
+


1184258
ususa

g

TA







AD-
ususcug(Chd)UfaUfUfCfcuuguuu
1049
VPusUfsaaaAfcaaggaaUfaGfcagaasas
1404
CTTTCTGCTATTCCTTGTTTT
1761
+


1184259
usasa

g

AA







AD-
uscsugc(Uhd)AfuUfCfCfuuguuuu
1050
VPusUfsuaaAfacaaggaAfuAfgcagasas
1405
TTTCTGCTATTCCTTGTTTTA
1762
+


1184260
asasa

a

AT







AD-
csusgcu(Ahd)UfuCfCfUfuguuuua
1051
VPusAfsuuaAfaacaaggAfaUfagcagsas
1406
TTCTGCTATTCCTTGTTTTAA
1763
+


1184261
asusa

a

TT







AD-
cscsaag(Ghd)UfuUfAfCfccaauaa
1052
VPusUfsauuAfuuggguaAfaCfcuuggsg
1407
CCCCAAGGTTTACCCAATAA
1764
+


1184262
usasa

sg

TAC







AD-
asasggu(Uhd)UfaCfCfCfaauaauac
1053
VPusAfsguaUfuauugggUfaAfaccuusgs
1408
CCAAGGTTTACCCAATAATA
1765
+


1184263
susa

g

CTG







AD-
asgsguu(Uhd)AfcCfCfAfauaauac
1054
VPusCfsaguAfuuauuggGfuAfaaccusus
1409
CAAGGTTTACCCAATAATAC
1766
+


1184264
usgsa

g

TGC







AD-
ascscca(Ahd)UfaAfUfAfcugcguc
1055
VPusAfsagaCfgcaguauUfaUfugggusas
1410
TTACCCAATAATACTGCGTC
1767
+


1184265
ususa

a

TTG







AD-
asgsucc(Ahd)GfaUfGfAfccaaauu
1056
VPusCfscaaUfuuggucaUfcUfggacusgs
1411
GCAGTCCAGATGACCAAATT
1768
+


1184266
gsgsa

c

GGC







AD-
gsuscca(Ghd)AfuGfAfCfcaaauug
1057
VPusGfsccaAfuuuggucAfuCfuggacsus
1412
CAGTCCAGATGACCAAATTG
1769
+


1184267
gscsa

g

GCT







AD-
asusugc(Chd)AfaAfAfGfgcuucua
1058
VPusCfsguaGfaagccuuUfuGfgcaausgs
1413
ACATTGCCAAAAGGCTTCTA
1770
+


1184268
csgsa

u

CGC







AD-
ususgcc(Ahd)AfaAfGfGfcuucuac
1059
VPusGfscguAfgaagccuUfuUfggcaasus
1414
CATTGCCAAAAGGCTTCTAC
1771
+


1184269
gscsa

g

GCA







AD-
gsgscag(Uhd)CfaAfGfCfcucuucu
1060
VPusCfsgagAfagaggcuUfgAfcugccsgs
1415
GCGGCAGTCAAGCCTCTTCT
1772
+


1184270
csgsa

c

CGT







AD-
asascug(Uhd)CfaCfUfAfagaaauc
1061
VPusCfsagaUfuucuuagUfgAfcaguusus
1416
CAAACTGTCACTAAGAAATC
1773
+


1184271
usgsa

g

TGC







AD-
usgsuca(Chd)UfaAfGfAfaaucugc
1062
VPusCfsagcAfgauuucuUfaGfugacasgs
1417
ACTGTCACTAAGAAATCTGC
1774
+


1184272
usgsa

u

TGC







AD-
asascug(Uhd)GfaCfUfCfuucuucc
1063
VPusCfsaggAfagaagagUfcAfcaguusus
1418
CAAACTGTGACTCTTCTTCCT
1775
+


1184273
usgsa

g

GC







AD-
uscsuug(Uhd)GfcAfGfAfaugaauu
1064
VPusAfsgaaUfucauucuGfcAfcaagasgs
1419
ACTCTTGTGCAGAATGAATT
1776
+


1184274
csusa

u

CTC







AD-
csusugu(Ghd)CfaGfAfAfugaauuc
1065
VPusGfsagaAfuucauucUfgCfacaagsas
1420
CTCTTGTGCAGAATGAATTC
1777
+


1184275
uscsa

g

TCG







AD-
ususgug(Chd)AfgAfAfUfgaauucu
1066
VPusCfsgagAfauucauuCfuGfcacaasgs
1421
TCTTGTGCAGAATGAATTCT
1778
+


1184276
csgsa

a

CGT







AD-
usgsugc(Ahd)GfaAfUfGfaauucuc
1067
VPusAfscgaGfaauucauUfcUfgcacasas
1422
CTTGTGCAGAATGAATTCTC
1779
+


1184277
gsusa

g

GTA







AD-
gsusgca(Ghd)AfaUfGfAfauucucg
1068
VPusUfsacgAfgaauucaUfuCfugcacsas
1423
TTGTGCAGAATGAATTCTCG
1780
+


1184278
usasa

a

TAA







AD-
usgscag(Ahd)AfuGfAfAfuucucgu
1069
VPusUfsuacGfagaauucAfuUfcugcascs
1424
TGTGCAGAATGAATTCTCGT
1781
+


1184279
asasa

a

AAC







AD-
gscsaga(Ahd)UfgAfAfUfucucgua
1070
VPusGfsuuaCfgagaauuCfaUfucugcsas
1425
GTGCAGAATGAATTCTCGTA
1782
+


1184280
ascsa

c

ACT







AD-
csasgaa(Uhd)GfaAfUfUfcucguaa
1071
VPusAfsguuAfcgagaauUfcAfuucugscs
1426
TGCAGAATGAATTCTCGTAA
1783
+


1184281
csusa

a

CTA







AD-
usasauc(Uhd)CfaCfAfUfagcaauc
1072
VPusAfsagaUfugcuaugUfgAfgauuasas
1427
TTTAATCTCACATAGCAATC
1784
+


1184282
ususa

a

TTT







AD-
uscsuca(Chd)AfuAfGfCfaaucuuu
1073
VPusUfsuaaAfgauugcuAfuGfugagasus
1428
AATCTCACATAGCAATCTTT
1785
+


1184283
asasa

u

AAT







AD-
csuscac(Ahd)UfaGfCfAfaucuuua
1074
VPusAfsuuaAfagauugcUfaUfgugagsas
1429
ATCTCACATAGCAATCTTTA
1786
+


1184284
asusa

u

ATC







AD-
uscsaca(Uhd)AfgCfAfAfucuuuaa
1075
VPusGfsauuAfaagauugCfuAfugugasgs
1430
TCTCACATAGCAATCTTTAA
1787
+


1184285
uscsa

a

TCA







AD-
csascau(Ahd)GfcAfAfUfcuuuaau
1076
VPusUfsgauUfaaagauuGfcUfaugugsas
1431
CTCACATAGCAATCTTTAAT
1788
+


1184286
csasa

g

CAG







AD-
gsasgcc(Chd)UfaAfUfGfuguaaaa
1077
VPusAfsauuUfuacacauUfaGfggcucsus
1432
AAGAGCCCTAATGTGTAAAA
1789
+


1184287
ususa

u

TTA







AD-
gscsccu(Ahd)AfuGfUfGfuaaaauu
1078
VPusUfsuaaUfuuuacacAfuUfagggcsus
1433
GAGCCCTAATGTGTAAAATT
1790
+


1184288
asasa

c

AAT







AD-
cscscua(Ahd)UfgUfGfUfaaaauua
1079
VPusAfsuuaAfuuuuacaCfaUfuagggscs
1434
AGCCCTAATGTGTAAAATTA
1791
+


1184289
asusa

u

ATT







AD-
cscsuaa(Uhd)GfuGfUfAfaaauuaa
1080
VPusAfsauuAfauuuuacAfcAfuuaggsgs
1435
GCCCTAATGTGTAAAATTAA
1792
+


1184290
ususa

c

TTT







AD-
cscscau(Ghd)UfgAfUfUfuuaauag
1081
VPusAfsgcuAfuuaaaauCfaCfaugggsgs
1436
TCCCCATGTGATTTTAATAG
1793
+


1184291
csusa

a

CTT
















TABLE 4







Unmodified Sense and Antisense Strand Coronavirus dsRNA Sequences























Strand










of the










Viral










RNA





Start in
End in



targeted





BetaCoV/Wuhan-Hu-
BetaCoV/Wuhan-Hu-
Targets
Targets
Targets
by the


Duplex Id
Sense Sequence 5′ to 3′
Antisense Sequence 5′ to 3′
1/2019|EP1_ISL_402125
1/2019|EP1_ISL_402125
SARS2?
SARS?
MERS?
Agent





AD-1231467
UACUCAUUCGUUUCGGAAGAA
UUCUUCCGAAACGAAUGAGUACA
26246
26268
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1794)
(SEQ ID NO: 1834)











AD-1231468
CUUCUGGUCUAAACAAACUAA
UUAGUUUGUUUAGACCAGAAGAU
26459
26481
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1795)
(SEQ ID NO: 1835)











AD-1231469
UUCUUGCUUUCGUGGUAUUCA
UGAAUACCACGAAAGCAAGAAAA
26301
26323
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1796)
(SEQ ID NO: 1836)











AD-1231470
UUAAUCUCACAUAGUAAUCUA
UAGAUUACUAUGUGAGAUUAAAG
29659
29681
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1797)
(SEQ ID NO: 1837)











AD-1231471
UAACUUUAAUCUCAUAUAGCA
UGCUAUAUGAGAUUAAAGUUAAC
29654
29676
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1798)
(SEQ ID NO: 1838)











AD-1231472
CCUGAUCUUCUGGUUUAAACA
UGUUUAAACCAGAAGAUCAGGAA
26453
26475
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1799)
(SEQ ID NO: 1839)











AD-1231473
UGUACAGUAAGUGAUAACAGA
UCUGUUAUCACUUACUGUACAAG
27180
27202
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1800)
(SEQ ID NO: 1840)











AD-1231474
UUCCUGAUCUUCUGGUCUAAA
UUUAGACCAGAAGAUCAGGAACU
26451
26473
TRUE
TRUE
FALSE
+



(SEQ ID NO: 143)
(SEQ ID NO: 498)











AD-1231475
UAUGUACUCAUUCGUUUCGGA
UCCGAAACGAAUGAGUACAUAAG
26242
26264
TRUE
TRUE
FALSE
+



(SEQ ID NO: 296)
(SEQ ID NO: 651)











AD-1231476
ACUGUACAGUCUAAAAUGUCA
UGACAUUUUAGACUGUACAGUGG
11832
11854
TRUE
TRUE
FALSE
+



(SEQ ID NO: 7)
(SEQ ID NO: 8)











