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WO2009143345A2 - Conjugués agent-protéine désactivant des acides nucléiques et leur utilisation pour traiter des troubles liés au vhc - Google Patents

Conjugués agent-protéine désactivant des acides nucléiques et leur utilisation pour traiter des troubles liés au vhc Download PDF

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Publication number
WO2009143345A2
WO2009143345A2 PCT/US2009/044840 US2009044840W WO2009143345A2 WO 2009143345 A2 WO2009143345 A2 WO 2009143345A2 US 2009044840 W US2009044840 W US 2009044840W WO 2009143345 A2 WO2009143345 A2 WO 2009143345A2
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WIPO (PCT)
Prior art keywords
conjugate
rna
antibody
protein
hcv
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PCT/US2009/044840
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English (en)
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WO2009143345A3 (fr
Inventor
Gregory J. Babcock
Donna M. Ambrosino
Robert W. Finberg
Phillip D. Zamore
Teresa Broering
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University Of Massachusetts
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Publication of WO2009143345A3 publication Critical patent/WO2009143345A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • HCV Hepatitis C virus
  • NANBH non-A non-B hepatitis
  • HCV Hepatitis C virus
  • the polyprotein is processed by host cell and viral proteases into three major structural proteins and several non-structural proteins necessary for viral replication.
  • the nucleotide sequence of HCV is highly variable; the most divergent isolates sharing only 60% nucleotide sequence homology. Several different genotypes of HCV with slightly different genomic sequences have been identified.
  • Genotypes 1-3 account for almost all infections in Europe, genotype 4 is prevalent in Egypt and Zaire, genotype 5 in South Africa and genotype 6 in Hong Kong. The virus is transmitted primarily by blood and blood products. The majority of infected individuals have either received blood transfusions prior to 1990 (when screening of the blood supply for HCV was implemented) or have used intravenous drugs. Approximately 200 to 400 million people worldwide are chronically infected with HCV and of those infected about 2 to 5 million reside in the United States. Chronic HCV infection occurs in approximately 80% of those infected and often leads to the development of cirrhosis and liver cancer.
  • the present invention solves the foregoing problems by providing a method of using a complex or a molecule comprising a targeting moiety (e.g., a therapeutic targeting moiety) to deliver therapeutic RNA molecules effectively to HCV or to a cell being infected or having the potential to be infected by HCV, resulting in the prevention, immunization, inhibition, cure, or other beneficial effects towards HCV infection.
  • a targeting moiety e.g., a therapeutic targeting moiety
  • the agents of the invention are not limited to those useful for the treatment of HCV, however.
  • the invention includes any agent (e.g., any complex or conjugate) comprising a targeting moiety (e.g., a therapeutic targeting moiety) and a nucleic acid molecule, wherein the agent delivers the nucleic acid molecule to a target cell of interest (e.g., a tumor cell, immune cell, or a cell infected with a virus other than HCV).
  • a targeting moiety e.g., a therapeutic targeting moiety
  • nucleic acid molecule e.g., a target cell of interest
  • a target cell of interest e.g., a tumor cell, immune cell, or a cell infected with a virus other than HCV.
  • the agent is a conjugate formed by a nucleic acid and a protein (e.g., a therapeutic protein or non-therapeutic (i.e., carrier) protein).
  • the protein is a binding protein.
  • the binding protein binds (e.g., binds to a target molecule) and modulates the activity of said target molecule.
  • the binding protein in the conjugate is a fusion protein.
  • One or more nucleic acid molecules e.g., about 2, about 5, about 10, about 15, about 20, about 25, or about 30 nucleic acid molecules
  • the nucleic acid and protein may be covalently linked, e.g., via an intervening linker.
  • the protein of the conjugate comprises a sulfhydryl moiety.
  • the nucleic acid is a double stranded nucleic acid
  • the nucleic acid is preferably bound to the the protein via a terminal nucleotide of the sense strand, more preferably the 3' terminus.
  • the nucleic acid of the conjugate comprises a carbonyl moiety at the 3' or 5' terminus.
  • the carbonyl moiety is present in a modified terminal ribonucleotide.
  • the nucleic acid molecules are linked to modified lysine residues within the protein.
  • the nucleic acid molecules do not interfere with the binding affinity of the binding protein when conjugated to the binding protein.
  • the nucleic acid molecules may be conjugated to residues located within the constant domains of an (e.g., within the Fc domain, e.g., hinge, CH2, and/or CH3 domains, or within the CHl or CL domains of the Fv domain) or within portions of the 5 variable region frameworks which are not critical for binding.
  • the protein and the nucleic acid are linked by a heterobifunctional crosslinker to form the conjugate.
  • the heterobifunctional crosslinker links the sulfhydryl and carbonyl moieties to form the conjugate.
  • the crosslinker is bonded (e.g., via a hydrazide moiety) o to the carbonyl moiety of the nucleic acid (e.g., a terminal ribonucleotide comprising a carbonyl group, e.g., an aldehyde-modified ribonucleotide) via a hydrazone bond.
  • the crosslinker is bonded to an amino moiety of the nucleic acid (e.g., a terminal nucleotide comprising an amino group, e.g., a terminal nucleotide comprising a primary amine (e.g., an amino alkyl phosphoester) at the 2' or 3' position).5
  • the crosslinker e.g., the maleimide moiety of a crosslinker
  • the sulfhydryl moiety of the protein e.g., a modified lysine residue comprising a sulfhydryl moiety
  • the crosslinker comprises a maleimide moiety and a hydrazide moiety, linked by a spacer moiety.
  • the linker is a0 heterobifunctional crosslinker, selected from the group consisting of N-( ⁇ - maleimidocaproic acid) hydrazide, N-( ⁇ -maleimidopropionic acid) hydrazide and N-( ⁇ - maleimidoundecanoic acid) hydrazide, including EMCH, BPMH, KMUH and other linkers containing a maleimide and a hydrazide.
  • the heterobifunctional linker comprises a hydrozone bond formed by a SANH moiety (S-5 HyNic) (Solulink, http://www.solulink.com/products/solulink-cross-linkers-s-1002- 105.php) and a SFB moiety.
  • SANH moiety S-5 HyNic
  • SFB moiety is covalently bonded to an amino moiety of the nucleic acid.
  • the agent of the invention comprises a protein which is a binding protein, e.g., an antigen binding protein, e.g., an antibody or fragment thereof,0 which binds to a target molecule target and thereby recruits the nucleic acid to the target molecule.
  • the binding protein is a human, humanized or chimeric antibody.
  • the binding protein is a fusion protein.
  • the binding protein is an antibody variant selected from the group consisting of a multispecific antibody, a multivalent antibody, a nanobody and a single chain antibody.
  • the binding protein in the conjugate is an antibody or antigen binding portion that specifically binds to one or more protein epitopes within HCV or a HCV-related hepacivirus.
  • the protein is an antibody or antigen binding portion that specifically binds to one or more epitopes within the E2 protein of HCV or a HCV-related hepacivirus.
  • the protein in the conjugate is an antibody or antigen binding portion thereof that inhibits the ability of the virus to infect cells.
  • the protein in the conjugate is a human, humanized or chimeric antibody.
  • the antibody competes therewith or binds other HCV-associated antigens.
  • the antibody or antigen binding portion binds an epitope within amino acid residues 412-464, 412-423, or 413- 420 of the E2 protein.
  • the binding protein is an antibody variant selected from the group consisting of a multispecific antibody, a multivalent antibody, a nanobody and a single chain antibody.
  • the conjugate binds to two or more HCV genotypes of an HCV E2 protein or fragment thereof.
  • the conjugate binds an HCV E2 protein from a HCV virus of the Ia, Ib, 2b, 3a, 4a, 5, 5a, 6a, 6g or 6k genotypes, or combinations thereof.
  • the binding protein is a fusion protein.
  • the protein, antibody or antigen binding portion thereof in the conjugate specifically binds to the E2 protein, or fragment thereof, of HCV with a K D of at least about 1 x 10 ⁇ 7 M, 1 x 10 ⁇ 8 M, 1 x 10 ⁇ 9 M, 1 x 10 ⁇ 10 M, 1 x 10 "11 M, 1 x 10 " 12 M or better.
  • the antibody is one of the following human monoclonal antibodies: 95-2, 83-128, 95-2, 83-128, 95-14, 95-15, 95-18, 95-20, 95-21, 95-25, 95- 26, 95-30, 95-38, 95-39, 95-42, 95-43, 95-48, 95-49, 95-52, 95-54, 95-58, and 95-62.
  • the antibody is the 95-2 or the 83-128 human monoclonal antibody.
  • Some of the embodiments of the invention feature antibodies with a heavy chain variable region that is encoded from gene VH 3-33.
  • the light chain variable region of the antibody is encoded from gene VK L6.
  • the RNA in the conjugate inhibits replication of HCV.
  • the RNA in the conjugate is a target mRNA is encoded by the genome of HCV.
  • the nucleic acid in this conjugate is modified to comprise a free carbonyl moiety at the 3' or 5' terminus.
  • the nucleic acid is a nucleic acid silencing agent.
  • the protein and nucleic acid are linked by a heterobifunctional crosslinker linking the sulfhydral and carbonyl moieties to form the RNA-protein conjugate.
  • the nucleic acid of an agent of the invention is a nucleic acid with gene silencing activity (e.g., a nucleic acid silencing agent).
  • exemplary nucleic acid silencing agents include DNA and RNA silencing agents.
  • the nucleic acids employed in the invention are preferably modified to comprise one or more nucleotide analogs (e.g., 2'0Me, LNA, and/or phosphorothiate modifications) which, for example, stabilize the nucleic acids from degradation in vivo and/or improve delivery to a target antigen, target RNA, or target cell in vivo).
  • the nucleic acids may be single- stranded (e.g., ssRNA) or double- stranded (e.g., dsRNA).
  • the agent RNA silencing agent comprises sense and antisense strands of 18-30 nucleotides in length.
  • the RNA silencing agent comprises sense and antisense strands of 21-23, 25-30, and preferably 27 nucleotides in length.
  • the RNA silencing agent comprises sense and antisense strands of 21-23 nucleotides in length.
  • RNA silencing agents may be selected from the group consisting of an siRNA, an miRNA, and an shRNA.
  • the nucleic acid is an inhibitor of RNA silencing (e.g., a DNA or RNA antisense oligonucleotide with complementarity to a miRNA, i.e., an antagomir).
  • the nucleic acid (e.g., a dsRNA) comprises a terminal ribonucleotide (e.g., at the 3' end of a sense strand).
  • the agent of the invention is internalized by receptor-mediated endocytosis.
  • the nucleic acid of the agent is released by acid hydrolysis following internalization.
  • the nucleic acid of the agent is not released following internalization.
  • a nucleic acid of the agent is released via the loss of the annealing of the nucleic acid to another nucleic acid of the agent.
  • a nucleic acid of the agent remains bound to the protein of the agent after the release of another nucleic acid of the agent.
  • the RNA exhibits greater than 50% silencing efficiency (e.g., 60%, 70%, 80%, 90% or more) of the target mRNA in a silencing assay relative to a control RNA.
  • the agents of the invention comprise a nucleic acid having a sequence that is substantially complementary to a target RNA (e.g., target mRNA or miRNA) associated with HCV to silence the target mRNA in a cell infected with the virus.
  • a target RNA e.g., target mRNA or miRNA
  • the nucleic acid inhibits replication of HCV.
  • the nucleic acid inhibits the ability of the virus to infect cells.
  • the nucleic acid targets an RNA encoded by the genome of HCV.
  • the nucleic acid targets the genome of HCV itself.
  • the nucleic acid targets an mRNA encoding the El and/or E2 glycoprotein of HCV.
  • the nucleic acid exhibits greater than 50% silencing efficiency of the target mRNA in a HCV infection assay relative to a control RNA.
  • the RNA in the conjugate inhibits HCV infection in vitro.
  • the RNA in the conjugate inhibits HCV replication in vivo.
  • the RNA in the conjugate treats HCV- mediated disease in vivo in a mammal.
  • the RNA protects from or inhibits HCV-mediated hepatocyte pathology in a subject.
  • the conjugate is used to treat HCV-mediated pathology in vivo in a mammal.
  • the agents of the invention further comprise at least a second nucleic acid (e.g., a second siRNA) linked to the protein of the agent, e.g., by a heterobifunctional crosslinker.
  • the second nucleic acid comprises a sequence that is substantially complementary to a different HCV-associated target mRNA than the first nucleic acid.
  • the crosslinker linking the first nucleic acid to the protein and the crosslinker linking the second nucleic acid to the protein may be the same or different.
  • the nucleic acid silencing agent-binding protein conjugate comprises a binding protein that specifically binds to one or more protein epitopes within a HCV or HCV-related hepacivirus and a nucleic acid silencing agent having a sequence that is substantially complementary to a target mRNA associated with HCV or a HCV-related hepacivirus to silence the target mRNA in a cell infected with the virus.
  • the RNA silencing agent is selected from the group consisting of a siRNA, a miRNA, a shRNA and an antagomir.
  • the protein and nucleic acid are linked by a heterobifunctional crosslinker to form the conjugate.
  • the binding protein in the conjugate comprises a free sulfhydryl moiety, while the nucleic acid silencing agent is modified to contain a free carbonyl moiety at the 3' or 5' terminus, and the heterobifunctional crosslinker links the sulfhydryl and carbonyl moieties to form the RNA-protein.
  • the nucleic acid silencing agent in the conjugate is a RNA silencing agent.
  • the protein and the nucleic acid in the conjugate is linked together by a crosslinker.
  • the crosslinker comprises a maleimide moiety and a hydrazide moiety, linked by a spacer moiety.
  • a maleimide moiety of a crosslinker reacts with a free sulfhydryl group on the antibody, or antigen binding portion thereof, to form a thioester bond.
  • the free sulfhydryl group on the antibody, or antigen binding portion thereof is added to lysines via a thiolation agent.
  • the thiolation agent is 2-Iminothiolane (Traut's Reagent).
  • the linker is a heterobifunctional crosslinker, selected from the group consisting of N-( ⁇ -maleimidocaproic acid) hydrazide, N-( ⁇ - maleimidopropionic acid) hydrazide and N-( ⁇ -maleimidoundecanoic acid) hydrazide, including EMCH, BPMH, KMUH and other linkers containing a maleimide and a hydrazide.
  • the hydrazide moiety of the crosslinker reacts with a carbonyl group on the RNA to form a hydrazone bond.
  • the carbonyl group on the 3 ' or 5' end of the RNA is added by chemical means.
  • the carbonyl group on the 3' end of the RNA is formed by reaction with sodium periodate.
  • the carbonyl group on the 5' is introduced by standard RNA synthesis techniques, e.g. chemically synthesized phosphoramidites.
  • the carbonyl group on the 5' is introduced by a chemical cross linker containing a carbonyl at one end and an NHS-ester at the other in which the NHS ester is reactive with an amine group at the 5' end of RNA generated by standard RNA synthesis techniques.
  • the RNA is double stranded.
  • the RNA comprises sense and antisense strands of 18-30 nucleotides in length.
  • the RNA comprises sense and antisense strands of 21-23, 25-30, and preferably 27 nucleotides in length.
  • the RNA comprises sense and antisense strands of 21-23 nucleotides in length and contains 2 nucleotide 3' overhangs or blunt-ended.
  • the RNA comprises an antisense strand that is complimentary to target sequences in the mRNA encoding E2 protein from HCV isolates.
  • the conjugate is internalized by receptor-mediated endocytosis.
  • the RNA silencing agent is released from the conjugate by acid hydrolysis.
  • the RNA exhibits greater than 50% silencing efficiency of the target mRNA in a silencing assay relative to a control RNA.
  • the conjugate further comprises at least a second RNA linked to the protein, antibody or antigen binding portion by a heterobifunctional crosslinker, wherein the second RNA silencing agent has a sequence that is substantially complementary to a second HCV-associated target mRNA in the HCV- infected cell to mediate cleavage of the second target mRNA.
  • the crosslinker in the conjugate linking the first RNA silencing agent and the crosslinker linking the second RNA silencing agent are the same. In another embodiment, the crosslinker linking the first RNA silencing agent and the crosslinker linking the second RNA silencing agent are different.
  • the RNA in the conjugate inhibits HCV infection in vitro. In another embodiment, the RNA in the conjugate inhibits HCV replication in vivo. In one preferred embodiment, the RNA in the conjugate treats HCV-mediated disease in vivo in a mammal. In another embodiment, the RNA protects from or inhibits HCV- mediated hepatocyte pathology in a subject. In one embodiment, the conjugate is used to treat HCV-mediated pathology in vivo in a mammal.
  • the invention also includes a method of treating or preventing a HCV-associated disease or disorder in a subject comprising, administering to the subject the RNA- protein conjugate of any of the preceding claims in an amount effective to treat or prevent a symptom or disorder associated with HCV infection.
  • the conjugate is administered in combination with a second therapeutic agent.
  • the second agent is an antiviral agent.
  • said second therapeutic agent is administered sequentially with the conjugate.
  • said second therapeutic agent is administered prior to the administration of the conjugate.
  • the subject is resistant to anti-viral therapy.
  • the subject has received a liver transplant.
  • This invention also features a pharmaceutical composition suitable for treating a HCV-mediated disease or disorder in a subject comprising the conjugate in a pharmaceutically acceptable carrier.
  • the composition further comprises a second therapeutic agent.
  • the conjugate in the composition is present in the composition in an amount that is less than 50% (e.g., less than about 20%, about 10%, or about 5%) than the amount of the second therapeutic agent.
  • This invention also feasures a method of making an agent of the invention (e.g., nucleic acid-binding protein conjugate).
  • a method includes, for example, a method providing a binding protein comprising a free sulfhydryl group, a method providing a nucleic acid comprising a modified ribonucleotide at a 3' or 5' terminus of the nucleic acid, wherein the nucleic acid comprises a nucleic acid strand that is sufficiently complementary to a target mRNA to silence expression of the target mRNA, and wherein the modified ribonucleotide comprises a modified carbonyl moiety, and a method combining the binding protein and the nucleic acid with a heterobifunctional linker under condition such that the binding protein and the nucleic acid are linked by the heterobifunctional crosslinker to form the nucleic acid-binding protein conjugate, etc.
  • the method includes, for example, (i) providing a binding protein comprising a free sulfhydryl group, (ii) providing a nucleic acid comprising a modified ribonucleotide at a 3' or 5' terminus of the nucleic acid, wherein the nucleic acid comprises a nucleic acid strand that is sufficiently complementary to a target mRNA to silence expression of the target mRNA, and wherein the modified ribonucleotide comprises a free carbonyl group (e.g., an aldehyde-modified ribonucletide), and (iii) combining the binding protein and the nucleic acid with a heterobifunctional linker under condition such that the binding protein and the nucleic acid are linked by the heterobifunctional crosslinker to form the nucleic acid-binding protein conjugate.
  • a binding protein comprising a free sulfhydryl group
  • a nucleic acid comprising a modified ribonucleot
  • the crosslinker comprises a maleimide moiety and a hydrazide moiety, linked by a spacer moiety.
  • a maleimide moiety of a crosslinker reacts with the free sulfhydryl group on the binding protein to form a thioester bond.
  • the free sulfhydryl group is added to a lysine residue of the binding protein via a thiolation agent.
  • the thiolation agent is 2-Iminothiolane (Traut's Reagent).
  • the heterobifunctional crosslinker is selected from the group consisting of N-( ⁇ - maleimidocaproic acid) hydrazide, N-( ⁇ -maleimidopropionic acid) hydrazide and N-( ⁇ - maleimidoundecanoic acid) hydrazide, including EMCH, BPMH, KMUH and other linkers containing a maleimide and a hydrazide.
  • the hydrazide moiety of the crosslinker reacts with the free carbonyl group of the nucleic acid to form a hydrazone bond.
  • the free carbonyl group is formed by oxidizing the modified ribonucleotide with sodium periodate.
  • the modified ribonucleotide is at the 3' terminus of the nucleic acid.
  • the free carbonyl group is introduced by chemically synthesis with phophoramidites.
  • the free carbonyl group is introduced by a chemical cross linker containing a carbonyl at one end and an NHS-ester at the other in which the NHS ester is reactive with an amine group at the 5' end of RNA generated by standard RNA synthesis techniques.
  • the modified ribonucleotide is at the 5' terminus of the nucleic acid.
  • Figures IA and IB are graphs showing binding of human antibody 95-2 ( Figure IA) and human antibody 83-128 (Figure IB) to soluble HCV genotype Ia E2-660 (triangles) and soluble HCV genotype Ib E2-661 (squares).
  • Figure 2A and 2B are graphs showing neutralization of infection of Hep3B cells with various HCV genotype pseudovirus in the presence of human antibodies 95-2, 83- 128, and 17C7 (a control human mAb in these experiments).
  • Figure 3A and 3B are Western blots showing antibody binding to purified soluble mammalian expressed E2 protein (E2-661) from HCV genotype Ia and Ib subjected to reducing ( Figure 3A) or non-reducing ( Figure 3B) SDS-PAGE followed by transfer to PVDF membrane.
  • E2-661 purified soluble mammalian expressed E2 protein
  • Figure 4 is is a table showing the affinity of human antibodies 95-2 and 83-128 to HCV E2 412-423 epitope expressed as a bacterial fusion protein.
  • Figure 5 is a schematic representation of the succinimidyl 4-formylbenzoate (SFB) modification of the oligo with a N6 linker.
  • SFB succinimidyl 4-formylbenzoate
  • Figure 6 shows the modification of the oligonucleotide with SFB.
  • Figure 7 shows the comparison between the duplex oligo and the single strand oligo in modifications with SFB.
  • Figure 8 shows that the oligo modification with SFB can be detected by the 2- HP method, while the antibody modification with succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH) can be detected by 4-NB method.
  • SAH succinimidyl 4-hydrazinonicotinate acetone hydrazone
  • Figure 9 shows that 2-HP results are comparable with electrophoresis in analyzing oligo modification with SFB.
  • Figure 10 shows the IC 50 of SFB modification of GFPz Stable 6 oligo
  • Figure 11 is a schematic representation of the formation of a hydrazone bond by conjugation of SFB -modified oligo and SANH-modified antibody.
  • Figure 12 is a schematic representation of the 2-day conjugation protocol.
  • Figure 13 is a schematic representation of the detection protocol of conjugates.
  • Figure 14 shows early non-optimized data for antibody-DNA/RNA duplex conjugation.
  • Figure 15 shows improved conjugation efficiency with fully modified DNA- SFB.
  • Figure 16 shows optimal conjugation with modified oligo.
  • Figure 17 shows optimization data for conjugation, detected by nucleic acid staining.
  • Figure 18 shows optimization data for conjugation, detected by protein staining.
  • Figure 19 shows the antibody-GFPz Stable 2 siRNA conjugation.
  • Figure 20 is a schematic representation of 3' oxidation of siRNA.
  • Figure 21 shows the optimization of oxidation reaction.
  • Figure 22 shows 3' oxidation of siRNA under varying pH conditions.
  • Figure 23 shows that 25 mM sodium periodate is sufficient to oxidize up to at least 100 uM siRNA.
  • Figure 24 is a schematic representation of reactions of oxidized siRNA with the linker, 3,3'-N-[e-Maleimidocaproic acid] hydrazide, trifluoroacetic acid salt (EMCH).
  • EMCH trifluoroacetic acid salt
  • Figure 25 shows the reaction of oxidized siRNA with the EMCH linker.
  • Figure 26 shows the optimization of oxidation of siRNA and reaction with EMCH in a single tube.
  • Figure 27 is a schematic representation of HPLC purification of siRNA-EMCH conjugate.
  • Figure 28 shows retention time for different component samples.
  • Figure 29 shows HPLC elution map of different components.
  • Figure 30 is a schematic representation of modification of lysine residues on the antibody by Traut' s reagent.
  • Figure 31 shows modification of lysine residues on the antibody with different amounts of Traut' s reagent.
  • Figure 32 shows that antibody modification does not significantly alter its antigen recognition.
  • Figure 33 is a schematic representation of conjugation of siRNA to a human monoclonal antibody.
  • Figure 34 shows the antibody-siRNA conjugation and detection on a native gel.
  • Figure 35 shows the detection of the antibody-siRNA conjugation using different staining methods.
  • Figure 36 shows the detection of the antibody-siRNA conjugation using isoelectric focusing (IEF) gel and staining.