AD-1231477
CCCAUGUGAUUUUAAUAGCUA
UAGCUAUUAAAAUCACAUGGGGA
29834
29856
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1801)
(SEQ ID NO: 1841)











AD-1231478
UUCGUGGUAUUCUUACUAGUA
UACUAGUAAGAAUACCACGAAAG
26309
26331
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1802)
(SEQ ID NO: 1842)











AD-1231479
UCUUCUGGUCUAAAUGAACUA
UAGUUCAUUUAGACCAGAAGAUC
26458
26480
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1803)
(SEQ ID NO: 1843)











AD-1231480
GUACUGCUGCAAUAUUGUUAA
UUAACAAUAUUGCAGCAGUACGC
26365
26387
TRUE
TRUE
FALSE
+



(SEQ ID NO: 310)
(SEQ ID NO: 665)











AD-1231481
UUUAAUCUCACAUAGCAAUCA
UGAUUGCUAUGUGAGAUUAAAGU
29658
29680
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1804)
(SEQ ID NO: 1844)











AD-1231482
UACAACUUCCUCAAGGAACAA
UUGUUCCUUGAGGAAGUUGUAGC
28747
28769
TRUE
TRUE
FALSE
+



(SEQ ID NO: 162)
(SEQ ID NO: 517)











AD-1231483
CUGCUAUUCCUUGUUUUAAUA
UAUUAAAACAAGGAAUAGCAGAA
27802
27824
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1805)
(SEQ ID NO: 1845)











AD-1231484
CUCACAUAGCAAUCUUUAAUA
UAUUAAAGAUUGCUAUGUGAGAU
29664
29686
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1806)
(SEQ ID NO: 1846)











AD-1231485
UCGUGCUACAACUUUCUCAAA
UUUGAGAAAGUUGUAGCACGAUU
28741
28763
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1807)
(SEQ ID NO: 1847)











AD-1231486
CAGAUGGUACACUUAUGAUUA
UAAUCAUAAGUGUACCAUCUGUU
15987
16009
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1808)
(SEQ ID NO: 1848)











AD-1231487
UAACAAUGUUGCUUUUCAAAA
UUUUGAAAAGCAACAUUGUUAGU
14644
14666
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1809)
(SEQ ID NO: 1849)











AD-1231488
UAAUAGUUAAUAGCGUACUUA
UAAGUACGCUAUUAACUAUUAAC
26277
26299
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1810)
(SEQ ID NO: 1850)











AD-1231489
CAUACAAUGCUAGUUAAACAA
UUGUUUAACUAGCAUUGUAUGUU
15884
15906
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1811)
(SEQ ID NO: 1851)











AD-1231490
UUUUCAAACUGUCAAACCCGA
UCGGGUUUGACAGUUUGAAAAGC
14656
14678
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1812)
(SEQ ID NO: 1852)











AD-1231491
CUUAUGUACUCAUUCGUUUCA
UGAAACGAAUGAGUACAUAAGUU
26240
26262
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1813)
(SEQ ID NO: 1853)











AD-1231492
GUUGUGUACACACAUUGGUAA
UUACCAAUGUGUGUACACAACAU
13154
13176
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1814)
(SEQ ID NO: 1854)











AD-1231493
AACUUAUGUACUCAUUCGUUA
UAACGAAUGAGUACAUAAGUUCG
26238
26260
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1815)
(SEQ ID NO: 1855)











AD-1231494
CUGCUGCAAUAUUGUUAACGA
UCGUUAACAAUAUUGCAGCAGUA
26368
26390
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1816)
(SEQ ID NO: 1856)











AD-1231495
UUCUUCGUAAGAACGGUAAUA
UAUUACCGUUCUUACGAAGAAGA
628
650
TRUE
TRUE
FALSE
+



(SEQ ID NO: 27)
(SEQ ID NO: 382)











AD-1231496
CGUGCUACAACUUCUUCAAGA
UCUUGAAGAAGUUGUAGCACGAU
28742
28764
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1817)
(SEQ ID NO: 1857)











AD-1231497
UGAAAUUGUUGACAUUGUGAA
UUCACAAUGUCAACAAUUUCAGC
17572
17594
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1818)
(SEQ ID NO: 1858)











AD-1231498
ACAAUGUUGCUUUUUAAACUA
UAGUUUAAAAAGCAACAUUGUUA
14646
14668
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1819)
(SEQ ID NO: 1859)











AD-1231499
AAAUUGUUGACACUAUGAGUA
UACUCAUAGUGUCAACAAUUUCA
17574
17596
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1820)
(SEQ ID NO: 1860)











AD-1231500
UCUUCGUAAGAACGGUAAUAA
UUAUUACCGUUCUUACGAAGAAG
629
651
TRUE
TRUE
FALSE
+



(SEQ ID NO: 28)
(SEQ ID NO: 383)











AD-1231501
CUGCAAUAUUGUUAACGUGAA
UUCACGUUAACAAUAUUGCAGCA
26371
26393
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1821)
(SEQ ID NO: 1861)











AD-1231502
GCUGAAAUUGUUGAUACUGUA
UACAGUAUCAACAAUUUCAGCAG
17570
17592
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1822)
(SEQ ID NO: 1862)











AD-1231503
CUGCUGAAAUUGUUGACACUA
UAGUGUCAACAAUUUCAGCAGGA
17568
17590
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1823)
(SEQ ID NO: 1863)











AD-1231504
UUGUGAUGAUGAUUAUUUCAA
UUGAAAUAAUCAUCAUCACAACA
17578
17600
TRUE
TRUE
FALSE
+



(SEQ ID NO: 228)
(SEQ ID NO: 583)











AD-1231505
UGUUGACACUGUGAAUGCUUA
UAAGCAUUCACAGUGUCAACAAU
26268
26290
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1824)
(SEQ ID NO: 1864)











AD-1231506
CAGGUACGUUAAUAGUUAAUA
UAUUAACUAUUAACGUACCUGUC
28767
28789
TRUE
TRUE
FALSE
+



(SEQ ID NO: 300)
(SEQ ID NO: 655)











AD-1231507
ACAUUGCCAAAAGGUUUCUAA
UUAGAAACCUUUUGGCAAUGUUG
26370
26392
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1825)
(SEQ ID NO: 1865)











AD-1231508
GCUGCAAUAUUGUUAACGUGA
UCACGUUAACAAUAUUGCAGCAG
29666
29688
TRUE
TRUE
FALSE
+



(SEQ ID NO: 313)
(SEQ ID NO: 668)











AD-1231509
CACAUAGCAAUCUUUAAUCAA
UUGAUUAAAGAUUGCUAUGUGAG
14824
14846
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1826)
(SEQ ID NO: 1866)











AD-1231510
AAUGUGUGAUAUCAGACAACA
UGUUGUCUGAUAUCACACAUUGU
26451
26473
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1827)
(SEQ ID NO: 1867)











AD-1231511
GUUCCUGAUCUUCUGGUCUAA
UUAGACCAGAAGAUCAGGAACUC
26450
26472
TRUE
TRUE
FALSE
+



(SEQ ID NO: 142)
(SEQ ID NO: 497)











AD-1231512
UUGCCAAAAGGCUUCUACGCA
UGCGUAGAAGCCUUUUGGCAAUG
28770
28792
TRUE
TRUE
FALSE
+



(SEQ ID NO: 349)
(SEQ ID NO: 704)











AD-1231513
UUCUGCUAAUCUUGUUGCUAA
UUAGCAACAAGAUUAGCAGAAGC
24620
24642
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1828)
(SEQ ID NO: 1868)











AD-1231514
ACCUAGUAAUAGGUUUCCUAA
UUAGGAAACCUAUUACUAGGUUC
26582
26604
TRUE
TRUE
FALSE
+



(SEQ ID NO: 1829)
(SEQ ID NO: 1869)











AD-1231515
CUGCUAAUCUUGCUGCUACUA
UAGUAGCAGCAAGAUUAGCAGAA
24622
24644
TRUE
TRUE
FALSE
+



(SEQ ID NO: 121)
(SEQ ID NO: 476)











AD-1231516
CACUCAUAAAGUCUGUGUUGA
UCAACACAGACUUUAUGAGUGUC
15609
15631
TRUE
FALSE
FALSE




(SEQ ID NO: 1830)
(SEQ ID NO: 1870)











AD-1231517
CAUAGGAAUAAAAUCUUCUAA
UUAGAAGAUUUUAUUCCUAUGGA
20413
20435
TRUE
FALSE
FALSE




(SEQ ID NO: 281)
(SEQ ID NO: 636)











AD-1231518
UCUACACAAACUCUUAAAGAA
UUCUUUAAGAGUUUGUGUAGAUA
3745
3767
TRUE
FALSE
FALSE




(SEQ ID NO: 210)
(SEQ ID NO: 565)











AD-1231519
AUCUUGUUUUCUCUAUUCAAA
UUUGAAUAGAGAAAACAAGAUGA
3929
3951
TRUE
FALSE
FALSE




(SEQ ID NO: 1831)
(SEQ ID NO: 1871)











AD-1231520
CAGUCAUAAUCUAUAUUAAAA
UUUUAAUAUAGAUUAUGACUGUG
10501
10523
TRUE
FALSE
FALSE




(SEQ ID NO: 1832)
(SEQ ID NO: 1872)











AD-1231521
UGGUAACACUAAUAAUAAAAA
UUUUUAUUAUUAGUGUUACCACA
23712
23734
TRUE
FALSE
FALSE




(SEQ ID NO: 1833)
(SEQ ID NO: 1873)
















TABLE 5







Modified Sense and Antisense Strand dsRNA Sequences Targeting COVID-19













SEQ ID

SEQ ID


Duplex Id
Sense Sequence 5′ to 3′
NO:
Antisense Sequence 5′ to 3′
NO:














AD-1231467
usascuca(Uhd)uCfGfUfuucggaagsasa
1874
VPusUfscudTc(Cgn)gaaacgAfaUfgaguascsa
1925





AD-1231468
csusuc(Uhd)gGfuCfUfAfaacaaacusasa
1875
VPusUfsagdTudTguuuagAfcCfagaagsasu
1926





AD-1231469
ususcuugCfuUfUfCfgugg(Uhd)auuscsa
1876
VPusGfsaadTa(Cgn)cacgaaAfgCfaagaasasa
1927





AD-1231470
ususaau(Chd)UfcAfCfAfuaguaaucsusa
1877
VPusAfsgadTudAcuauguGfaGfauuaasasg
1928





AD-1231471
usasacu(Uhd)UfaAfUfCfucauauagscsa
1878
VPusGfscudAudAugagauUfaAfaguuasasc
1929