  • Figure 37 depicts the alignment of the heavy chain variable region of antibodies derived from clones 83-128 (SEQ ID NO:1), 95-2 (SEQ ID NO:3), 95-14 (SEQ ID NO:32), 95-38 (SEQ ID NO:33), 95-25 (SEQ ID NO:34), 95-42 (SEQ ID NO:35), 95- 43 (SEQ ID NO:36), 95-49 (SEQ ID NO:37), 95-54 (SEQ ID NO:38), 95-58 (SEQ ID NO:39) and 95-62 (SEQ ID NO:40) and which are reactive against epitope 412-423 of E2-660 HCV glycoprotein.
  • Figure 38 depicts the alignment of light chain variable region of antibodies derived from clones 83-128 (SEQ ID NO:2), 073-1 (SEQ ID NO:6), 95-2 (SEQ ID NO:4), 95-14 (SEQ ID NO:44) and 95-38 (SEQ ID NO:53).
  • RNA silencing refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein-coding gene.
  • RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
  • target gene is a gene whose expression is to be substantially inhibited or "silenced.” This silencing can be achieved by RNA silencing, e.g. by cleaving the mRNA of the target gene or translational repression of the target gene.
  • non-target gene is a gene whose expression is not to be substantially silenced.
  • the polynucleotide sequences of the target and non-target gene e.g. mRNA encoded by the target and non-target genes
  • the target and non-target genes can differ by one or more polymorphisms.
  • the target and non-target genes can share less than 100% sequence identity.
  • the non-target gene may be a homolog (e.g. an ortholog or paralog) of the target gene.
  • RNA silencing agent refers to an RNA which is capable of inhibiting or “silencing" the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post- transcriptional silencing mechanism.
  • RNA silencing agents include small ( ⁇ 50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, and dual-function oligonucleotides as well as precursors thereof.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • nucleoside refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar.
  • Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine.
  • nucleosides include inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2 N-methylguanosine and 2 ' 2 N,N-dimethylguanosine (also referred to as "rare" nucleosides).
  • nucleotide refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety.
  • Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.
  • polynucleotide and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester linkage between 5' and 3' carbon atoms.
  • RNA or "RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides.
  • DNA or “DNA molecule” or deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized.
  • DNA and RNA can be single- stranded (i.e., ssRNA and ssDNA, respectively) or multi- stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively).
  • mRNA or “messenger RNA” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.
  • rare nucleotide refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine.
  • rare nucleotides include, but are not limited to, inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2 iV-methylguanosine and 2 ' 2 iV,iV-dimethylguanosine.
  • nucleotide analog or altered nucleotide or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides.
  • Preferred nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of preferred modified nucleotides include, but are not limited to, 2-amino-guanosine, 2-amino-adenosine, 2,6-diamino- guanosine and 2,6-diamino-adenosine.
  • positions of the nucleotide which may be derivatized include the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2- amino)propyl uridine; the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc.
  • Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310. Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides.
  • the 2' OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH 2 , NHR, NR 2 , COOR, or OR, wherein R is substituted or unsubstituted Ci -C 6 alkyl, alkenyl, alkynyl, aryl, etc.
  • R is substituted or unsubstituted Ci -C 6 alkyl, alkenyl, alkynyl, aryl, etc.
  • Other possible modifications include those described in U.S. Patent Nos. 5,858,988, and 6,291,438.
  • the phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions which allow the nucleotide to perform its intended function such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr.
  • oligonucleotide refers to a short polymer of nucleotides and/or nucleotide analogs.
  • RNA analog refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA.
  • the oligonucleotides may be linked with linkages which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages.
  • the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, and/or phosphorothioate linkages.
  • exemplary RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA).
  • RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate (mediates) RNA silencing (e.g. RNA interference).
  • oligonucleotides comprise Locked Nucleic Acids (LNAs) or Peptide Nucleic Acids (PNAs).
  • the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the nucleic acid silencing agent (e.g., RNA silencing agent) has a sequence (e.g. in the antisense strand, mRNA targeting moiety or miRNA recruiting moiety) which is sufficient to bind the desired target RNA, respectively, and to trigger the silencing of the target mRNA.
  • RNA interference refers to a type of RNA silencing which results in the selective intracellular degradation of a target RNA.
  • RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs).
  • Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences.
  • RISC RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.
  • translational repression refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.
  • RNA silencing agent having a strand which is "sequence sufficiently complementary to a target mRNA sequence to direct target- specific RNA interference (RNAi)" means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • siRNA small interfering RNA
  • siRNA refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNA interference.
  • a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, more preferably between about 16-25 nucleotides (or nucleotide analogs), even more preferably between about 18-23 nucleotides (or nucleotide analogs), and even more preferably between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs).
  • the term "short" siRNA refers to a siRNA comprising -21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides.
  • long siRNA refers to a siRNA comprising -24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides.
  • Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi.
  • long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.
  • miRNA small temporal RNAs
  • shRNAs small temporal RNAs
  • stRNAs small temporal RNAs
  • a “miRNA disorder” shall refer to a disease or disorder characterized by an aberrant expression or activity of a miRNA.
  • RNA silencing agent e.g. an siRNA or RNAi agent
  • RNA silencing agent refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing.
  • the antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target- specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA.
  • sense strand or “second strand” of an RNA silencing agent, e.g. a siRNA or RNAi agent, refers to a strand that is complementary to the antisense strand or first strand.
  • Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand.
  • miRNA duplex intermediates or siRNA-like duplexes include a RNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and another RNA strand having sufficient complementarity to form a duplex with the RNA strand that has complementarity with the mRNA.
  • guide strand refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA.
  • an RNA silencing agent e.g., an antisense strand of an siRNA duplex or siRNA sequence
  • engineered indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by man. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell.
  • an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor.
  • isolated nucleic acid molecule or sequence is a nucleic acid molecule or sequence that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA or RNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.
  • isolated RNA refers to RNA molecules which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • transgene refers to any nucleic acid molecule, which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell.
  • a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
  • transgene also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNAs, that encode one or more engineered RNA precursors, to be expressed in a transgenic organism, e.g., animal, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal's genome at a location which differs from that of the natural gene.
  • a transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence.
  • therapeutic moiety refers to a moiety (e.g., a biomolecule) capable of eliciting a therapeutic effect.
  • a gene "involved” in a disease or disorder includes a gene, the normal or aberrant expression or function of which effects or causes the disease or disorder or at least one symptom of said disease or disorder.
  • hepatitis C virus “HCV,” “non-A non-B hepatitis,” or “NANBH” are used interchangeably herein, and include any “genotype” or “subgenotype” (also termed “subtype”) of the virion, or portion thereof (e.g., a portion of the E2 protein of genotype Ia of HCV), that is encoded by the RNA of hepatitis C virus or that occurs by natural allelic variation.
  • HCV-mediated disorder includes disease states and/or symptoms associated with HCV infection.
  • HCV-mediated disorder refers to any disorder, the onset, progression or the persistence of the symptoms of which requires the participation of HCV.
  • Exemplary HCV-mediated disorders include, but are not limited to, for example, cirrhosis and liver cancer.
  • HCV is organized by a 5 '-untranslated region that is followed by an open reading frame (ORF) that codes for about 3,010 amino acids. The ORF runs from nucleotide base pair 342 to 8,955 followed by another untranslated region at the 3' end.
  • ORF open reading frame
  • the amino acids are subdivided into ten proteins in the order from 5' to 3' as follows: C; El; E2; NSl; NS2; NS3; NS4 (a and b); and NS5 (a and b). These proteins are formed from the cleavage of the larger polyprotein by both host and viral proteases.
  • the C, El, and E2 proteins are structural and the NS1-NS5 proteins are nonstructural proteins.
  • the C region codes for the core nucleocapsid protein.
  • El and E2 are glycosylated envelope proteins that coat the virus.
  • NS2 may be a zinc metalloproteinase.
  • NS3 is a helicase.
  • NS4a functions as a serine protease cofactor involved in cleavage between NS4b and NS5a.
  • NS5a is a serine phosphoprotein whose function is unknown.
  • the NS5b region has both RNA-dependent RNA polymerase and terminal transferase activity.
  • genotypes 1, 2, 3, 4, 5, and 6 there are about six (6) distinct genotypes (e.g., genotypes 1, 2, 3, 4, 5, and 6) that are categorized by variations in the core protein and over 80 subgenotypes which exhibit further variation within each genotype, some of which include: Ia; Ib; Ic; 2a; 2b; 2c; 3a; 3b; 4a; 4b; 4c; 4d; 4e; 5a; and 6a.
  • antibody as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen -binding portion") or single chain thereof.
  • An “antibody” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V H ) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, C H I, C H 2 and C H 3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as V L ) and a light chain constant region.
  • the light chain constant region is comprised of one domain, C L .
  • the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • human antibody is an antibody that has variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
  • HCV refers to the virion or portion thereof, for example, a protein portion, such as the HCV El or E2 glycoprotein that is encoded by the RNA of HCV, or a fragment or portion of an HCV gene product.
  • anti-HCV antibody is an antibody that interacts with (e.g., binds to) a HCV or a protein, carbohydrate, lipid, or other component produced by or associated with HCV.
  • a "HCV glycoprotein antibody” is an antibody that binds a glycoprotein of HCV or a fragment thereof.
  • An anti-HCV or glycoprotein antibody may bind to an epitope, e.g., a conformational epitope or a linear (non-conformational) epitope, or to a portion or fragment of the virus or component thereof.
  • an anti- HCV antibody is a neutralizing antibody.
  • an anti-HCV antibody is a binding antibody.
  • An anti-HCV antibody, or antigen-binding portion thereof can be administered alone or in combination with a second agent.
  • the subject can be a patient infected or suspected to be infected with HCV or having a symptom of HCV-mediated disease (e.g., liver inflammation, jaundice, tender hepatomegaly, or loss of appetite).
  • the treatment can be used to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve, or affect the infection and the disease associated with the infection, the symptoms of the disease, or a predisposition toward the disease.
  • Treatment may be understood to mean the prophylaxis or prevention of a productive infection before the onset of illness.
  • an amount of an anti-HCV antibody effective to treat HCV infection is an amount of the antibody that is effective, upon single or multiple dose administration to a subject, in inhibiting HCV infection, disease, or sequelae thereof, in a subject.
  • a therapeutically effective amount of the antibody or antibody fragment may vary according to factors such as the disease state, severity of symptoms, degree of liver inflammation, HCV strain or isolate, route of infection or exposure to HCV, whether the route of infection or exposure is known, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion is outweighed by the therapeutically beneficial effects.
  • an antibody to inhibit a measurable parameter can be evaluated in an animal model system predictive of efficacy in humans.
  • this property of an antibody or antibody composition can be evaluated by examining the ability of the compound to modulate HCV/cell interactions, e.g., binding, infection, virulence, and the like, by in vitro assays known to the skilled practitioner.
  • In vitro assays include binding assays, such as ELISA, and neutralization assays.
  • an amount of an antibody effective to prevent a disorder, or a "prophylactically effective amount,” of the antibody is an amount that is effective, upon single- or multiple-dose administration to the subject, in preventing or delaying the occurrence of the onset or recurrence of the disorder, or inhibiting a symptom thereof. However, if longer time intervals of protection are desired, increased doses or more frequent doses can be administered.
  • telomere binding' refers to the ability of an antibody to bind to its antigen (e.g., HCV, or a portion thereof), with an affinity of at least 1 x 10 ⁇ 6 M, and/or bind to its antigen (e.g., HCV, or a portion thereof), with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.
  • An "antibody” is a protein including at least one or two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one or two light (L) chain variable regions (abbreviated herein as VL).
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (CDRs), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, 1991, and Chothia, C. et al., J. MoI. Biol. 196:901-917, 1987, which are incorporated herein by reference).
  • each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRl, CDRl, FR2, CDR2, FR3, CDR3, FR4.
  • the VH or VL regions of the antibody can further include all or part of a heavy or light chain constant region.
  • the antibody is a tetramer of two heavy immunoglobulin chains and two light immunoglobulin chains, wherein the heavy and light immunoglobulin chains are inter-connected by, e.g., disulfide bonds.
  • the heavy chain constant region includes three domains, CHl, CH2 and CH3.
  • the light chain constant region is comprised of one domain, CL.
  • the variable region of the heavy and light chains contains a binding domain that interacts with an antigen.
  • the constant regions of the antibodies typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (CIq) of the classical complement system.