AD-1231472
cscsuga(Uhd)CfulifCfUfgguuuaaascsa
1879
VPusGfsuudTadAaccagaAfgAfucaggsasa
1930





AD-1231473
usgsua(Chd)aGfuAfAfGfugauaacasgsa
1880
VPusCfsugdTudAucacuuAfcUfguacasasg
1931





AD-1231474
ususccugAfuCfUfUfcugg(Uhd)cuasasa
1881
VPusUfsuadGa(Cgn)cagaagAfuCfaggaascsu
1932





AD-1231475
usasuguaCfuCfAfUfucgu(Uhd)ucgsgsa
1882
VPusCfscgaAfacgaaugAfgUfacauasasg
1361





AD-1231476
ascsugua(Chd)aGfUfCfuaaaauguscsa
1883
VPusGfsacaUfuuuagacUfgUfacagusgsg
12





AD-1231477
cscscaugUfgAfUfUfuuaa(Uhd)agcsusa
1884
VPusAfsgcuAfuuaaaauCfaCfaugggsgsa
1436





AD-1231478
ususcgugGfuAfUfUfcuua(Chd)uagsusa
1885
VPusAfscudAgdTaagaauAfcCfacgaasasg
1933





AD-1231479
uscsuuc(Uhd)GfgUfCfUfaaaugaacsusa
1886
VPusAfsgudTcdAuuuagaCfcAfgaagasusc
1934





AD-1231480
gsusacugCfuGfCfAfauau(Uhd)guusasa
1887
VPusUfsaacAfauauugcAfgCfaguacsgsc
1375





AD-1231481
ususuaa(Uhd)CfuCfAfCfauagcaauscsa
891
VPusGfsaudTg(Cgn)uaugugAfgAfuuaaasgsu
1935





AD-1231482
usascaa(Chd)UfuCfCfUfcaaggaacsasa
872
VPusUfsgudTc(Cgn)uugaggAfaGfuuguasgsc
1936





AD-1231483
csusgcuaUfuCfCfUfuguu(Uhd)uaasusa
1888
VPusAfsuuaAfaacaaggAfaUfagcagsasa
1406





AD-1231484
csuscacaUfaGfCfAfaucu(Uhd)uaasusa
1889
VPusAfsuuaAfagauugcUfaUfgugagsasu
1429





AD-1231485
uscsgug(Chd)UfaCfAfAfcuuucucasasa
1890
VPusUfsugdAgdAaaguugUfaGfcacgasusu
1937





AD-1231486
csasgaugGfuAfCfAfcuua(Uhd)gaususa
1891
VPusAfsaucAfuaaguguAfcCfaucugsusu
1320





AD-1231487
usasacaaUfgUfUfGfcuuu(Uhd)caasasa
1892
VPusUfsuugAfaaagcaaCfaUfuguuasgsu
10





AD-1231488
usasauagUfuAfAfUfagcg(Uhd)acususa
1893
VPusAfsaguAfcgcuauuAfaCfuauuasasc
1367





AD-1231489
csasua(Chd)aAfuGfCfUfaguuaaacsasa
1894
VPusUfsguuUfaacuagcAfuUfguaugsusu
1318





AD-1231490
ususuu(Chd)aAfaCfUfGfucaaacccsgsa
1895
VPusCfsgggUfuugacagUfuUfgaaaasgsc
1296





AD-1231491
csusuaug(Uhd)aCfUfCfauucguuuscsa
1896
VPusGfsaaaCfgaaugagUfaCfauaagsusu
1359





AD-1231492
gsusugugUfaCfAfCfacau(Uhd)ggusasa
1897
VPusUfsacdCadAugugugUfaCfacaacsasu
1938





AD-1231493
asascuuaUfgUfAfCfucau(Uhd)cgususa
1898
VPusAfsacgAfaugaguaCfaUfaaguuscsg
1357





AD-1231494
csusgcugCfaAfUfAfuugu(Uhd)aacsgsa
1899
VPusCfsguuAfacaauauUfgCfagcagsusa
1376





AD-1231495
ususcuu(Chd)GfuAfAfGfaacgguaasusa
737
VPusAfsuudAc(Cgn)guucuuAfcGfaagaasgsa
1939





AD-1231496
csgsugc(Uhd)AfcAfAfCfuucuucaasgsa
1900
VPusCfsuudGadAgaaguuGfuAfgcacgsasu
1940





AD-1231497
usgsaaa(Uhd)UfgUfUfGfacauugugsasa
1901
VPusUfscadCadAugucaaCfaAfuuucasgsc
1941





AD-1231498
ascsaaug(Uhd)uGfCfUfuuuuaaacsusa
1902
VPusAfsgudTudAaaaagcAfaCfauugususa
1942





AD-1231499
asasauugUfuGfAfCfacua(Uhd)gagsusa
1903
VPusAfscudCadTagugucAfaCfaauuuscsa
1943





AD-1231500
uscsuucgUfaAfGfAfacgg(Uhd)aausasa
1904
VPusUfsaudTa(Cgn)cguucuUfaCfgaagasasg
1944





AD-1231501
csusgcaaUfaUfUfGfuuaa(Chd)gugsasa
1905
VPusUfscacGfuuaacaaUfaUfugcagscsa
1379





AD-1231502
gscsugaaAfuUfGfUfugaua(Chd)ugsusa
1906
VPusAfscadGudAucaacaAfuUfucagcsasg
1945





AD-1231503
csusgc(Uhd)gAfaAfUfUfguugacacsusa
1907
VPusAfsgudGu(Cgn)aacaauUfuCfagcagsgsa
1946





AD-1231504
ususgugaUfgAfUfGfauua(Uhd)uucsasa
1908
VPusUfsgaaAfuaaucauCfaUfcacaascsa
1293





AD-1231505
usgsuugaCfaCfUfGfugaa(Uhd)gcususa
1909
VPusAfsagdCadTucacagUfgUfcaacasasu
1947





AD-1231506
csasgguaCfgUfUfAfauag(Uhd)uaasusa
1910
VPusAfsuuaAfcuauuaaCfgUfaccugsusc
1365





AD-1231507
ascsauugCfcAfAfAfaggu(Uhd)ucusasa
1911
VPusUfsagdAadAccuuuuGfgCfaaugususg
1948





AD-1231508
gscsug(Chd)aAfuAfUfUfguuaacgusgsa
1912
VPusCfsacgUfuaacaauAfuUfgcagcsasg
1378





AD-1231509
csasca(Uhd)aGfcAfAfUfcuuuaaucsasa
1913
VPusUfsgauUfaaagauuGfcUfaugugsasg
1431





AD-1231510
asasugug(Uhd)gAfUfAfucagacaascsa
1914
VPusGfsuudGu(Cgn)ugauauCfaCfacauusgsu
1949





AD-1231511
gsusucc(Uhd)GfaUfCfUfucuggucusasa
852
VPusUfsagdAc(Cgn)agaagaUfcAfggaacsusc
1950





AD-1231512
ususgccaAfaAfGfGfcuuc(Uhd)acgscsa
1915
VPusGfscguAfgaagccuUfuUfggcaasusg
1414





AD-1231513
ususcug(Chd)UfaAfUfCfuuguugcusasa
1916
VPusUfsagdCadAcaagauUfaGfcagaasgsc
1951





AD-1231514
ascscuagUfaAfUfAfgguu(Uhd)ccusasa
1917
VPusUfsaggAfaaccuauUfaCfuaggususc
1388





AD-1231515
csusgcuaAfuCfUfUfgcug(Chd)uacsusa
1918
VPusAfsgudAg(Cgn)agcaagAfuUfagcagsasa
1952





AD-1231516
csascucaUfaAfAfGfucug(Uhd)guusgsa
1919
VPusCfsaacAfcagacuuUfaUfgagugsusc
1315





AD-1231517
csasuaggAfaUfAfAfaauc(Uhd)ucusasa
1920
VPusUfsagaAfgauuuuaUfuCfcuaugsgsa
1346





AD-1231518
uscsuaca(Chd)aAfAfCfucuuaaagsasa
1921
VPusUfscuuUfaagaguuUfgUfguagasusa
1275





AD-1231519
asuscuugUfuUfUfCfucua(Uhd)ucasasa
1922
VPusUfsugdAadTagagaaAfaCfaagausgsa
1953





AD-1231520
csasgucaUfaAfUfCfuaua(Uhd)uaasasa
1923
VPusUfsuudAadTauagauUfaUfgacugsusg
1954





AD-1231521
usgsguaaCfaCfUfAfauaa(Uhd)aaasasa
1924
VPusUfsuudTadTuauuagUfgUfuaccascsa
1955
















TABLE 6







Single Dose In Vitro Screens in Cos-7 Cells












% of Message

% of Message




Remaining-

Remaining-




10 nM

10 nM



Duplex ID
Concatenate-02
STDEV
Concatenate-21
STDEV














AD-1184048.1
2.63
0.37
10.90
2.58


AD-1184130.1
4.52
0.51
15.30
3.51


AD-1184064.1
5.13
2.78
18.41
12.53


AD-1184050.1
5.58
2.20
14.85
7.91


AD-1184202.1
5.58
2.27
7.20
2.28


AD-1184100.1
6.41
1.86
1.63
1.97


AD-1184098.1
6.46
0.74
1.51
1.09


AD-1184123.1
6.48
0.59
19.79
1.49


AD-1184223.1
6.63
0.25
8.39
1.65


AD-1184219.1
6.66
0.52
6.38
2.51


AD-1184062.1
6.67
4.16
4.12
0.32


AD-1184070.1
6.72
0.66
16.86
4.27


AD-1184061.1
6.78
0.65
10.62
2.56


AD-1184216.1
6.82
0.44
11.90
0.70


AD-1184255.1
6.88
0.67
2.84
0.96


AD-1184237.1
6.90
0.16
5.26
0.45


AD-1184218.1
7.05
0.15
10.23
0.16


AD-1184215.1
7.15
0.17
15.26
3.28


AD-1184137.1
7.17
1.30
18.05
4.06


AD-1184271.1
7.19
0.52
4.76
2.43


AD-1184166.1
7.57
0.13
N/A
N/A


AD-1184291.1
7.60
0.68
3.12
1.19


AD-1184256.1
7.68
2.71
4.95
0.80


AD-1184260.1
7.69
1.24
10.95
3.08


AD-1184051.1
7.71
0.32
17.00
2.76


AD-1184063.1
7.71
3.85
10.87
3.90


AD-1184213.1
7.71
0.22
14.68
9.77


AD-1184124.1
7.85
1.08
18.09
1.81


AD-1184230.1
7.85
0.71
11.08
1.15


AD-1184099.1
7.94
0.91
1.24
0.61


AD-1184189.1
7.94
0.98
6.87
1.66


AD-1184152.1
8.01
0.63
45.16
29.64


AD-1184080.1
8.03
3.13
20.44
9.44


AD-1184261.1
8.04
0.33
10.46
5.96


AD-1184138.1
8.11
1.81
34.34
17.00


AD-1184221.1
8.28
0.89
14.34
3.37


AD-1184284.1
8.28
0.72
0.55
0.15


AD-1184144.1
8.31
0.21
19.46
14.43


AD-1184272.1
8.43
0.68
2.04
0.80


AD-1184285.1
8.55
2.60
N/A
N/A


AD-1184167.1
8.55
0.22
26.83
12.68


AD-1184110.1
8.57
0.27
32.02
18.77


AD-1184113.1
8.67
0.36
42.10
37.37


AD-1184141.1
8.67
0.76
32.39
13.05


AD-1184226.1
8.70
0.62
10.59
3.78


AD-1184077.1
8.72
0.18
16.74
1.13


AD-1184175.1
8.79
0.38
17.35
1.43


AD-1184053.1
8.85
1.05
26.88
9.12


AD-1184114.1
8.96
0.95
35.26
15.77


AD-1184091.1
9.06
0.96
35.70
15.75


AD-1184228.1
9.10
1.23
9.24
4.10


AD-1184150.1
9.15
2.15
11.22
2.79


AD-1184222.1
9.27
1.29
11.23
2.32


AD-1184173.1
9.32
3.48
12.47
5.60


AD-1184040.1
9.46
3.27
53.64
29.25


AD-1184125.1
9.62
2.10
27.24
1.20


AD-1184151.1
9.65
5.07
20.35
6.60


AD-1184214.1
9.66
0.52
13.01
2.02


AD-1184235.1
9.74
2.34
4.91
0.68


AD-1184217.1
9.79
0.95
15.78
4.08


AD-1184055.1
9.83
0.63
38.63
12.62


AD-1184169.1
9.92
2.16
28.77
14.24


AD-1184227.1
10.11
4.84
9.70
1.00


AD-1184195.1
10.11
8.02
45.22
21.80


AD-1183971.1
10.43
1.92
4.64
1.24


AD-1184085.1
10.45
0.59
17.89
2.27


AD-1184212.1
10.59
1.87
14.04
0.85


AD-1184231.1
10.67
4.28
16.42
4.86


AD-1184148.1
10.87
8.58
18.19
3.78


AD-1183945.1
10.95
0.72
19.06
1.18


AD-1184268.1
11.00
0.54
6.03
2.13


AD-1184054.1
11.12
2.65
34.41
9.52


AD-1184071.1
11.14
2.81
23.38
8.83


AD-1184232.1
11.16
1.04
9.87
2.12


AD-1184078.1
11.19
2.29
16.35
2.82


AD-1184229.1
11.44
0.22
15.36
1.87


AD-1184018.1
11.92
2.94
11.18
5.66


AD-1184004.1
11.93
0.06
30.41
15.72


AD-1183979.1
12.07
0.81
20.29
8.12


AD-1184259.1
12.15
3.41
7.92
1.24


AD-1184252.1
12.20
6.36
9.64
1.21


AD-1184224.1
12.21
0.77
11.43
2.61


AD-1184037.1
12.26
2.11
36.01
8.76


AD-1184112.1
12.57
1.41
53.76
36.30


AD-1184019.1
12.74
5.26
6.57
3.27


AD-1184211.1
12.84
0.95
15.37
1.00


AD-1183946.1
13.10
2.42
16.50
4.85


AD-1184270.1
13.15
0.69
17.48
13.18


AD-1184234.1
13.16
1.74
12.12
2.73


AD-1184111.1
13.20
0.51
27.70
3.35


AD-1184017.1
13.22
6.55
7.64
1.71


AD-1183944.1
13.24
7.54
22.04
3.53


AD-1184045.1
13.26
2.03
26.78
12.50


AD-1184016.1
13.28
0.81
18.27
7.54


AD-1184146.1
13.56
5.67
24.87
17.23


AD-1184168.1
13.78
11.08
14.98
1.72


AD-1184136.1
13.78
0.38
18.39
3.38


AD-1184147.1
13.87
8.89
16.79
1.97


AD-1184020.1
13.90
5.93
4.01
0.50


AD-1183981.1
14.01
5.51
45.10
24.43


AD-1184220.1
14.03
2.59
14.74
1.03


AD-1184086.1
14.29
1.54
20.17
3.50


AD-1184282.1
14.32
4.56
1.75
0.61


AD-1184106.1
14.34
0.80
2.75
0.47


AD-1184047.1
14.47
0.30
45.66
19.02


AD-1184174.1
14.51
9.37
34.31
12.21


AD-1184008.1
14.69
0.12
41.14
11.84


AD-1184283.1
14.78
4.90
1.91
0.22


AD-1184052.1
14.90
0.63
58.07
17.97


AD-1184209.1
15.39
0.52
14.16
2.43


AD-1184233.1
15.40
3.88
20.15
2.08


AD-1183962.1
15.63
1.31
54.76
30.80


AD-1184083.1
15.83
4.95
22.37
1.47


AD-1184286.1
16.01
2.27
2.00
0.03


AD-1184015.1
16.20
11.40
29.61
8.40


AD-1183989.1
16.34
6.20
13.86
3.56


AD-1184057.1
16.41
3.28
49.66
9.68


AD-1184088.1
17.10
6.67
33.62
21.10


AD-1184060.1
17.11
10.60
15.28
14.57


AD-1184044.1
17.75
1.12
45.51
28.15


AD-1184210.1
18.20
2.59
18.08
2.59


AD-1184172.1
18.41
11.26
30.65
27.58


AD-1183938.1
18.45
3.98
38.39
12.64


AD-1184097.1
19.01
1.36
23.80
10.22


AD-1183982.1
19.21
9.72
47.72
16.24


AD-1184269.1
19.30
8.09
13.74
1.68


AD-1184056.1
19.38
5.22
29.08
13.13


AD-1184038.1
19.44
1.60
11.35
2.52


AD-1184139.1
19.53
1.18
27.71
12.16


AD-1184243.1
19.77
1.94
19.11
1.57


AD-1184140.1
19.85
5.99
48.11
27.84


AD-1184254.1
20.28
1.40
12.15
1.63


AD-1184039.1
20.38
0.98
12.30
6.12


AD-1184242.1
20.41
0.99
33.51
12.48


AD-1184102.1
20.41
3.10
4.37
1.78


AD-1184082.1
21.24
0.58
33.45
16.23


AD-1184236.1
21.31
0.68
9.04
1.66


AD-1184006.1
21.33
14.37
17.11
3.28


AD-1184205.1
21.55
1.62
4.37
1.37


AD-1184042.1
21.83
5.52
33.36
12.05


AD-1184065.1
21.90
2.60
58.16
25.42


AD-1183969.1
21.90
10.09
5.17
2.16


AD-1184225.1
23.07
3.35
17.73
2.00


AD-1184034.1
23.11
0.49
7.40
3.24


AD-1184075.1
23.96
0.96
12.73
6.25


AD-1184207.1
24.80
2.68
14.52
3.96


AD-1184258.1
25.77
5.86
18.80
1.87


AD-1184103.1
26.50
4.94
3.15
1.78


AD-1184180.1
27.78
2.01
4.08
2.97


AD-1184238.1
28.23
0.71
20.35
2.33


AD-1183980.1
28.37
20.86
27.84
18.41


AD-1184142.1
28.71
0.80
14.04
2.14


AD-1184043.1
29.12
4.83
26.00
10.19


AD-1184198.1
29.63
8.04
9.62
1.92


AD-1184132.1
29.71
7.08
3.65
1.37


AD-1183935.1
30.45
1.06
13.92
7.44


AD-1183970.1
30.50
22.12
16.61
16.60


AD-1184109.1
30.85
2.81
3.10
0.65


AD-1184002.1
31.09
2.55
30.36
6.41


AD-1184129.1
31.47
6.67
33.15
11.16


AD-1183998.1
32.57
2.99
22.22
11.73


AD-1184251.1
33.06
0.66
2.68
1.11


AD-1184120.1
33.85
4.79
9.82
3.79


AD-1184032.1
33.94
3.79
10.30
1.57