  • antibody includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda.
  • immunoglobulin refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes.
  • the recognized human immunoglobulin genes include the kappa, lambda, alpha (IgAl and IgA2), gamma (IgGl, IgG2, IgG3, IgG4), delta (IgD), epsilon (IgE), and mu (IgM) constant region genes, as well as the myriad immunoglobulin variable region genes.
  • Full-length immunoglobulin "light chains” (about 25 K D and 214 amino acids) are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus.
  • Full-length immunoglobulin "light chains" (about 25 K D and 214 amino acids) are encoded by a variable region gene at the NH 2 -terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH-terminus.
  • immunoglobulin includes an immunoglobulin having: CDRs from a human or non- human source.
  • the framework of the immunoglobulin can be human, humanized, or non-human, e.g., a murine framework modified to decrease antigenicity in humans, or a synthetic framework, e.g., a consensus sequence.
  • a mature immunoglobulin / antibody variable region is typically devoid of a leader sequence.
  • Immunoglobulins / antibodies can be further distinguished by their constant regions into class (e.g., IgA, IgD, IgE, IgG, or IgM) and subclass or isotype (e.g., IgGl, IgG2, IgG3, or IgG4).
  • class e.g., IgA, IgD, IgE, IgG, or IgM
  • subclass or isotype e.g., IgGl, IgG2, IgG3, or IgG4
  • an antibody refers to a portion of an antibody that specifically binds to an antigen (e.g., HCV or a component thereof, e.g., G glycoprotein), e.g., a molecule in which one or more immunoglobulin chains is not full length, but which specifically binds to an antigen.
  • an antigen e.g., HCV or a component thereof, e.g., G glycoprotein
  • binding portions encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341:544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR) having sufficient framework to specifically bind, e.g., an antigen binding portion of a variable region.
  • CDR complementarity determining region
  • An antigen binding portion of a light chain variable region and an antigen binding portion of a heavy chain variable region e.g., the two domains of the Fv fragment, VL and VH
  • a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science
  • Such single chain antibodies are also encompassed within the term "antigen binding portion" of an antibody.
  • antibody portions are obtained using conventional techniques known to those with skill in the art, and the portions are screened for utility in the same manner as are intact antibodies.
  • the term “monospecific antibody” refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody” or “monoclonal antibody composition,” which as used herein refer to a preparation of antibodies or portions thereof with a single molecular composition.
  • recombinant antibody refers to antibodies that are prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes or antibodies prepared, expressed, created, or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences.
  • recombinant antibodies include humanized, CDR grafted, chimeric, in vitro generated (e.g., by phage display) antibodies, and may optionally include constant regions derived from human germline immunoglobulin sequences.
  • substantially identical refers to a first amino acid or nucleotide sequence that contains a sufficient number of identical or equivalent (e.g., with a similar side chain, e.g., conserved amino acid substitutions) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have similar activities.
  • the second antibody has the same specificity and has at least 50% of the affinity of the first antibody.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at o least 50% of the length of the reference sequence.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid5 "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent homology between0 two sequences can be accomplished using a mathematical algorithm.
  • the percent homology between two amino acid sequences is determined using the Needleman and Wunsch, J. MoI. Biol. 48:444-453, 1970, algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.5
  • the term "hybridizes under low stringency, medium stringency, high stringency, or very high stringency conditions” describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y.
  • the antibodies and antigen binding portions thereof described herein may have additional conservative or non-essential amino acid substitutions, which do not have a substantial effect on the polypeptide functions. Whether or not a particular substitution will be tolerated, i.e., will not adversely affect desired biological properties, such as binding activity, can be determined as described in Bowie et al., Science, 247:1306-1310, 1990.
  • a "conservative amino acid substitution” is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of a polypeptide, such as a binding agent, e.g., an antibody, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change.
  • nucleic Acid Silencing Agent - Binding Protein Conjugates The present invention is based on the discovery, that a polypeptide or protein can be chemically conjugated to a nucleic acid silencing agent without substantially reducing the silencing activity of the nucleic acid silencing agent. Furthermore, such a conjugate can be used to effectively deliver the nucleic acid silencing agent into a cell which expresses a target RNA to which the nucleic acid silencing agent is complementary. In exemplary embodiments, the target RNA accesses the endosomes of a cell. Without wishing to be bound in theory, the acidic environment of the endosome can facilitate release of the silencing agent from the conjugate in certain embodiments in order to silence the target RNA.
  • a carbonyl group may be introduced into the terminal ribonucleotide of a nucleic acid silencing agent and conjugated via a heterobifunctional linker to a sufhydryl group in an antibody or other polypeptide.
  • the conjugates of the invention have the following basic formula:
  • L is one or a linking moiety
  • n is a positive integer (e.g., 1, 2, 3, 4 or more)
  • Y is a polypeptide.
  • nucleic acid silencing agent (X) is any one of the nucleic acid silencing agents described infra. Said nucleic acid silencing agents may be conjugated to any one of the polypeptides ("Y") described infra.
  • the linking moiety (“L”) may cross-link the polypeptide (Y) to one or more nucleic acid silencing agents ("X") via covalent or non-covalent binding interactions.
  • the linking moiety can be formed by a non-covalent binding interaction between a first functional group (e.g., a tag or domain) on the nucleic silencing agent and a second, complementary, functional group on the binding protein (e.g., a complementary tag or domain).
  • the linking moiety is formed by a more stable, covalent binding interaction between an amino acid present in the polypeptide and a nucleotide (e.g., a ribonucleotide) present in the nucleic acid silencing agent.
  • the linking moiety may comprise a heterobifunctional cross-linker which is bonded (e.g., covalently bonded) to both an amino acid in the polypeptide and a nucleotide in the nucleic acid silencing agent.
  • the linking moiety may comprise a cross-linker bonded to a free amine (e.g., a lysine ⁇ -amine group or an N-terminal ⁇ -amine groups) or a carboxylate group (e.g., at the C-terminus, an aspartic acid, or a glutamic acid residues) in the polypeptide.
  • the linking moiety comprises cross-linker bonded to a sulfhydryl group in the polypeptide.
  • linking moieties may be formed by inter alia by NHS ester-maleimide-mediated conjugation, glutaraldehyde-mediated conjugation, or reductive-amination-mediated conjugation.
  • NHS ester-maleimide-mediated conjugations may be formed, for example, by the reduction (e.g., with 2- mercaptoethylamine (MEA)) of disulfide groups (e.g., in the hinge region of an antibody).
  • the polyeptide may be first treated with 2-iminothiolane (Traut's reagent) which reacts with amino groups in the antibody to introduce the sulfhydryl residues.
  • glutaraldehyde-mediated conjugation involves Schiff base formation with possible rearrangement to a stable product or through a Michael-type addition reaction.
  • the reductive-amination-mediated conjugations include, for example, the activation of antibodies with sodium periodate, conjugation of periodate-oxidized HRP (horseradish peroxidase) to antibodies, or conjugation of periodate-oxidized antibodies with amine or hydrazide derivatives.
  • the conjugations using antibody fragments include, for example, preparation of F(ab') 2 fragments using pepsin, or preparation of Fab fragments using papain.
  • Preparation of labeled antibodies involves, for example, fluorescently labeled antibodies, radiolabeled antibodies, or biotinylated antibodies.
  • Toxins of many different types can be conjugated to monoclonal antibodies to create effective immunotoxins, including the proteins ricin from castor beans (Ricinus communis), abrin from Abrus precatorius, modeccin, gelonin from Gelonium multiflorum seeds, diphtheria toxin produced by Corynebacterium diphtheriae, pokeweed antiviral proteins (PAPs; three types: PAP, PAP II, and PAP-S) from Phytolacca americana seeds, cobra venom factor (CVF), Pseudomonas exotoxin, restrictocin from Aspergillus restrictus, momordin from Momordica charantia seeds, and saporin from Saponaria officinalis seeds, as well as other ribosome-inactivating proteins (RIPs).
  • proteins ricin from castor beans (Ricinus communis)
  • abrin from Abrus precatorius
  • modeccin gelonin from Gelonium multiflor
  • Preparation of immunotoxin conjugates can be achieved from disulfide exchange reactions, amino- and sulfhydryl-reactive heterobifunctional cross-linkers, or reductive amination.
  • the disulfide exchange reagents include, for example, pyridyldisulfide reagents (e.g. SPDP (N-succinimidyl 3- (2-pyridyldithio propionate), SMPT (succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2- pyridyldithio)toluene), 3-(2-pyridyldithio)propionate, etc.).
  • SPDP N-succinimidyl 3- (2-pyridyldithio propionate
  • SMPT succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2- pyridyldithio)toluene
  • the amino- and sulfhydryl- reactive heterobifunctional cross-linkers include, for example, SIAB (N-succinimidyl(4- iodoacetyl)aminobenzoate), SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), or SMPB (succinimidyl-4-(p-maleimidophenyl)butyrate).
  • SIAB N-succinimidyl(4- iodoacetyl)aminobenzoate
  • SMCC succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1-carboxylate
  • MBS m-maleimidobenzoyl-N-hydroxysuccinimide ester
  • SMPB succinimidyl-4-(p
  • Reductive amination can be utilized to prepare immunotoxin, exemplified in the periodate oxidation of glycoproteins followed by reductive conjugation, or periodate-oxidized dextran as cross-linking agent.
  • Antibodies may also be coupled through sulfhydryl residues to liposomes using liposomes containing PE groups derivatized with heterobifunctional cross-linkers such as SMCC, MBS, SMPB, SIAB, and SPDP.
  • Antibodies also may be coupled through their amine groups using reductive amination to periodate-oxidized glycolipids.
  • Antibodies or fragments of antibodies can be covalently linked to PEG (polyethylene glycols), which is a non-immunogenic polymer stabilizing these fragments in vivo.
  • PEG polyethylene glycols
  • PEG polyethylene glycols
  • Two Fabs can also be attached at one end of PEG or antibody-dextrans conjugates.
  • the most common activation methods for PEG include, for example, trichloro-s-triazine activation and coupling, NHS ester and NHS carbonate activation and coupling, carbodiimide coupling of carboxylate-PEG derivatives, or CDI (N',N'-Carbonyldiimidazole) activation and coupling.
  • Antibodies can also be modified with activated dextran and the major methods for this modification include, for example, polyaldehyde activation and coupling, carbodiimide-mediated reaction to form an amide bond for carboxyl, amine, and hydrazide derivatives, epoxy activation and coupling, or cross-linking of sulfhydryl-reactive derivatives.
  • the therapeutic RNA interference agent e.g. siRNAs, shRNAs, etc.
  • the targeting antibody or fragment of the said antibody e.g. Fab, F(ab') 2 , scFv. etc.
  • L binding or linking moiety
  • This conjugation is able to improve stability of the antibody/fragment of the antibody and/or the RNA interference agent, target the RNA interference agent to a particular tissue or cell-type, or increase cell permeability of the antibody/fragment of the antibody and/or the RNA interference agent, e.g., by an endocytosis-dependent or -independent mechanism.
  • this conjugation can also increase the interaction specificity and/or efficiency between the said antibody/fragment of the antibody and their target antigens/proteins .
  • the binding or linking moiety can be a functional group(s) on the RNA molecule ("X") and/or the targeting molecule ("Y") to mediate the non-covalent interaction between "X” and "Y".
  • "L" is the nucleic acid binding domain of a protein selected from the group consisting of protamine, GCN4, Fox, Jun, TFIIS, FMRI, yeast protein HX, Vigillin, Merl, bacterial polynucleotide phosphorylase, ribosomal protein S3, and heat shock protein.
  • the binding or linking moiety can include biotin-avidin or streptavidin conjugates, prepared by NHS ester-maleimide-mediated conjugation, periodate oxidation/reductive amination conjugation, glutaraldehyde conjugation, hydrazide activation, or modified by fluorescent labels (e.g. FITC (fluorescein isothiocyanate)), lissamine rhodamine B sulfonyl chloride, AMCA-NHS, or phycobiliproteins.
  • fluorescent labels e.g. FITC (fluorescein isothiocyanate)
  • lissamine rhodamine B sulfonyl chloride e.g., AMCA-NHS, or phycobiliproteins.
  • the binding or linking moiety can be a binding moiety(s) to mediate the non-covalent interaction between "X" and "Y".
  • "L” is the nucleic acid binding domain of a protein selected from the group consisting of protamine, GCN4, Fox, Jun, TFIIS, FMRI, yeast protein HX, Vigillin, Merl, bacterial polynucleotide phosphorylase, ribosomal protein S3, and heat shock protein.
  • the binding or linking moiety (“L”) can include biotin-avidin or streptavidin conjugates, prepared by NHS ester-maleimide-mediated conjugation, periodate oxidation/reductive amination conjugation, glutaraldehyde conjugation, hydrazide activation, or modified by fluorescent labels (e.g. FITC (fluorescein isothiocyanate)), lissamine rhodamine B sulfonyl chloride, AMCA-NHS, or phycobiliproteins .
  • the binding or linking moiety (“L”) can be a functional group(s) on the RNA molecule ("X”) and/or the targeting molecule ("Y”) to mediate the covalent bond between "X" and "Y”.
  • the binding or linking moiety can be a cross-linking reagent or multiple reagents to mediate the covalent bond between "X" and "Y".
  • the covalent linkage forms between the antibody/fragment of the antibody and the RNA interference agent through a cross-linker.
  • the cross-linker can be a liposome-based complex.
  • the cross-linker can be one of so-called zero-length cross -linking agents that bring about the direct formation of covalent bonds between existing amino acid side chain groups.
  • the zero-length cross-linking agents can initiate the formation of at least three types of bonds: an amide linkage made by the condensation of a primary amine with a carboxylic acid, a phosphoramidate linkage made by the reaction of an organic phosphate group with a primary amine, and a secondary or tertiary amine linkage made by the reductive amination of a primary or secondary amine with an aldehyde group.
  • the zero-length cross-linking agents can be, for example, carbodiimides, a Woodward's Reagent K (N-ethyl-3-phenylisoxazolium- 3 '-sulfonate), N',N'-Carbonyldiimidazole (CDI; Aldrich), or Schiff bases formed with or without reductive amination (or alkylation).
  • a Woodward's Reagent K N-ethyl-3-phenylisoxazolium- 3 '-sulfonate
  • CDI N',N'-Carbonyldiimidazole
  • Carbodiimides includes, for example, EDC (or EDAC; l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) with or without supplement of sulfo-NHS (N-Hydroxysulfo-succinimide; Pierce), CMC (1- Cyclohexyl-3-(2-morpholinoethyl) carbodiimide, or its metho p-toluene sulfonate salt; Aldrich), or DIC (diisopropyl carbodiimide), or its by-produces diisopropylurea and diisopropyl-N-acylurea).
  • EDC or EDAC; l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
  • sulfo-NHS N-Hydroxysulfo-succinimide; Pierce
  • Disulfide bonds obtained from existing thiol groups would also, presumably, be considered zero-length cross-links (Korodi et al., Biochemistry 25, 6895-6900 (1986); Huston et al., Biochemistry 27, 8945-8952 (1988)). Such linkages appear to be formed only when the reacting groups are in close proximity.
  • the cross-linking agents may be organized according to the type(s) of reactive groups, their side chain reactivity, their hydrophobicity or hydrophilicity, and the length or distance between the reactive groups; whether the two, or in some cases more (as illustrated in Hiratsuka, Biochemistry 27, 4110-4114 (1988)) reactive groups are the same or different (i.e., "homobifunctional”, “heterobifunctional” “trifunctional”, or “multifunctional” reagents), whether the structure connecting the reactive groups is readily cleavable, and whether the groups are membrane permeable or impermeable, and according to various other criteria.
  • a much more extensive list of cross-linking agents includes those presented in Ji, Methods Enzymol. 91, 580-609 (1983).
  • homobifunctional linkers are used for bioconjugates. Most of these homobifunctional reagents are symmetrical in design with a carbon chain spacer connecting the two identical reactive ends. Like molecular rope, these reagents could tie one protein to another by covalently reacting with the same common groups on both molecules.
  • Homobifunctional linkers include, for example, homobifunctional NHS (N-hydroxysuccinimide) esters, sulfo-NHS (N-hydroxysulfo-succinimide) esters, homobifunctional imidoesters, homobifunctional Sulfhydryl-reactive cross-linkers, difluorobenzene derivatives, homobifunctional photoreactive cross-linkers, homobifunctional aldehydes, bis-epoxides, homobifunctional hydrazides, bis-diazonium derivatives, or bis-alkylhalides.
  • NHS N-hydroxysuccinimide
  • sulfo-NHS N-hydroxysulfo-succinimide
  • homobifunctional imidoesters homobifunctional Sulfhydryl-reactive cross-linkers
  • difluorobenzene derivatives homobifunctional photoreactive cross-linkers
  • homobifunctional aldehydes bis-epoxides
  • Homobifunctional NHS (N-hydroxysuccinimide) esters or sulfo-NHS (N-hydroxysulfo-succinimide) esters include, for example, Lomant's reagent or DSP (dithiobis(succinimidyl propionate) ; Pierce), DTSSP (dithiobis(sulfosuccinimidyl propionate; Pierce), DSS (disuccinimidyl suberate; Pierce), BS 3 (bis(sulfosuccinimidyl)suberate; Pierce), DST (disuccinimidyl tartarate; Pierce), sulfo-DST (disulfosuccinimidyl tartarate; Pierce), BSOCOES (bis[2- (succinimidyloxycarbonyloxy)ethyl]sulfone; Pierce), sulfo-BSOCOES (bis[2- (sulfosuccinimidyloxy
  • Homobifunctional imidoesters include, for example, DMA (dimethyl adipimidate; Pierce), DMP (dimethyl pimelimidate; Pierce), DMS (dimethyl suberimidate), or DTBP (dimethyl 3,3'- dithiobispropionimidate; Pierce).
  • Homobifunctional Sulfhydryl-reactive cross-linkers include, for example, DPDPB (l,4-di-[3'-(2'-pyridyldithio)propionamido]butane), or BMH (bismaleimidohexane; Pierce).
  • Difluorobenzene derivatives include, for example, DFDNB (l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6-dinitrobenzene; Pierce), or DFDNPS (4,4'-difluoro-3,3'-dinitrophenylsulfone).
  • Homobifunctional photoreactive cross-linkers include, for example, BASED (bis-[ ⁇ -(4- azidosalicylamido)ethyl]disulfide; Pierce).
  • Homobifunctional aldehydes include, for example, formaldehyde, or glutaraldehyde.
  • Bis-exoxides include, for example, 1,4- butanediol diglycidyl ether.
  • Homobifunctional hydrazides include, for example, adipic acid dihydrazide (Aldrich), or carbohydrazide.
  • Bis-diazonium derivatives include, for example, diazotized o-Tolidine (or 3,3'-dimethylbenzidine), or bis-diazotized benzidine (or p-diaminodiphenyl).
  • heterobifunctional linkers are preferred to form bioconjugates.
  • These heterobifunctional conjugation reagents contain two different reactive groups that can couple to two different functional targets on the antibody/fragment of antibody and the RNA interference reagent of this invention.
  • the cross-bridge or spacer that ties the two reactive ends together may also govern the overall hydrophilicity of the reagent, the reactivity of functional groups, and the immunogenic ability and stability of the conjugate.
  • Some heterobifunctional linkers can contain cleavable groups within their cross-bridges.
  • Heterobifunctional linkers include, for example, amine-reactive and sulfhydryl-reactive cross-linkers, carbonyl-reactive and sulfhydryl-reactive cross-linkers, amine-reactive and photoreactive cross-linkers, sulfhydryl-reactive and photoreactive cross -linkers, carbonyl-reactive and photoreactive cross -linkers, carboxylate-reactive and photoreactive cross-linkers, or arginine-reactive and photoreactive cross -linkers.
  • Amine-reactive and sulfhydryl-reactive cross -linkers include, for example, SPDP (N-succinimidyl 3-(2-pyridyldithio propionate, including standard SPDP, a long-chain version designated LC-SPDP, and a water-soluble, sulfo- NHS form also containing an extended chain, called sulfo-LC-SPDP; all from Pierce), SMPT (succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene), sulfo-LC- SMPT (sulfosuccinimidyl-6-[ ⁇ -methyl- ⁇ -(2-pyridyldithio)toluamido]hexanoate), SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l-carboxylate), sulfo-SMCC (sul
  • Carbonyl -reactive and sulfhydryl-reactive cross-linkers include, for example, MPBH (4-(4-N- maleimidophenyl)butyric acid hydrazide; Pierce), M 2 C 2 H (4-(N- maleimidomethy ⁇ cyclohexane-l-carboxyl-hydrazide; Pierce), or PDPH (3-(2- pyridyldithio)propionyl hydrazide; Pierce).
  • Amine-reactive and photoreactive cross- linkers include, for example, NHS-ASA (N-hydroxysuccinimidyl-4-azidosaliucylic acid; Pierce), sulfo-NHS-ASA, sulfo-NHS-LC-ASA, SASD (sulfosuccinimidyl-2-(p- azidosalicylamido)ethyl-l,3'-dithiopropionate; Pierce), HSAB (N-hydroxysuccinimidyl- 4-azidobenzoate; Pierce), sulfo-HSAB (N-hydroxysulfosuccinimidyl-4-azidobenzoate; Pierce), SANPAH (N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate; Pierce), sulfo-SANPAH (sulfosuccinimidyl-6-(4'-azido-2
  • Sulfhydryl-reactive and photoreactive cross-linkers include, for example, ASIB (l-(p-azidosalicylamido)-4-(iodoacetamido)butane; Pierce), APDP (N-[4-(p- azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide; Pierce), Benzophenone-4- iodoacetamide (Molecular Probes), or Benzophenone-4-maleimide (Molecular Probes).
  • Carbonyl-reactive and photoreactive cross-linkers include, for example, ABH (p- azidobenzoyl hydrazide; Pierce).
  • Carboxylate-reactive and photoreactive cross-linkers include, for example, ADBA (4-(p-azidosalicylamido)butylamine; Pierce).
  • Arginine- reactive and photoreactive cross-linkers include, for example, APG (p-azidophenyl glyoxal; Pierce).
  • trifunctional cross-linkers are preferred to form bioconjugates.
  • Trifunctional linkers possess three reactive or complexing groups per molecule and utilizes two reactive groups at two ends to couple with the antibody/fragment of the antibody and the RNA interference agent of this invention, while the third arm branching out with a reactive group to link with another molecule of the said antibody/fragment of the antibody, the RNA interference agent, or a third chemical or biological target.
  • trifunctional linkers are built from the amino acid L-lysine. Its three functional groups, ⁇ -carboxy, ⁇ -amino, and ⁇ -amino, are derivatized independently to contain three arms.
  • Each arm can be designed to terminate in a complexing group able to participate in a particular type of conjugation reaction.
  • trifunctional linkers are built from biocytin, the lysine derivative of biotin with its valeric acid side chain amide-bonded to the ⁇ -amino group of the amino acid.
  • trifunctional cross-linkers include, for example, 4-azido-2- nitrophenylbiocytin-4-nitrophenyl ester (ABNP) or sulfo-SBED (sulfosuccinimidyl-2- [6-(biotinamido)-2-(p-azidobenzamido)hexanoamido]ethyl-l,3'-dithiopropionate).
  • one molecule of same RNA silencing agent of this invention may be linked with the third arm of the trifunctional linker.
  • one molecule of a different RNA silencing agent against the same target gene may be linked with the third arm of the trifunctional linker.
  • one molecule of a different RNA silencing agent against a different target gene may be linked with the third arm of the trifunctional linker.
  • one molecule of same antibody or fragment of the antibody of this invention may be linked with the third arm of the trifunctional linker.
  • one molecule of a different antibody or fragment of the antibody recognizing the same target antigen or protein may be linked with the third arm of the trifunctional linker.
  • one molecule of a different antibody or fragment of the antibody recognizing a different target antigen or protein may be linked with the third arm of the trifunctional linker.
  • one molecule of a moiety selected from the group of metal ions, nucleotides, amino acids, polyamines, peptides, peptide mimics, proteins, protein binding agents, toxins, antibiotics, lipids, lipophiles, steroids, terpenes, vitamins, carbohydrates, synthetic polymers, or organic compounds (e.g., a dye) may be linked with the third arm of the trifunctional linker.
  • Preferred moieties linked with the third arm of the trifunctional linker can improve transport, target-binding, or specificity properties and may also improve nuclease resistance of the resultant bioconjugate.