AD-1184186.1
34.00
11.85
9.38
1.87


AD-1184067.1
34.35
11.07
14.02
8.43


AD-1184161.1
34.50
0.31
25.68
15.70


AD-1184084.1
34.60
5.69
43.25
4.15


AD-1184119.1
34.69
3.51
16.92
11.78


AD-1184026.1
34.98
4.17
20.63
4.42


AD-1184003.1
34.98
24.54
24.07
1.36


AD-1183959.1
35.01
0.67
17.15
10.10


AD-1184108.1
35.13
1.60
14.10
6.45


AD-1184164.1
35.44
12.85
20.94
6.85


AD-1184277.1
35.50
4.03
6.23
1.22


AD-1184116.1
36.10
7.48
4.91
3.04


AD-1184184.1
36.25
2.52
14.56
7.78


AD-1184249.1
36.26
6.98
2.06
0.48


AD-1183972.1
36.48
24.57
16.50
10.24


AD-1184143.1
37.10
4.12
60.26
47.27


AD-1184279.1
37.93
0.75
3.81
2.02


AD-1184191.2
37.93
0.64
13.99
0.87


AD-1184278.1
38.05
2.23
5.47
2.54


AD-1184160.1
38.14
10.62
48.46
19.14


AD-1184046.1
38.51
1.35
60.87
21.93


AD-1184206.1
38.57
7.93
4.42
0.27


AD-1183984.1
38.80
5.36
9.39
1.60


AD-1184247.1
39.35
2.55
2.45
0.56


AD-1184133.1
39.92
7.07
5.26
1.58


AD-1184154.1
40.25
8.75
15.70
4.99


AD-1184101.1
40.47
5.58
17.96
3.62


AD-1184192.1
40.75
3.21
47.06
22.51


AD-1184204.1
41.20
5.72
1.46
0.62


AD-1184094.1
41.25
8.86
15.40
3.72


AD-1184281.1
41.82
2.91
5.81
1.47


AD-1184248.1
41.98
1.67
1.41
0.85


AD-1184115.1
42.06
2.95
19.78
11.59


AD-1184250.1
42.26
4.20
2.73
1.36


AD-1184041.1
42.48
3.19
7.06
0.96


AD-1184027.1
42.83
6.09
30.80
14.97


AD-1184122.1
42.88
2.78
21.18
3.28


AD-1183956.1
43.02
4.15
27.77
0.81


AD-1183936.1
43.22
5.77
14.20
7.54


AD-1183983.1
43.73
2.34
23.48
6.77


AD-1184239.1
43.79
11.20
65.40
24.69


AD-1184117.1
43.91
2.03
33.46
19.25


AD-1184090.1
44.17
10.78
17.42
3.89


AD-1183996.1
44.48
2.38
18.83
6.96


AD-1184159.1
44.54
3.99
71.46
60.74


AD-1183986.1
45.32
11.06
15.98
1.72


AD-1184171.1
45.52
3.49
15.87
9.86


AD-1184107.1
45.87
2.08
10.22
2.57


AD-1184265.1
46.01
9.43
12.04
2.84


AD-1184275.1
46.21
6.74
16.22
7.54


AD-1184014.1
46.37
5.74
14.22
1.87


AD-1184263.1
47.01
8.63
9.79
3.27


AD-1184183.1
47.07
6.75
9.20
4.87


AD-1184012.1
47.42
3.84
11.60
0.86


AD-1183999.1
47.62
2.37
33.42
15.40


AD-1184203.1
47.81
2.60
2.76
0.77


AD-1184118.1
47.81
1.54
14.60
1.57


AD-1183940.1
48.07
4.28
15.84
5.02


AD-1184191.1
48.37
18.93
13.85
2.40


AD-1184127.1
48.59
4.03
9.19
2.45


AD-1184049.1
48.71
1.62
77.51
44.26


AD-1183993.1
48.74
9.36
19.86
1.34


AD-1184121.1
48.98
21.86
19.80
13.18


AD-1184165.1
49.21
1.14
6.48
2.56


AD-1184093.1
49.84
3.10
15.27
2.93


AD-1184280.1
49.97
1.27
5.25
0.01


AD-1184073.1
50.08
1.97
37.32
14.73


AD-1184182.1
50.14
0.70
32.68
11.12


AD-1183943.1
50.21
4.56
18.62
2.35


AD-1184276.1
50.40
10.18
7.22
0.40


AD-1184163.1
50.64
1.71
25.14
15.54


AD-1184179.1
50.74
11.32
9.27
4.91


AD-1184135.1
50.93
8.21
13.80
2.70


AD-1183992.1
51.42
8.33
42.18
23.25


AD-1183967.1
51.71
6.86
57.35
28.94


AD-1184158.1
52.02
1.89
14.09
2.71


AD-1184157.1
52.16
6.95
58.46
31.63


AD-1184266.1
52.38
6.11
25.86
8.79


AD-1184246.1
52.39
12.83
2.66
0.61


AD-1184190.1
52.42
12.47
35.31
12.80


AD-1184092.1
53.02
2.85
24.54
13.96


AD-1184264.1
53.03
2.78
6.81
0.99


AD-1184005.1
53.20
11.03
71.42
11.07


AD-1184177.1
53.21
14.56
20.72
15.48


AD-1184104.1
53.27
10.42
26.83
14.02


AD-1183978.1
53.56
2.47
24.02
16.27


AD-1184194.1
54.03
4.33
249.38
121.89


AD-1184033.1
54.25
14.79
16.32
7.81


AD-1183975.1
54.47
21.12
42.79
19.29


AD-1184290.1
54.65
3.02
5.15
0.39


AD-1184176.1
54.77
2.23
18.76
13.79


AD-1184030.1
54.81
1.50
69.90
25.45


AD-1184105.1
54.84
8.20
9.56
4.30


AD-1184187.1
55.39
15.91
42.43
31.40


AD-1183990.1
56.00
9.90
9.63
5.29


AD-1184196.1
56.24
11.11
21.62
1.09


AD-1184188.1
56.26
22.13
19.16
4.83


AD-1184024.1
56.46
11.22
28.76
10.49


AD-1184087.1
56.55
13.61
32.17
11.27


AD-1184193.1
56.61
4.42
85.01
31.27


AD-1183995.1
56.69
5.48
53.57
47.57


AD-1184074.1
56.98
9.01
33.24
13.16


AD-1184267.1
57.34
2.47
14.58
3.09


AD-1183966.1
57.41
3.21
22.22
9.46


AD-1183977.1
57.68
4.84
19.98
15.03


AD-1184273.1
57.94
2.34
3.97
0.64


AD-1183985.1
58.54
1.43
12.79
1.24


AD-1184011.1
58.67
6.24
31.15
5.26


AD-1184021.1
58.73
12.50
19.65
8.36


AD-1184066.1
58.74
2.53
9.67
6.29


AD-1184178.1
58.81
21.97
23.61
18.10


AD-1184274.1
59.03
3.54
10.66
9.31


AD-1184208.1
59.10
17.95
21.32
1.63


AD-1184149.1
59.11
24.90
42.83
36.12


AD-1184245.1
59.13
10.08
3.51
0.74


AD-1183937.1
59.99
5.18
29.11
16.30


AD-1183963.1
60.24
8.46
53.73
20.46


AD-1183942.1
60.39
11.40
26.33
8.27


AD-1184069.1
60.49
1.93
5.64
0.53


AD-1184289.1
60.67
1.35
20.43
17.67


AD-1184023.1
61.62
18.34
57.01
30.89


AD-1184185.1
62.17
11.09
64.99
62.12


AD-1184096.1
62.37
2.21
23.80
9.93


AD-1184197.1
62.64
17.03
57.02
9.62


AD-1184156.1
62.66
5.82
33.70
14.37


AD-1184013.1
62.74
2.59
27.30
20.05


AD-1184162.1
62.79
17.70
14.73
1.96


AD-1184240.1
63.69
3.43
58.69
22.89


AD-1183997.1
63.76
10.95
42.99
22.78


AD-1183957.1
65.30
19.84
39.22
5.07


AD-1183952.1
65.47
3.48
13.78
2.17


AD-1184076.1
65.58
17.26
28.83
2.54


AD-1184181.1
66.59
12.10
100.82
84.44


AD-1184068.1
67.28
16.70
12.10
4.59


AD-1184089.1
67.93
15.70
25.15
2.99


AD-1183941.1
67.94
1.68
36.29
6.11


AD-1184244.1
68.07
9.26
7.51
3.41


AD-1183965.1
69.26
9.89
25.51
8.59


AD-1184001.1
69.46
10.20
19.18
5.13


AD-1184145.1
72.78
31.76
79.63
52.62


AD-1183958.1
72.98
22.53
24.20
4.22


AD-1184134.1
73.15
13.38
68.22
25.29


AD-1183987.1
73.70
32.04
33.56
10.90


AD-1184153.1
75.12
42.83
132.33
72.85


AD-1183974.1
75.42
24.31
48.58
13.88


AD-1184262.1
77.77
12.69
10.06
1.53


AD-1184000.1
78.82
13.60
61.86
32.12


AD-1184241.1
78.94
8.87
46.74
4.99


AD-1184288.1
81.27
21.61
13.77
4.01


AD-1184095.1
82.94
23.57
22.66
5.47


AD-1183939.1
85.76
3.23
40.92
18.61


AD-1184022.2
86.03
4.66
48.90
2.36


AD-1184287.1
86.16
15.61
4.36
2.08


AD-1183994.1
87.40
24.94
91.29
76.63


AD-1184007.1
88.09
33.36
31.42
3.87


AD-1184155.1
88.35
42.97
20.59
2.82


AD-1184028.1
88.83
19.76
87.84
3.75


AD-1184025.1
90.53
20.18
54.27
23.14


AD-1183991.1
90.88
42.68
30.34
4.53


AD-1184025.2
92.98
12.32
50.82
18.55


AD-1184022.1
93.46
48.39
52.21
8.69


AD-1183960.1
97.09
73.05
27.29
2.02


AD-1184031.1
108.42
12.44
87.48
8.47


AD-1183988.1
110.39
51.89
98.51
74.09


AD-1183976.1
137.05
47.58
10.82
0.28
















TABLE 7