  • the said moieties may be a cell or tissue targeting agent or to enhance cellular uptake by target cells.
  • multifunctional cross-linkers are preferred to form bioconjugates.
  • Multifunctional cross-linkers possess three or more reactive or complexing groups per molecule.
  • Multifunctional cross-linkers include, for example, the polyaldehyde dextran or small organic molecules like trichloro-S-triazine (TsT).
  • TsT trichloro-S-triazine
  • the dextran polymer contains adjacent hydroxyl groups on each glucose monomer. These diols may be oxidized with sodium periodate to cleave the associated carbon-carbon bonds and produce aldehydes. This procedure results in two aldehyde groups formed per glucose monomer, thus producing a highly reactive, multifunctional polymer able to couple with numerous amine-containing molecules (Bernstein et al., J. Natl. Cancer Inst. 60, 379-384, (1978)).
  • a heterobifunctional linker is utilized to form a bioconjugate comprising of the antibody or fragment of the antibody and the RNA interference agent of this invention.
  • a heterobifunctional linker contains one or more thiol-reactive groups, including haloacetyl and alkyl halide derivatives, maleimides, aziridines, acryloyl derivatives, arylating agents, or thiol- disulfide exchange reagents (e.g. pyridyl disulfides, TNB-thiol, disulfide reductants, etc.).
  • the heterobifunctional linker comprises of a maleimide moiety and a hydrazide moiety.
  • the heterobifunctional linker can include but is not limited to N-( ⁇ -maleimidocaproic acid) hydrazide (EMCH, with the formula as C 10 H 16 N 3 O 3 CI), N-( ⁇ -maleimidopropionic acid) hydrazide (MPH, with the formula as C 8 H 12 N 3 O 3 CI), N-( ⁇ -maleimidoundecanoic acid) hydrazide (KMUH, with the formula as C 15 H 25 N 3 O 3 , and 4-(4-N-maleimidophenyl) butyric acid hydrazide (MPBH).
  • EMCH N-( ⁇ -maleimidocaproic acid) hydrazide
  • MPH N-( ⁇ -maleimidopropionic acid) hydrazide
  • KMUH N-( ⁇ -maleimidoundecanoic acid) hydrazide
  • MPBH 4-(4-N-maleimidophenyl) butyric acid hydra
  • the maleimide end of the heterobifunctional linker is linked to a thiolated antibody or fragment of the antibody of this invention through a thioether bond.
  • the thiol group is added by 2-Iminothiolane (Traut' s Reagent) to a NH 2 side chain of a Lysine residue on a heavy chain of the antibody or fragment of the antibody of this invention.
  • the hydrazide end of the heterobifunctional linker is linked to the RNA interference agent of this invention.
  • the heterobifunctional linker may contain cleavable reagents.
  • cleavable reagents are typically built into the cross-bridge or reactive ends of a reagent using disulfides, glycol groups, diazo bonds, esters, sulfone groups, or acetal linkages.
  • the cleavage element is built in as a pH-liable hydrazone bond between the hydrazide end of the heterobifunctional linker and the aldehyde group on an alkylated RNA interference agent of this invention.
  • the said hydrazone bond in the linker is stable in serum at pH7.4 but hydrolyzes at pH5.0 in the endosomes, leading to the release of the siRNA conjugated to the antibody.
  • diamine or bis-hydrazide modification results in the formation of an alkyl spacer arm terminating in a primary amine group or a hydrazide functional group, respectively, via bisulfite activation of cytosine, bromine activation of thymine, guanine, and cytosine, or carbodiimide reaction with 5' phosphates of DNA
  • the sulfhydryl modification allows conjugation with sulfhydryl-reactive heterobifunctional cross-linkers and includes, for example, cystamine modification of 5' phosphate groups using EDC (l-ethyl-3-(3- dimethylamino-propyl)carbodiimide hydrochloride; Pierce), SPDP (N-succinimidyl 3- (2-pyridyldithio)propionate) modification of amines on nucleotides, or SATA (N- succinimidyl S-acetylthioacetate) modification of amines on nucleotides.
  • EDC l-ethyl-3-(3- dimethylamino-propyl)carbodiimide hydrochloride; Pierce
  • SPDP N-succinimidyl 3- (2-pyridyldithio)propionate modification of amines on nucleotides
  • SATA N- succinimidyl S-acetylthi
  • Biotin- labeling includes, for example, the linkage of Biotin-LC-dUTP, photo-Biotin modification, reaction of NHS-LC-Biotin with diamine-modified nucleotide probes, Biotin-diazonium modification, reaction of Biotin-BMCC with sulfhydryl-modified nucleotides, or Biotin-hydrazide modification of bisulfite-activated cytosine groups.
  • Enzyme conjugations include, for example, alkaline phosphatase conjugation to cystamine-modified nucleotides using amine- and sulfhydryl-reactive heterobifunctional cross-linkers, alkaline phosphatase conjugation to diamine-modified nucleotides using DSS (disuccinimidyl suberate), enzyme conjugation to diamine-modified nucleotides using PDITC, or conjugation of SFB-modified alkaline phosphatase to bis-hydrazide- modified oligonucleotides.
  • DSS disuccinimidyl suberate
  • enzyme conjugation to diamine-modified nucleotides using PDITC or conjugation of SFB-modified alkaline phosphatase to bis-hydrazide- modified oligonucleotides.
  • the fluorescent labeling includes, for example, the conjugation of amine-reactive fluorescent probes to diamine-modified nucleotides, or conjugation of sulfhydryl-reactive fluorescent probes to sulfhydryl-modified nucleotides.
  • a few more examples of chemical modifications include the introduction of phosphorothioate linkages, boranophosphate linkage, 2'-O-methylation, 2'-fluoro nucleotides, 2'-O-(2-methoxyethyl) nucleotides, locked nucleic acid (LNA) nucleotides, or terminal modification on sense and/or antisense strand, including adding protein transduction domain (PTDs), a Tat peptide containing the cationic peptide YGRKKRRQRRR, partial phosphorothioate linkages, 2'-O-methyl sugars, or cholesterol molecules.
  • PTDs protein transduction domain
  • the methods to create new specific functional groups to the RNA molecules of this invention include, for example, the introduction of sulfhydryl residues (thiolation), carboxylate groups, primary amine groups, aldehyde residues, or hydrazide functional groups.
  • sulfhydryl residues includes, for example, modification of amines with 2-iminothiolane (Traut's Reagent), modification of amines with SATA (N-succinimidyl S-acetylthioacetate), modification of amines with SATP (succinimidyl acetylthiopropionate), modification of amines with SPDP (N- succinimidyl 3-(2-pyridyldithio)propionate), modification of amines with SMPT (succinimidyloxycarbonyl- ⁇ -methyl- ⁇ -(2-pyridyldithio)toluene), modification of amines with N-acetylhomocysteinethiolactone, modification of amines with SAMSA (S-acetylmercaptosuccinic anhydride), modification of aldehydes or ketones with AMBH (2-acetamido-4-mercaptobutyric acid
  • carboxylate groups includes, for example, the modification of amines with anhydrides, including succinic anhydride, glutaric anhydride, maleic anhydride, or citraconic anhydride, modification of sulfhydryls with iodoacetate, or modification of hydroxyls with chloroacetic acid.
  • anhydrides including succinic anhydride, glutaric anhydride, maleic anhydride, or citraconic anhydride, modification of sulfhydryls with iodoacetate, or modification of hydroxyls with chloroacetic acid.
  • the introduction of primary amine groups includes, for example, the modification of carboxylates with diamines, modification of sulfhydryls with N-( ⁇ - iodoethyl)trifluoroacetamide), modification of sulfhydryls with ethylenimine, modification of sulfhydryls with 2-bromoethylamine, modification of carbohydrates with diamines, modification of alkylphosphates with diamines, or modification of aldehydes with ammonia or diamines.
  • aldehyde residues includes, for example, the periodate oxidation of glycols and carbohydrates, oxidase modification of sugar residues, modification of amines with NHS-aldehydes (SFB (succinimidyl p- formylbenzoate) and SFPA (succinimidyl p-formylphenoxyacetate), or modification of amines with glutaraldehyde.
  • SFB succinimidyl p- formylbenzoate
  • SFPA succinimidyl p-formylphenoxyacetate
  • the introduction of hydrazide functional groups includes, for example, the modification of aldehydes with bis-hydrazide compounds, modification of carboxylates with bis-hydrazide compounds, or modification of alkylphosphates with bis-hydrazide compounds.
  • the conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.:47(l), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).
  • the compositions and methods of the invention feature nucleic acid silencing agents (e.g., RNA or DNA silencing agents).
  • the nucleic acid silencing agents are silencing agents having sufficient complementarity to a target RNA (e.g., a HCV-related target mRNA or microRNA) to mediate gene silencing of the target RNA.
  • gene silencing is such that the target gene level is reduced or decreased by at least 30% (e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more).
  • the nucleic acid silencing agents may be single or double stranded.
  • the nucleic acid silencing agents may be RNA silencing agents comprising or consisting of an antisense RNA strand (or portions thereof) comprising a sequence with sufficient complementarity to the target mRNA to silence expression of a target mRNA via an RNA silencing mechanism (e.g. RNA interference (RNAi) or translational repression).
  • RNA silencing mechanism e.g. RNA interference (RNAi) or translational repression
  • the nucleic acid silencing agent may be DNA silencing agents comprising or consisting of an antisense strand comprising a sequence with sufficient complementarity to the target RNA to silence expression via an antisense mechanism (e.g., via RNase H mediated cleavage).
  • the nucleic acid silencing agents may be inhibitors of RNA silencing.
  • the nucleic acid silencing agents may comprise or consist of an antisense strand comprising a sequence with sufficient complementarity to a small, non-coding RNA (e.g., a miRNA, pre-miRNA, pri-miRNA, rasi-RNA, or piRNA) to inhibit RNA silencing by the RNA (e.g., so-called "antagomiRs").
  • a small, non-coding RNA e.g., a miRNA, pre-miRNA, pri-miRNA, rasi-RNA, or piRNA
  • Candidate nucleic acid silencing agents are designed based on the sequence of a selected target site in a target RNA, e.g., a target mRNA or small non-coding RNA (e.g., a miRNA).
  • the targeted sequence can be selected by performing, for example, a gene walk analysis of the target RNA. Overlapping, adjacent, or closely spaced candidate agents corresponding to all or some of the transcribed region can be generated and tested. Each of the silencing agents can be tested and evaluated for the ability to mediate gene silencing.
  • a target site can be selected using the method of Elbashir et al. (see e.g., Elbashir et al., Methods, 26: 199-213 (2002)).
  • the RNA e.g., target mRNA
  • the RNA can be searched for regions that are likely to be free of translational or regulatory proteins (e.g., at least 100 bases downstream of the start codon).
  • a sequence of about 10-50 contiguous nucleotides e.g., about 18-30 contiguous nucleotides, about 20-25 contiguous nucleotides, or about 21-23 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides
  • the sequence has between 30-70% G/C content.
  • sequences characterized by an AA (or NA) dinucleotide at their 5' terminus may be selected.
  • in silico design programs are available to those of skill in the art including, for example, RFRCDB-siRNA (Jiang et al., Comput Methods Programs Biomed. 2007 Sep;87(3):230-8), sIR (Shah et al., BMC Bioinformatics. 2007 May 31;8:178), and DQOR (Henschel et al., Nucleic Acids Res. 2004 JuI 1;32:W113-20).
  • the antisense (or guide) strand sequence employed in the nucleic acid silencing agent may be designed based on the sequence of the selected target site in the target RNA.
  • the antisense sequence is sufficiently complementary to target sequence such that the nucleic silencing agent can mediate silencing of the target RNA (e.g., via RNAi, translational represession, or other gene silencing mechanisms).
  • the antisense strand may have perfect complementarity (ie. 100% complementarity) identity to the target site in a target RNA (e.g., a target mRNA).
  • perfect complementarity is not required. Greater than 80% complementarity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity, between the antisense sequence and the target RNA sequence is preferred.
  • the antisense sequence forms one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatched base pair(s)) with a target region Moreover, antisense sequences with small insertions or deletions of 1 or 2 nucleotides (or nucleotide analogs) may also be effective for mediating gene silencing (e.g., via RNAi or translational repression).