Single Dose In Vitro Screens in Cos-7 Cells














10 nM
10 nM
1 nM
1 nM
0.1 nM
0.1 nM


Duplex ID
Avg
STDEV
Avg
STDEV
Avg
STDEV
















AD-1231493.1
25.3
6.2
47.6
10.4
103.4
12.8


AD-1231505.1
21.6
11.7
18.4
6.3
72.0
26.6


AD-1231488.1
13.3
1.3
48.3
2.9
116.3
9.3


AD-1231486.1
11.5
4.3
25.9
3.9
98.8
33.2


AD-1231502.1
7.9
2.9
37.5
8.2
100.4
5.6


AD-1231478.1
30.9
18.8
53.0
9.9
75.0
6.4


AD-1231499.1
46.4
26.3
51.4
24.0
77.9
12.6


AD-1231470.1
8.0
6.3
15.7
7.4
78.4
15.8


AD-1231477.1
13.7
7.4
36.0
9.5
104.2
12.0


AD-1231515.1
51.9
6.6
79.3
15.4
113.3
24.4


AD-1231503.1
58.9
14.7
91.2
11.9
139.0
7.0


AD-1231479.1
15.4
3.7
50.2
13.1
123.5
24.1


AD-1231498.1
15.5
9.0
17.0
2.8
78.8
22.3


AD-1231483.1
16.7
3.0
31.0
11.8
78.9
2.7


AD-1231484.1
14.7
7.3
28.2
4.7
80.1
19.1


AD-1231506.1
45.8
13.2
52.3
5.1
112.1
14.4


AD-1231495.1
20.4
8.1
51.1
6.8
98.9
25.0


AD-1231516.1
109.2
32.1
129.8
28.9
102.8
22.3


AD-1231508.1
30.6
5.6
65.3
8.9
121.5
8.5


AD-1231475.1
8.7
4.3
14.8
4.0
70.9
12.4


AD-1231490.1
23.2
19.6
32.0
5.9
86.5
11.7


AD-1231494.1
42.3
9.1
89.6
9.6
128.1
16.4


AD-1231473.1
12.1
5.5
35.2
4.6
108.2
18.5


AD-1231496.1
8.5
3.6
51.5
10.9
112.9
11.9


AD-1231491.1
33.4
26.2
38.9
17.4
88.1
4.4


AD-1231469.1
46.2
8.2
37.9
5.2
82.2
14.7


AD-1231476.1
18.3
17.0
34.7
7.4
89.7
21.5


AD-1231481.1
10.6
1.0
36.2
6.6
107.7
6.3


AD-1231512.1
61.1
37.0
63.7
9.3
94.9
11.2


AD-1231471.1
12.1
5.3
26.7
7.9
99.6
24.4


AD-1231510.1
12.5
5.9
49.9
1.8
106.0
19.4


AD-1231472.1
11.3
6.2
29.0
7.1
80.3
19.1


AD-1231480.1
49.7
25.7
73.5
12.6
107.0
19.1


AD-1231492.1
36.8
24.7
73.8
15.3
105.4
40.6


AD-1231507.1
61.0
22.1
73.5
1.5
100.8
10.4


AD-1231517.1
87.4
18.0
99.2
25.5
109.4
25.0


AD-1231511.1
13.7
4.8
39.3
10.6
108.1
7.9


AD-1231513.1
60.8
12.9
70.9
11.0
98.8
13.1


AD-1231514.1
57.8
18.2
86.1
15.6
91.7
11.5


AD-1231468.1
8.9
0.1
46.8
18.4
93.5
19.1


AD-1231500.1
43.4
16.2
50.8
7.8
85.0
16.7


AD-1231497.1
15.1
13.6
24.9
2.8
80.1
18.9


AD-1231501.1
20.7
7.8
65.5
10.7
122.5
5.7


AD-1231467.1
35.9
8.6
62.0
15.1
102.7
10.7


AD-1231518.1
126.4
19.5
131.1
15.7
141.9
22.7


AD-1231504.1
9.6
6.0
29.1
12.9
89.7
16.4


AD-1231509.1
28.9
25.3
44.1
13.6
87.6
13.3


AD-1231482.1
28.0
10.4
54.0
3.5
136.1
31.3


AD-1231489.1
12.9
3.0
21.4
2.0
90.5
9.6


AD-1231474.1
59.0
26.0
66.1
17.5
86.9
19.8


AD-1231519.1
85.2
19.5
107.1
12.6
116.2
24.4


AD-1231485.1
22.8
17.8
42.3
3.9
80.5
21.0


AD-1231520.1
111.6
15.3
103.8
24.6
99.7
15.5


AD-1231487.1
22.5
9.9
51.5
14.2
96.5
17.8


AD-1231521.1
107.1
20.2
100.7
30.3
115.6
24.2









Example 3. In Vivo Screening of dsRNA Duplexes in Mice

siRNA molecules targeting the coronavirus genome, identified from the above in vitro studies, are evaluated in vivo.


Mice previously infected with a coronavirus, e.g., severe acute respiratory syndrome-2 (SARS-2)-CoV-2, are administered, via pulmonary system or subcutaneous delivery, a dsRNA molecule at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg. Uptake of dsRNA in bronchioles and alveoli and expression level of target gene in whole lung of treated mice are measured. Expression level of coronavirus target genes are further evaluated by in situ hybridization in mice bronchus and bronchiole.


Example 4. In Vitro SARS-CoV-2 Replication Screening of siRNA Duplexes

In Vitro screening of siRNAs in SARS-CoV-2 Infection Assay


Vero E6 cells cultured in DMEM supplemented with 10% FBS (VWR) and 1× Penicillin/Streptomycin (Thermo Fisher Scientific) were reverse transfected with siRNAs in white 96-well plates using RNAiMax transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Twenty-four hours later, cells were infected with SARS-CoV-2 (isolate USA-WA1/2020, passage 2, MOI 0.001) in a BSL-3 facility. The viral inoculum was removed after 4 hours, cells were washed with phosphate-buffered saline (PBS, pH 7.4) and media was replaced. At 48 hours post-infection, the cell supernatant was collected for RNA extraction and RT-qPCR analysis. The cells were fixed for in-cell ELISA analysis (below).


Quantification of Extracellular SARS-CoV-2 Genomes by RT-qPCR

Viral RNA was extracted from the cell culture supernatant using the NucleoSpin 96 Virus kit (Macherey-Nagel). Quantification of viral genomes was performed using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) with a primer/probeset binding in the M region (forward: CACTATTCTGACCAGACCGCTTCT (SEQ ID NO: 1956), reverse: CCTGCTACACGCTGCGAAG (SEQ ID NO: 1957), probe: TCACGAACGCTTTCTT (SEQ ID NO: 1958))). A standard curve of defined dilutions of a synthetic SARS-CoV-2 RNA (Twist Bioscience) was used for normalization.


Quantification of Intracellular Viral Nucleocapsid Protein by In-Cell ELISA

Cells were fixed with 4% paraformaldehyde for 30 minutes, followed by two PBS (pH 7.4) washes and permeabilization with 0.125% Triton X-100 in PBS for 30 min. After blocking in 2% milk powder/PBS for 30 minutes, cells were incubated with a primary antibody targeting SARS-CoV-2 nucleocapsid protein (Sino Biological, cat. 40143-R001) at a 1:3000 dilution for 1 hour. After washing and incubation with a secondary HRP-labeled antibody for 1 hour, SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific) was added to the cells and luminescence signal was read on an Ensight plate reader (Perkin-Elmer). After subtraction of background (uninfected) controls, all signals were normalized to the mock transfected controls.


Results

The RT-qPCR assay quantifying viral genome copy number based on the M (membrane) ORF, identified 47 siRNAs capable of reducing viral RNA ≥2-log10s at 10 nM, with 10 siRNAs reducing viral RNA ≥2-log10s at 0.1 nM. Reduction in viral RNA correlated with reduction in intracellular nucleocapsid protein. A total of 11 siRNAs had >80% reduction of viral nucleocapsid at a 0.1 nM dose. As expected, the control siRNA (targeting firefly luciferase transcripts) had no significant effect on SARS-CoV2 viral replication. The results of these assays are provided in Table 8 and FIG. 3.









TABLE 8







Single Dose In Vitro SARS-COV-2 Replication Screens











RT-qPCR
in-cell ELISA




(% mock transfected)
(% mock transfected)















Duplex ID
10 nM
1 nM
0.1 nM
10 nM
1 nM
0.1 nM
Target

















AD-1184106.1
0.12
0.21
3.46
8.5
8.6
24.0



AD-1184102.1
0.13
0.17
2.65
8.5
8.4
25.0



AD-1184136.1
0.17
41.04
25.26
9.6
77.7
54.9



AD-1184151.1
0.05
0.10
16.96
10.5
10.0
33.8



AD-1184130.1
0.14
0.05
36.95
11.0
9.5
63.4



AD-1184137.1
0.06
0.04
0.03
10.2
10.6
10.2
ORF1ab


AD-1184144.1
0.30
0.26
66.10
9.9
9.4
118.1



AD-1184189.1
0.05
0.06
33.02
7.7
7.6
84.5



AD-1184123.1
0.13
0.80
81.37
6.3
13.2
95.6



AD-1184168.1
0.08
0.08
33.20
8.4
7.5
70.2



AD-1184124.1
0.06
0.07
174.39
9.0
9.2
110.3



AD-1184131.1
18.05
45.93
48.79
47.1
78.8
81.0
(-)strand


AD-1184147.1
5.74
0.03
0.04
26.7
10.2
10.2
ORF1ab


AD-1184170.1
26.55
38.87
150.04
74.2
66.1
123.1
(-)strand


AD-1184199.1
12.44
38.26
185.16
47.2
91.5
119.0
(-)strand


AD-1184126.1
8.75
103.16
32.55
42.9
120.5
80.3
(-)strand


AD-1184148.1
0.08
0.08
27.76
8.8
9.4
74.0



AD-1184200.1
15.87
75.29
120.16
51.7
101.5
105.0
(-)strand


AD-1184128.1
26.30
93.02
225.36
71.9
94.9
112.0
(-)strand


AD-1184150.1
0.05
0.07
0.20
10.3
10.2
10.0
ORF1ab


AD-1184173.1
0.95
3.84
166.25
8.9
9.4
119.1



AD-1184202.1
0.74
30.76
51.06
10.7
61.0
87.7



AD-1184210.1
0.09
0.10
2.41
9.7
8.1
25.5
ORF3AE


AD-1184218.1
0.07
0.71
79.43
11.9
15.5
98.8



AD-1184232.1
0.29
0.10
22.09
9.1
10.5
46.1



siLuc
24.50
79.00
351.04
73.3
98.0
131.0



mock tr
116.89
35.49
79.73
102.3
72.8
116.8



mock tr
118.33
83.41
166.15
118.5
81.5
108.1



AD-1184255.1
5.15
33.33
50.18
47.0
87.9
88.6



AD-1184270.1
0.23
2.32
0.03
9.1
22.6
88.9
N


AD-1184211.1
0.09
0.39
28.21
10.1
13.9
89.8



AD-1184219.1
12.94
1.54
75.97
82.3
19.9
96.7



AD-1184226.1
0.06
0.60
22.51
9.3
14.4
81.1



AD-1184233.1
1.04
5.11
0.72
37.5
27.9
10.6
E


AD-1184256.1
89.78
45.86
31.97
148.0
76.4
83.1



AD-1184271.1
0.03
0.02
1.26
7.5
8.0
22.5
N


AD-1184286.1
0.26
2.20
38.59
9.3
19.5
86.2



AD-1184212.1
0.07
0.57
3.56
8.5
14.9
22.1
ORF3AE


AD-1184234.1
0.18
1.01
27.60
12.9
19.2
72.6



AD-1184213.1
0.04
0.04
10.47
7.4
8.8
55.0



AD-1184220.1
0.06
1.47
35.19
8.7
17.9
70.4



AD-1184227.1
0.10
1.02
29.57
7.8
13.4
80.7



AD-1184235.1
0.16
0.09
110.96
9.3
8.6
108.8



AD-1184272.1
0.04
0.03
4.82
7.7
7.6
50.5



AD-1184221.1
0.06
0.09
23.30
12.3
9.2
68.5



AD-1184228.1
0.10
0.56
1.75
7.3
15.6
15.4
E


AD-1184259.1
11.19
11.00
43.81
50.6
45.4
92.3



AD-1184215.1
0.09
0.81
15.71
9.8
24.5
59.9



AD-1184229.1
0.07
0.20
8.25
9.2
9.6
65.3



AD-1184237.1
0.04
0.07
37.98
8.5
8.8
88.0



AD-1184252.1
1.63
9.54
45.25
38.0
82.0
111.9



AD-1184260.1
53.62
25.57
6.91
117.5
65.9
119.8



AD-1184282.1
0.10
0.04
48.94
8.9
6.9
99.7



siLuc
6.59
8.32
72.62
45.3
32.0
104.8



mock tr
1.98
50.59
65.01
12.4
65.0
99.3



mock tr
132.78
85.04
166.58
114.7
103.5
117.6



AD-1184216.1
20.99
0.27
9.84
52.2
7.4
48.0



AD-1184223.1
0.38
0.02
16.14
8.4
10.1
32.1
E


AD-1184261.1
323.03
96.87
107.35
178.0
98.1
92.5



AD-1184268.1
88.37
no result
95.18
112.9
31.9
83.7



AD-1184283.1
0.09
3.19
99.85
8.3
17.7
93.3



AD-1184209.1
15.92
60.57
138.32
78.1
92.9
92.0



AD-1184217.1
0.18
1.82
38.03
12.6
14.0
79.4



AD-1184224.1
0.52
2.86
257.68
7.6
20.8
95.8



AD-1184254.1
156.78
31.46
109.25
121.2
45.3
83.7



AD-1184269.1
1.67
3.94
31.99
16.6
26.5
71.1



AD-1184284.1
0.36
0.07
25.81
7.1
7.1
42.5



AD-1231467.1
0.19
0.12
21.93
12.7
7.7
52.6



AD-1231475.1
0.06
no result
37.33
8.4
13.3
73.6



AD-1231482.1
0.11
no result
44.60
10.3
9.8
78.7



AD-1231490.1
0.14
0.07
0.06
7.6
7.5
6.3



AD-1231512.1
0.23
4.74
241.32
8.5
25.2
90.0



AD-1231520.1
113.26
146.18
54.93
107.1
90.5
58.9



AD-1231468.1
1.29
18.06
161.56
23.3
56.2
84.8



AD-1231476.1
0.22
0.23
75.68
8.8
7.6
57.7



AD-1231491.1
0.39
0.92
16.39
8.9
10.2
40.8



AD-1231506.1
0.45
1.96
194.85
13.0
16.6
104.6



AD-1231513.1
0.10
1.12
1.76
8.5
8.7
9.4



AD-1231521.1
130.20
106.72
399.69
114.6
85.9
101.9



AD-1231469.1
12.77
21.10
66.09
50.1
44.0
63.3



AD-1231484.1
0.08
0.11
72.20
7.2
9.1
107.4



siLuc
55.81
27.22
143.92
117.5
52.2
95.6



mock tr
102.03
78.63
199.25
88.9
95.0
126.0



mock tr
66.37
55.42
98.31
80.1
89.1
120.9



AD-1231492.1
0.28
0.78
70.62
9.4
11.0
86.6



AD-1231514.1
0.01
132.16
79.46
39.0
116.7
84.5



AD-1231485.1
0.16
no result
0.50
11.3
11.5
9.4



AD-1231500.1
56.30
85.87
10.97
108.2
108.1
42.2



AD-1231507.1
0.62
0.23
2.89
8.8
9.5
20.2



AD-1231471.1
0.13
0.22
0.16
8.1
8.0
8.3



AD-1231494.1
0.14
0.17
1.98
10.1
11.0
100.5



AD-1231501.1
0.11
no result
53.02
11.7
26.2
72.7



AD-1231508.1
0.25
2.32
no result
9.5
21.7
58.9



AD-1231516.1
32.57
50.07
119.43
50.4
77.9
86.8



AD-1231472.1
0.23
1.00
53.86
10.2
17.6
81.8



AD-1231480.1
0.18
12.95
52.14
12.2
60.2
90.8



AD-1231487.1
0.13
0.14
8.03
8.4
8.1
24.8



AD-1231495.1
10.79
no result
109.05
44.8
8.1
94.1



AD-1231509.1
0.58
2.36
46.14
9.7
16.1
61.0



AD-1231517.1
50.31
51.27
42.30
105.6
82.7
69.1



AD-1231473.1
0.46
20.26
55.45
19.6
48.4
73.3



AD-1231496.1
0.23
0.19
0.85
8.4
9.2
10.8



AD-1231510.1
0.15
12.10
57.03
10.3
41.1
70.3



AD-1231518.1
49.92
92.40
57.36
122.1
103.1
69.1



AD-1231474.1
no result
59.84
42.40
105.4
70.6
88.3



AD-1231497.1
0.19
0.24
0.16
5.4
7.7
8.5



AD-1231504.1
1.71
0.15
41.15
8.3
6.4
60.3



AD-1231511.1
no result
0.48
12.27
8.6
9.0
27.4



AD-1231519.1
23.98
24.46
113.17
76.6
78.7
124.1



siLuc
35.83
46.93
121.44
89.0
74.5
115.7



mock tr
112.40
0.16
60.57
84.8
4.8
79.7



mock tr
116.06
99.56
111.85
107.6
105.2
117.5









Example 5. Evaluation of siRNA Duplex Against Resistance Mutants

Because of their high mutation rates, viruses, such as SARS-CoV-2, have the potential to elude host defense systems as well as antiviral drugs and vaccines.


Accordingly, in order to identify duplexes, or combinations or duplexes, that are able to evade or delay the emergence of antiviral resistance, a resistance selection analysis was performed and, subsequently, agents targeting the coronavirus genome were assessed for their ability to inhibit expression and inhibit infection of host cells and infectious virus particles in a focus-forming assay using these resistant cells.


The materials and methods for this example are described below.


Resistance Selection Using Fixed Concentrations

The selection of variants in the presence of fixed concentrations of AD-1184150 or a combination of AD-1184150 with AD-1184137 was conducted in VeroE6 cells cultured in DMEM supplemented with 10% FBS (VWR) and 1× Penicillin/Streptomycin (Thermo Fisher Scientific). 1.2×105 VeroE6 cells per well in a 24-well plate were reverse transfected with 5×, 10× or 20× EC50 of AD-1184150 (330 pM, 660 pM or 1320 pM, respectively) or of an equimolar mixture of AD-1184150 and AD-1184137 at the same combined total concentrations. As non-targeting control, VeroE6 cells were reverse transfected with the highest concentration (1320 pM) of an siRNA targeting the luciferase gene. All transfections used RNAiMax transfection reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. Six hours post reverse transfection, cells were incubated with SARS-CoV-2 (isolate USA-WA1/2020, passage 2) at an MOI (multiplicity of infection) of 0.01 in infection medium (DMEM, 10% FBS) for 1 hour at 37° C. After viral adsorption, cells were washed with DMEM and overlaid with infection media. For each condition, three independent wells were transfected and infected, and the supernatants were pooled together at the time of harvest. Infected cells were monitored visually for cytopathic effect (CPE) daily. When the cells in the luciferase-targeting control wells exhibited >50% CPE, the culture supernatants were harvested, diluted 1:10 and added to fresh VeroE6 cells in 24-well plates that had been reverse-transfected with the equivalent amounts of siRNA(s) as used for the initial passage. Selection continued for a total of 5 passages. At each passage, supernatant was aliquoted and frozen at −80° C. for further analyses.


Determination of Viral Titer by Focus-Forming Assay

Viral titers were determined using a focus-forming assay (FFA) on VeroE6 cells. One day prior to infection, 1.5×104 VeroE6 cells were plated in black-walled, clear bottomed 96-well plates. The next day, undiluted or 4-point 10-fold serially diluted virus samples using 10% FBS-containing media were adsorbed onto VeroE6 cells for one hour at 37° C. The cells were washed once and overlaid with 1% methylcellulose (Sigma-Aldrich) in serum-containing medium. At 24 hours post-infection, the methylcellulose overlay was removed and cells were washed with PBS. Cells were fixed with 4% PFA, incubated for 30 minutes at room temperature, then washed with PBS to remove residual PFA. The cells were permeabilized with 100 μL of 0.25% Triton X-100 (Sigma-Aldrich) in PBS for 30 minutes at room temperature. The Triton X-100 was removed, cells were washed twice with PBS, and incubated with 50 μL of SARS-CoV-2 nucleocapsid (N) antibody (Sino Biological) at 1:2,000 in blocking buffer (2% milk powder/PBS) for 1 hour at room temperature. Plates were washed three times with PBS and then incubated for 1 hour at room temperature with 50 μL/well of goat anti-rabbit-Alexa647 (Thermo Fisher Scientific) secondary antibody at 1:1,000 in blocking buffer along with 1 μg/ml Hoechst33342 (Thermo Fisher Scientific). After washing three times with PBS, plates were imaged on a Cytation5 (BioTek) plate reader (12 images at 4× magnification) with fluorescence detected in DAPI (377,447 nm) and Cy5 (628,685 nm) channels. Nucleocapsid-positive foci were counted from images and used to determine focus-forming units/mL supernatant (FFU/mL).


Evaluation of Antiviral Activity Against Selected Virus

Cell supernatants from viral passages containing detectable virus as determined by FFA were evaluated for a shift in EC50 values in an siRNA antiviral activity assay. 7-point 5-fold serial dilutions of AD-1184150 and AD-1184137 were prepared in PBS and VeroE6 cells were reverse transfected in 96-well plates with each dilution in duplicates (range: 41250 to 3 pM final concentration). Twenty-four hours later, cells were infected with 20 FFUs of the virus-containing cell supernatants generated during passaging. The viral inoculum was removed after 1 hour and media was replaced. At 24 hours post-infection, the cells were fixed with 4% PFA and viral nucleocapsid protein was immunostained as described above. For automated image acquisition and quantification, cells were imaged on a Cytation 5 Cell Imaging reader with 12 images per well to cover the complete well. Nuclei and AlexaFluor647-positive cells were counted using the manufacturer's provided software. After subtraction of background (uninfected) controls, all signals were normalized to the mock transfected controls.


Sequencing of siRNA Binding Sites within the SARS-CoV-2 Genome


To isolate nucleic acid from the supernatant of viral passages, 300 μL of cell supernatant was added to 900 μL of Trizol and stored at −80° C. for further analysis. Trizol collected samples were subjected to RNA isolation using PureLink RNA Mini Kit with the incorporation of on-column PureLink DNase Treatment, following manufacturer's instructions. Reverse transcription reactions were performed with 6 μL of purified RNA and random hexamer primers using the NEB ProtoScript II First Strand cDNA Synthesis kit, according to manufacturer's instructions. The resulting cDNA was used as a template for PCR amplification of the siRNA binding sites, two primer pairs per site, using KapaBiosystems polymerase (KAPA HiFi HotStart ReadyMix) with primers, 5′-acgctgcttctggtaatc-3′ (SEQ ID NO: 1959) plus 5′-agaaacccttagacacagc-3′ (SEQ ID NO: 1960), and 5′-ctagataaacgcactacgtg-3′ (SEQ ID NO: 1961) and 5′-cctgagcaaagaagaagtg-3′ (SEQ ID NO: 1962) for the AD-1184150 binding site, and 5′-agatgccttcaaactcaac-3′ (SEQ ID NO: 1963) plus 5′-ttctactctgagttgttgc-3′ (SEQ ID NO: 1964), and 5′-ttggtggcaaaccttgtatc-3′ (SEQ ID NO: 1965) plus 5′-tgtgtaactggacacattg-3′ (SEQ ID NO: 1966) for the AD-1184137 binding site. Each PCR with a primer pair was carried out independently, then PCR products from each treatment were pooled for purification and subsequent library preparation. Amplification conditions included an initial 3 minutes at 95° C., followed by 24 cycles with 20 seconds at 98° C., 15 seconds at 60° C. and 72° C. for 15 seconds, with a final 1 minute at 72° C. Pooled PCR products were purified using AMPure XP beads following manufacturer's instructions. The size of the amplicon was confirmed by analyzing 2 μL of PCR products using the Agilent D1000 ScreenTape System. Products were quantified by analyzing 1 μL with the Quant-iT dsDNA High-Sensitivity Assay Kit. Fifty ng of purified PCR product was used as input for library construction using the NEBNext Ultra II DNA Library Prep kit following manufacturer's instructions. NEBNext Multiplex Oligos for Illumina Dual Index Primer Set 1 was used for library construction, with a total of 4 PCR cycles. Libraries size was determined using the Agilent D1000 ScreenTape System and quantified with the Quant-iT dsDNA High-Sensitivity Assay Kit. Equal amounts of each library were pooled together for multiplexing and ‘Protocol A: Standard Normalization Method’ of the Illumina library preparation guide was used to prepare 8 pM final multiplexed library with 1% PhiX spike-in for sequencing. The MiSeq Reagent Kit v3 (600-cycle) was used for sequencing the libraries on the Illumina MiSeq platform, with 150 cycles for Read 1, 150 cycles for Read 2, 8 cycles for Index 1, and 8 cycles for Index 2.