  • mismatches e.g., 1, 2, 3, 4, or 5 mismatched base pair(s)
  • antisense sequences with small insertions or deletions of 1 or 2 nucleotides (or nucleotide analogs) may also be effective for mediating gene silencing (e.g., via RNAi or translational repression).
  • the antisense sequence is sufficiently complementary to sequence of about 18-30 contiguous nucleotides, about 20-25 contiguous nucleotides, or about 21-23 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides, such that the silencing agent can mediate silencing of the target RNA (e.g., silencing of a target mRNA via RNAi or translational repression or silencing of a target microRNA or other small, non-coding RNA via RISC inhibition).
  • the antisense strand may have perfect complementarity (ie. 100% complementarity) identity to the target site.
  • the antisense sequence forms one or more mismatches (e.g., 1, 2, 3, 4, or 5 mismatched base pair(s)) with a target region.
  • antisense sequences with small insertions or deletions of 1 or 2 nucleotides (or nucleotide analogs) may also be effective for mediating silencing (e.g., via RNAi).
  • a nucleic acid silencing agent can be designed to specifically target and silence the sequence of a target RNA that is encoded by only one allele of a gene (i.e., an allele- specific target sequence).
  • the silencing agent can be designed to have single-nucleotide specificity.
  • the silencing agent can be designed to specifically silence a heterozygous mutation (e.g., a point mutation) or a single- nucleotide polymorphism (SNP) found in a mutant allele (e.g., a disease-associated allele), but not a wild-type allele.
  • Targeted SNPs may be located in the coding region of a target mRNA or in its untranslated regions (e.g., a 5' UTR). Silencing of the target RNA at these sites (e.g., via RNAi) should allow for allele- specific gene silencing and elimination of the corresponding mutant protein.
  • the target RNA is a small, non-coding RNA
  • the SNP may be located in the guide strand of the RNA (e.g., the guide strand of the miRNA) encoded by the mutant allele, but not the wild-type allele. Polymorphisms from other regions of the mutant gene are also suitable for targeting.
  • Allele- specific agents may be designed such that the antisense strand has a greater degree of complementarity for the target allele sequence than the non-target allele sequence. Sufficient complementarity may exist between the agent and the allele- specific target sequence in the targeted allele such that there are a greater number of mismatches when the nucleic acid silencing agent is compared (e.g., aligned) to the corresponding sequence in the non-target allele (e.g., wild type allele or RNA sequence).
  • the nucleic acid silencing agent may have perfect complementarity with the target sequence in the desired allele and at least one mismatch with non-target allele sequence.
  • the antisense strand sequence may be designed such that the extra mismatch with the non-target allele is essentially in the middle of the antisense strand.
  • the mismatch may be located at a nucleotide position that is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 nucleotides (i.e., nucleotide position P6, P7, P8, P9, PlO, PIl, P12, P13, P14, P15 or P16) from the 5' end of the antisense or guide strand of an RNA silencing agent (e.g., an siRNA or miRNA).
  • an RNA silencing agent e.g., an siRNA or miRNA.
  • the mismatch with the non-target allele is present at position PlO or P16.
  • the nucleic acid silencing agent may be desirable to design the nucleic acid silencing agent such that the one or more mismatches between the antisense strand and non-target allele form a purine:purine mismatch.
  • the purine:purine pairing may be selected from among G:G, A:G, G:A and A:A pairings, with G:G being preferred.
  • a wobble base pairing may be employed instead of a mismatched based pairing.
  • Nucleic acid silencing agents may be chemically modified.
  • nucleic acid silencing agents may be substituted with at least one modified nucleotide analogue.
  • the nucleotide analogues are located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity, is not substantially effected, e.g., in a region at the 5'-end and/or the 3'-end of the molecule.
  • Chemical modifications, even to a large number of nucleotides, may be made without interfering with the gene silencing activity of the nucleic acid silencing agent.
  • RNA silencing agent will be recognized as RNA in that it will have "RNA-like" properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively of ribonucleotide-based content.
  • RNA-like properties i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively of ribonucleotide-based content.
  • some or all of the nucleotide sugars can contain e.g., 2'0Me, or 2' fluoro in place of 2' hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties.
  • RNA silencing agent will be recognized as being a RNA molecule if has a sufficient number of nucleotide sugars in a C 3 -endo pucker configuration (e.g., at least 50, 75, 80, 85, 90, or 95% of its sugars).
  • the RNA silencing agent will have no more than 20, 10, 5, 4, 3, 2, or 1 sugar(s) which are not a C 3 -endo pucker structure. These limitations are particularly preferred in the antisense strand of an RNA silencing agent.
  • Preferred 2 '-modifications with C3'-endo sugar pucker include: 2'-OH, 2'-0-Me, 2'-O-methoxyethyl, 2'-O-aminopropyl,2'-F, T- 0-CH2-C0-NHMe, 2'-O-CH2-CH2-O-CH2-CH2-N(Me)2, and LNA.
  • DNA molecules, or any molecule in which more than 50, 60, or 70 % of the nucleotides in the molecule are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2' position are excluded from the definition of RNA silencing agent.
  • Positions which are repeated within a nucleic acid silencing agent may be preferentially modified, e.g., a base, a phosphate moiety, or a non-linking O of a phosphate moiety.
  • the modification will occur at all of the subject positions in the RNA silencing agent but in many, and in fact in most, cases it will not.
  • a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal region, e.g. at a position (e.g., a non-linking O 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 stranded region of a double- stranded RNA silencing agent or may only occur in a single stranded region of an RNA silencing agent (e.g., a 5' overhang, a 3' overhang, the unpaired regions or regions of a hairpin structure (e.g., a loop region)).
  • both single-stranded and double- stranded regions are modified.
  • a moiety can be conjugated to the 3' end, the 5' end, or at an internal position, or at a combination of these positions.
  • a moiety is conjugated to the 3' end of the antisense strand or sense strands.
  • a modification may be made only on the sense strand, only on the antisense strand, or on both strands.
  • the sense and antisense strand will have the same modifications or the same class of modifications, but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it may be desirable to modify only one strand, e.g. the sense strand.
  • the nucleic acid silencing agents of the invention may be modified by the substitution of internal nucleotides with modified nucleotides.
  • an "internal" nucleotide is one occurring at any position other than the 5' end or 3' end of nucleic acid molecule, polynucleotide or oligonucleoitde.
  • An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double- stranded molecule.
  • the sense strand and/or antisense strand is modified by the substitution of at least one internal nucleotide.
  • the sense strand and/or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides.
  • the sense strand and/or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides.
  • the sense strand and/or antisense strand is modified by the substitution of all of the internal nucleotides.
  • the nucleic acid silencing agents of the present invention can be modified to improve stability in serum or in growth medium for cell cultures.
  • the 3'-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, particularly adenosine or guanosine nucleotides.
  • pyrimidine nucleotides may be substituted by modified analogues, e.g., substitution of uridine by 2'-deoxythymidine.
  • RNA silencing agent e.g., against exonuclease or endonucleolytic degradation by cellular nucleases.
  • a stabilizing modification is a 2' modification.
  • Exemplary 2' stabilizing modification include provision of a 2' OMe moiety (e.g, on a U in a sense or antisense strand, in a 3' overhang, or at the 3 'end or terminus of a sense or antisense strand).
  • a stabilizing modification is a backbone modification.
  • Exemplary backbone modifications include the replacement of an O or a P in the phosphate backbone with an S (e.g., a phosphorothioate backbone modification, e.g., on the U or the A or both) or the use of a methylated P in a 3' overhang.
  • Other exemplary stabilizing modifications include replacement of the U with a C5 amino linker; replacement of an A with a G; modification with a 3' alkyl; and modification with an abasic pyrolidine in a 3' overhang, e.g., at the 3' terminus.
  • Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
  • it is particularly preferred 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.
  • all or some of the bases in a 3' or 5' overhang will be modified, e.g., with a modification described herein.
  • Modifications can include, e.g., the use of modifications at the 2' OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate o modifications.
  • Overhangs need not be homologous with the target sequence.
  • RNA silencing agents may be modified with chemical moieties, for example, to enhance cellular uptake by target cells (e.g., neuronal cells).
  • target cells e.g., neuronal cells
  • the invention includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non-nucleic acid moiety5 such as a peptide), an organic compound (e.g., a dye), or the like.
  • the conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv.
  • RNA silencing agent may be modified to favor entry of the antisense or guide strand into RISC such that undesirable, off-target silencing by the corresponding sense strand is reduced. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent its function a guide strand.
  • Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5'-OH by H rather than O-Me.
  • nucleic acid silencing agents may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see US Application No. 11/698,689, filed January 25, 2007 which is incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g.
  • the RNA silencing agents of the invention are modified by the introduction of at least one universal nucleotide in the antisense strand thereof.
  • Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A,G,C,U).
  • a universal nucleotide is preferred because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA.
  • Exemplary universal nucleotide include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g. 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'- deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate- inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine.
  • the universal nucleotide is an inosine residue or a naturally occurring analong thereof.
  • RNA silencing agents of the invention are preferably modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (ie. the nucleotide which recognizes the disease- related polymorphism).
  • the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide.
  • the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (ie.
  • the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity- determining nucleotide.
  • the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.
  • cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body.
  • the invention includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked.
  • the invention also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3' terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like).
  • Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.
  • Preferred nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone).
  • the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom.
  • the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphothioate group.
  • the 2' OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or ON, wherein R is Ci-C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
  • the modifications are 2'-fluoro, 2'-amino and/or T- thio modifications.
  • Particularly preferred modifications include 2'-fluoro-cytidine, T- fluoro-uridine, 2'-fluoro-adenosine, 2'-fluoro-guanosine, 2'-amino-cytidine, 2'-amino- uridine, 2'-amino-adenosine, 2'-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino-allyl-uridine.
  • the 2'-fluoro ribonucleotides are every uridine and cytidine.
  • Additional exemplary modifications include 5-bromo- uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 2'-amino- butyryl-pyrene -uridine, 5-fluoro-cytidine, and 5-fluoro-uridine.
  • 2'-deoxy-nucleotides and 2'-0me nucleotides can also be used within modified RNA-silencing agent moieties of the instant invention.
  • Additional modified residues include, for example, deoxy- abasic, inosine, N3-methyl-uridine, N6, N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin.
  • the 2' moiety is a methyl group such that the linking moiety is a 2'-O-methyl oligonucleotide.
  • the RNA silencing agent of the invention comprises Locked Nucleic Acids (LNAs).
  • LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids, with possible modifications such as 2'-deoxy-2"-fluorouridine.
  • the RNA silencing agent of the invention comprises Peptide Nucleic Acids (PNAs).
  • PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).
  • nucleobase-modified ribonucleotides i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase.
  • Bases may be modified to block the activity of adenosine deaminase.
  • modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.
  • Exemplary chemical modifications can include one or more of: (i) alteration, e.g., replacement, of a phosphate group in the phosphate backbone of an RNA molecule (e.g., replacement of the phosphate group with a siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and/or methyleneoxymethylimino); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose; (iii) modification or replacement of a naturally occurring nucleotide base; (
  • modifications include: (a) 2' modification, e.g., provision of a 2' OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, or provision of a 2' OMe moiety in a 3' overhang, e.g., at the 3' terminus (3' terminus means at the 3' atom of the molecule or at the most 3' moiety, e.g., the most 3' P or 2' position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a P with an S; (c) replacement of the U with a C5 amino linker; (d) replacement of an A with a G (sequence changes are preferred
  • Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.
  • Yet other exemplary modifications include the use of a methylated P in a 3' overhang, e.g., at the 3' terminus; combination of a 2' modification, e.g., provision of a 2' O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3' overhang, e.g., at the 3' terminus; modification with a 3' alkyl; modification with an abasic pyrolidine in a 3' overhang, e.g., at the 3' terminus; modification with naproxen, ibuprofen, or other moieties which inhibit degradation at the 3' terminus.
  • nucleic acid silencing agents include, but are not limited to, the following agents: A. RNA Silencing Agents i. siRNA Molecules
  • An exemplary siRNA molecule of the invention is a duplex consisting of a sense strand and complementary antisense or guide strand
  • the antisense strand comprises a sequence (herein, "guide strand sequence” or “antisense strand sequence”) having sufficient complementary to an mRNA (e.g., a HCV-related mRNA) to mediate RNA silencing of the mRNA via an RNA silencing mechanism (e.g., RNAi).