Sequence Analysis of siRNA Binding Sites


Paired-end reads were trimmed to 2×150 upon inspection with FastQC to retain segments of high quality (>Q30). Illumina adapters were clipped using Trimmomatic. Read alignment to SARS-CoV-2 Wuhan-Hu-1 reference (NCBI: NC_045512.2) was performed with Burrows-Wheeler Aligner (BWA). Variants were called with LoFreq* upon indel realignment and base quality recalibration, using a frequency threshold of 1%. Primers were excluded from variant calling. A consensus sequence was generated, mapped back to reference sequence coordinates and used to align reads a second time. The incorporation of variants from the first mapping iteration into a consensus sequence facilitates read alignment in case of mismatches with the reference. Variants were called again against the reference sequence from reads mapped to the consensus sequence. A third iteration of consensus generation read alignment and variant calling was performed. The variant call set from this last iteration was retained as final. Nucleotide variants were annotated with SnpEff to generate amino acid variants. Extensive QCs were performed at read, alignment and variant level using FastQC, samtools, picard, mosdepth, bcftools and MultiQC. An end-to-end workflow was automated using NextFlow. All programs are available through the Bioconda Initiative (bioconda.github.io).


Quantification of Extracellular SARS-CoV-2 Genomes by RT-qPCR

Viral RNA was extracted from the cell culture supernatant using the NucleoSpin 96 Virus kit (Macherey-Nagel). Quantification of viral genomes was performed using the Luna Universal Probe One-Step RT-qPCR Kit (New England Biolabs) with a primer/probeset binding in the orflab region (forward: CCCTGTGGGTTTTACACTTAA (SEQ ID NO: 1967), reverse: ACGATTGTGCATCAGCTGA (SEQ ID NO: 1968), probe: CCGTCTGCGGTATGTGGAAAGGTTATGG (SEQ ID NO: 1969)). A standard curve of defined dilutions of a synthetic SARS-CoV-2 RNA (Twist Bioscience) was used for normalization.


Results

Duplexes AD-1184137; AD-1184150; AD-1184151; AD-1184284; AD-1184212; AD-1231490; and the combinations of duplexes AD-1184137 and AD-1184150; AD-1184137 and AD-1184151; and AD-1184137, AD-1184150, AD-1184284, and AD-1184212 were assessed for their ability to inhibit coronavirus genome expression and focus formation in resistance selected cells.


As depicted in FIG. 4, all of the duplexes and duplex combinations inhibited expression of the SARS-CoV2 genome in the resistance selected cells and, as depicted in FIG. 5, all of the duplexes and duplex combinations inhibited SARS-CoV2 cell infection of the resistance selected cells. FIG. 6 is an exemplary immunofluorescence assay confirming the results of the q-PCR and focus-forming assays that the duplexes inhibit formation of viral particles in resistance selected cells. The duplexes that were most effective were AD-1184137 and AD-1184150, as was the combination of duplexes AD-1184137 and AD-1184150 (see Table 9 below and FIGS. 4 and 5).













TABLE 9






EC50
EC50
Genome
Genome



EC95
EC95
Reactivity*
Reactivity*


siRNA
(pM; PCR)
(pM; IFA)
(0 mm)
(1 mm)







AD-1184150
 42
 66
99.91%
100.00%



1183
763




AD-1184137
 86
118
99.89%
 99.98%



 702
608





*N = 4386 genomes analyzed






Example 6. Intranasal Delivery of siRNA Duplexes Prevents Coronavirus Infection
Experimental Design

To determine the efficacy of dsRNA agents administered intranasally, fifty-four (54) Male Syrian Golden hamsters, approximately 6-8 weeks of age were divided among seven groups, according to Table 10, below, in groups of 6 animals. Group 1 was a control group administered PBS via intranasal (IN) dosing on day −7 pre-challenge. Group 2 was a control group administered a dsRNA agent targeting luciferase via intranasal (IN) dosing on day −7 pre-challenge. Groups 3-6 were administered either a combination of AD-1184150 and AD-1184137, both targeting COVID-19, or an iRNA agent targeting ACE2, (see Table 10) via intranasal (IN) dosing on day −7 pre-challenge. Group 7 was administered a combination of AD-1184150 and AD-1184137, via subcutaneous (SQ) dosing on day −7 pre-challenge.


Animals were challenged on study day 0 with SARS-CoV-2 via the intranasal route. Animals were monitored to Day 7 post-challenge. Oral swabs were collected in the post-challenge period, days 1, 3, and 5. Terminal oral swabs, blood, and tissue collection occured on day 7 post-challenge.









TABLE 10







Experimental Design
















Dose at







each
Treatment


Group
N
Treatment
Route
Treatment
Days





1
6
PBS
IN
 0 mg/kg
SD −7


2
6
iRNA agent targeting
IN
30 mg/kg
SD −7




Luciferase





3
6
AD-1184150 + AD-1184137
IN
30 mg/kg
SD −7


4
6
iRNA agent targeting ACE2
IN
30 mg/kg
SD −7


5
6
AD-1184150 + AD-1184137
IN
10 mg/kg
SD −7


6
6
AD-1184150 + AD-1184137
IN
 1 mg/kg
SD −7


7
5
AD-1184150 + AD-1184137
SQ
30 mg/kg
SD −7









For animals receiving a combination of AD-1184150 and AD-1184137, the two duplexes were mixed together and the weight administered to each animal, as indicated in Table 10, is the total weight of the mixture of the two duplexes.


Each animal received a dose volume for IN dosing of 100 μl per animal (50 μl per nostril) or 200 μl per animal for SQ dosing.


Virus Challenge with SARS-CoV-2


The intranasal inoculation (IN) was performed on Ketamine/Xylazine anesthetized hamsters. Administration of virus was conducted as follows: using a calibrated P200 pipettor, 50 μL of the viral inoculum was administered dropwise into each nostril, for a total of 100 μL per animal. Anesthetized animals were held upright such that the nostrils of the hamster were pointing towards the ceiling. The tip of the syringe was placed into the first nostril and virus inoculum was slowly injecting into the nasal passage, and then removed. This was repeated for the second nostril. The animal's head was tilted back for about 20 seconds and then returned to its housing unit and monitored until fully recovered.


Body weights were determined each day post-challenge through Day 7 post-challenge to assess the effectiveness of the duplexes as assessed by the weight of the animals.


The results are provided in FIGS. 7 and 8 and demonstrate that intranasal administration of a single 10 mg/kg dose or 30 mg/kg dose of the combination of AD-1184137 and AD-1184150 prevents SARS-CoV-2 infection as demonstrated by the maintenance of the weights of the hamsters (FIG. 7) and that intranasal administration of a single 30 mg/kg dose of the combination of AD-1184137 and AD-1184150 is superior to subcutaneous administration of a single 30 mg/kg dose of the combination of AD-1184137 and AD-1184150 in preventing SARS-CoV-2 infection (FIG. 8).


EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims
  • 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of coronavirus genome in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.
  • 2. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of coronavirus genome in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:2, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:2, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.
  • 3.-5. (canceled)
  • 6. The dsRNA agent of claim 1 or 2, wherein the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-5.
  • 7.-15. (canceled)
  • 16. The dsRNA agent of claim 1 or 2, wherein the dsRNA agent comprises at least one modified nucleotide.
  • 17.-24. (canceled)
  • 25. The dsRNA agent of claim 1 or 2, wherein each strand is no more than 30 nucleotides in length.
  • 26.-81. (canceled)
  • 82. A cell containing the dsRNA agent of claim 1 or 2.
  • 83. A pharmaceutical composition for inhibiting expression of a coronavirus genome, comprising the dsRNA agent of claim 1 or 2.
  • 84. (canceled)
  • 85. A composition comprising two or more of the double stranded RNAi agents of claim 1 or 2.
  • 86.-89. (canceled)
  • 90. The composition of claim 80 or 81, wherein each of the sense strands or each of the antisense strands is a sense strand or an antisense strand independently selected from the group consisting of any of the sense strands and antisense strands in any one of Table 2-5.
  • 91.-167. (canceled)
  • 168. A method of inhibiting expression of a coronavirus genome in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of claim 1 or 2; and(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the coronavirus genome, thereby inhibiting expression of the coronavirus genome in the cell.
  • 169.-177. (canceled)
  • 178. A method of treating a subject having a coronavirus infection, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1 or 2, thereby treating said subject.
  • 179.-182. (canceled)
  • 183. The method of claim 178, wherein the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.
  • 184. The method of claim 178, wherein the administration of the dsRNA is pulmonary system administration.
  • 185. (canceled)
  • 186. The method of claim 178, wherein the dsRNA agent is administered to the subject subcutaneously.
  • 187.-204. (canceled)
RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 17/321,561, filed May 17, 2021, which is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2021/024038, filed on Mar. 25, 2021, which, in turn, claims the benefit of priority to U.S. Provisional Application No. 62/994,907, filed on Mar. 26, 2020; U.S. Provisional Application No. 63/001,580, filed on Mar. 30, 2020; U.S. Provisional Application No. 63/019,481, filed on May 4, 2020; and U.S. Provisional Application No. 63/124,910, filed on Dec. 14, 2020. The entire contents of each of the foregoing applications are incorporated herein by reference.

Provisional Applications (4)
Number Date Country
63124910 Dec 2020 US
63019481 May 2020 US
63001580 Mar 2020 US
62994907 Mar 2020 US
Continuations (2)
Number Date Country
Parent 17321561 May 2021 US
Child 17523030 US
Parent PCT/US2021/024038 Mar 2021 US
Child 17321561 US