  • the antisense strand sequence may be perfectly complementary to the mRNA, although perfect complementarity to the mRNA is not required to mediate RNA silencing.
  • the antisense strand sequence has at least 80% sequence complementarity (e.g., 80%, 85%, 90%, 92%, 95%, or more sequence complementarity) to a corresponding sequence in a target mRNA.
  • the antisense sequence may form one or more mismatches (e.g., non- Watson Crick base pairs) with the target mRNA sequence (e.g., 1, 2, 3, 4, or 5 mismatches) and still mediate RNA silencing of the target mRNA.
  • the antisense strand sequence may comprise about 10-50 contiguous nucleotides (or nucleotide analogs). More preferably, the antisense sequence may comprise about 18-30 contiguous nucleotides, about 20-25 contiguous nucleotides, or about 21-23 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. In preferred embodiments, most or substantially all of the nucleotides of the antisense sequence of the siRNA moleucle are ribonucleotides (or ribonucleotide analogs).
  • the antisense strand of the siRNA molecule consists of antisense strand sequence without any additional nucleotides.
  • the antisense strand comprises an antisense strand sequence with one or more additional nucleotides flanking either end of the sequence. These additional nucleotides (termed “flanking nucleotides”) may lack complementarity to target mRNA sequence and are not necessary for mediating RNA silencing.
  • the additional nucleotides are processed from the siRNA by cellular enzymes leaving the antisense strand sequence intact.
  • the siRNA may comprise a sense strand of siRNA may comprise a sequence of contiguous nucleotides that is perfectly complementary to, and the same length as, the antisense strand sequence of the antisense strand.
  • sense strand sequence may, however, comprise greater or fewer number of nucleotides.
  • the sense strand sequence may comprise a small number of extra nucleotides (e.g., 1, 2, or 3) which form bulges in the duplexed siRNA molecule when the sense and antisense strands are aligned.
  • the sense strand sequence may form one or more mismatched base pairs (e.g., 1, 2, 3, 4, or 5 mismatches) with nucleotides in the antisense strand sequence.
  • the siRNA molecule comprises strands having a total length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). More preferably, the strands of the siRNA molecule have a length from about 18-30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target mRNA.
  • the sense and antisense strands are each about 20-25 nucleotides in length (e.g., 21-23 nucleotides in length).
  • most or substantially all of the nucleotides of the siRNA molecule are ribonucleotides (or ribonucleotide analogs).
  • at least 75% (more preferably 80%, 85%, 90%, 95%, or more) of the nucleotides are ribonucleotides (or ribonucleotide analogs).
  • the strands of an siRNA molecule may be aligned such that there are about 1-5 (e.g., 1, 2, or 3) bases at the end of the strands which do not align (i.e., for which no complementary bases occur in the opposing strand) such that an overhang of about 1-5 residues occurs at one or both ends of the duplex when strands are annealed.
  • the overhangs are present on one or both of the 3' ends of the duplex to form 3' overhangs. 5' overhangs are also contemplated.
  • the strands of the siRNA molecules are aligned such that one or both ends of the siRNA molecule are blunt-ended.
  • the total length of an siRNA molecule includes any overhangs which may be present.
  • an siRNA molecule comprises a sense strand of 18 nucleotides and an antisense strand of 20 nucleotides
  • the total length of the siRNA molecule is considered to be 20 nucleotides.
  • the siRNA molecule has a total length from about 10-50 or more nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a length from about 18 -30, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides (or nucleotide analogs).
  • siRNA molecules employed in the invention preferably include a 5' phosphate group (e.g., a 5 '-monophosphate) on the terminal nucleotide at the 5' end of the antisense strand. Accordingly, siRNA molecules may be synthesized to incorporate a 5' phosphate on one or both 5' ends of the molecule, or they may be phosphorylated in vitro or in vivo by cellular enzymes.
  • a 5' phosphate group e.g., a 5 '-monophosphate
  • siRNA molecules may be synthesized to incorporate a 5' phosphate on one or both 5' ends of the molecule, or they may be phosphorylated in vitro or in vivo by cellular enzymes.
  • siRNA and other dsRNA silencing agents, such as shRNAs
  • shRNAs may be designed to facilitate enhanced efficacy and specificity in mediating gene silencing (e.g., via RNAi).
  • sequence criteria may be employed to select dsRNA silencing agent with enhanced activity.
  • the silencing agent satisfies one or more of the following sequence criteria: (a) low internal stability at the 5' end of the antisense strand; (b) low propensity to form internal hairpins; (c) presence of adenosine (A) and/or absence at cytosine (C) at position 19 (P19) of the sense strand sequence; (d) presence of uridine (U) at position PlO; (e) presence of adenosine (A) at position P3; and (f) absence of guanosine (G) at position P13.
  • sequence criteria : (a) low internal stability at the 5' end of the antisense strand; (b) low propensity to form internal hairpins; (c) presence of adenosine (A) and/or absence at cytosine (C) at position 19 (P19) of the sense strand sequence; (d) presence of uridine (U) at position PlO; (e) presence of adenosine (A
  • dsRNA silencing agents with enhanced sequence asymmetry are preferably selected (see International Publication No. WO 2005/001045, US Publication No. 2005-0181382 Al).
  • the RNA silencing agent may have fewer G:C base pairs at the 5' end of the first or antisense strand than the 5' end of the sense strand.
  • RNA silencing agent may contain a greater number of mismatched or wobble base pairs at the 5' end of antisense strand than the 5' end of the sense strand.
  • the RNA silencing agent may modified such that its asymmetry is further enhanced.
  • sequence of one or both 5 'ends may be modified such there fewer G:C base pairs or a larger number of mismatches or wobbles at the 5' end of the antisense strand than the 5' end of the sense strand.
  • Such alterations favor entry of the antisense strand sequence of the RNA silencing agent into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there are fewer G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion than between the 3' end of the first or antisense strand and the 5' end of the sense strand portion.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion.
  • the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G,
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5' end of the first or antisense strand and the 3' end of the sense strand portion.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I).
  • the base pair is selected from the group consisting of an LA, LU and LC.
  • the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a modified nucleotide.
  • the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino- G, and 2,6-diamino-A.
  • the siRNA may be incubated with cDNA in a Drosophila-based in vitro mRNA expression system. Radiolabeled with 32 P, newly synthesized mRNAs are detected autoradiographically on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity.
  • Suitable controls include omission of siRNA and use of wild-type cDNA.
  • control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene.
  • negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome.
  • negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. ii. miRNA Molecules
  • the nucleic acid silencing agents are similar or identical in sequence to that of a miRNA.
  • miRNAs are naturally-occurring noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level during plant and animal development. At least 5000 miRNAs have been identified to date and together they are thought to comprise a significant fraction (1-5%) of all predicted genes in the genome.
  • miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos -Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., MoI. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai EC et al., Genome Bio., 2003).
  • homology-based searches Pasquinelli et al., 2000; Lagos -Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001
  • computer algorithms e.g. MiRScan, MiRSeeker
  • Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses (Lagos -Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos -Quintana et al.,Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr.
  • miRNAs can exist transiently in vivo as a double- stranded duplex but only one strand is taken up by the RISC complex to direct gene silencing.
  • Certain miRNAs e.g. plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs.
  • Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs.
  • the degree of complementarity between an miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism.
  • the miRNA sequence has substantial sequence identity (i.e., 80% or greater sequence identity, e.g., 80%, 85%, 90%, 95% or 100% sequence identity) to that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder.
  • exemplary miRNAs whose expression or activity is correlated with disease include lin-4, let-7, miR-10, mirR-15, miR-16, miR-122, miR-168, miR-175, miR-181, miR-196, miR-216, miR-217, miR-221, or miR-222 and their homologs.
  • the naturally-occurring miRNA sequence is derived from a viral miRNA, i.e., a viral miRNA expressed by a virus, e.g., HCV virus.
  • a viral miRNA i.e., a viral miRNA expressed by a virus, e.g., HCV virus.
  • exemplary viral miRNAs include miR-155 (miR-K12-ll) which is expressed by the Kaposis's Sarcoma associated Herpesvirus.
  • siRNA-like molecules of the invention have a sequence (i.e., have a strand having a sequence) that is "sufficiently complementary" to a heterozygous SNP of a htt mRNA to direct gene silencing either by RNAi or translational repression.
  • siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between the sense strand and target RNA approximates that observed between an miRNA and its target. In general, as the degree of sequence identity between a msiRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased.
  • the msiRNA sequence has partial complementarity with the target gene sequence.
  • the msiRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e.g. within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., MoI. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003).
  • complementarity sites e.g., 2, 3, 4, 5, or 6
  • the capacity of a siRNA-like duplex to mediate RNAi or translational repression may be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the site of complementarity.
  • At least one non-identical nucleotide is present in the central portion of the complementarity site so that duplex formed by the msiRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003).
  • 2, 3, 4, 5, or 6 contiguous or non-contiguous non- identical nucleotides are introduced.
  • the non-identical nucleotide may be selected such that it forms a wobble base pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G, A:A, C:C, U:U).
  • the "bulge" is centered at nucleotide positions 12 and 13 from the 5'end of the msiRNA molecule.
  • Short Hairpin RNA RNAs
  • engineered RNA precursors are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the invention).
  • desired RNA silencing agents e.g., siRNAs of the invention.
  • a shRNA molecule is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.
  • the requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion.
  • the two portions need not be fully or perfectly complementary.
  • the first and second "stem” portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a "loop" portion in the shRNA molecule.
  • the shRNA molecules are processed to generate siRNAs.
  • shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide "loop" in a portion of the stem, for example a one-, two- or three-nucleotide loop.
  • the stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides.
  • the overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.
  • one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the heterozygous SNP.
  • one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi).
  • RNAi RNA interference
  • engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions.
  • the antisense portion can be on the 5' or 3' end of the stem.
  • the stem portions of a shRNA are preferably about 15 to about 50 nucleotides in length.
  • the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length.
  • the length of the stem portions should be 21 nucleotides or greater.
  • the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway.
  • the stem can be longer than 30 nucleotides.
  • the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).
  • a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).
  • the two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem.
  • the two portions can be, but need not be, fully or perfectly complementary.
  • the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides.
  • the overhanging nucleotides can include, for example, uracils (Us), e.g., all Us.
  • the loop in the shRNAs or engineered RNA precursors may differ from natural pre-msiRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences.
  • the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.
  • the loop in the shRNAs or engineered RNA precursors may differ from natural pre-msiRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences.
  • the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.
  • a preferred loop consists of or comprises a "tetraloop" sequences.
  • Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.
  • shRNAs of the invention include the sequences of a desired siRNA molecule described supra.
  • the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotide, or longer, sequence from within the target RNA (e.g., SODl or htt mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation.
  • the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5' UTR (untranslated region), coding sequence, or 3' UTR, provided said portion is distant from the site of the gain-of-function muation.
  • This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides.
  • the last two nucleotides of the nucleotide sequence can be selected to be UU.
  • This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA.
  • This sequence can replace a stem portion of a wild-type pre-msiRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized.
  • DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-msiRNA.
  • Engineered RNA precursors include in the duplex stem the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo.
  • the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited.
  • the two 3' nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.
  • Embodiments of the invention feature a wide variety of possible nucleic acid- based and non-nucleic acid-based silencing agents.
  • the activity of a silencing agent may belong to one category of activity ascribed to such agents.
  • the activity of a silencing agent may belong to two or more categories of activity ascribed to such agents and/or to nucleic acids.
  • Some types of RNA silencing agents for use in preferred embodiments of the invention include RNAi, siRNA, miRNA, rasiRNA, shRNA, smRNA, dsRNA, tncRNA, piRNA.
  • RNA silencing agents for use with particular embodiments of the invention may have activity belonging to the categories of agents described above or may have activity ascribed to other categories of RNA agent. In some embodiments, the particular activity of a silencing agent may not be characterized.
  • a nucleic acid molecule employed in a composition of the invention is a nucleic acid molecule other than an RNA silencing agent.
  • said nucleic acid molecules may comprise any of the chemical modifications discussed supra.
  • a nucleic acid molecule employed in the invention is an antisense nucleic acid molecule that is complementary to a target mRNA or to a portion of said mRNA, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • Antisense nucleic acid molecules are generally single- stranded DNA, RNA, or DNA/RNA molecules which may comprise one or more nucleotide analogs.
  • the use of antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol.
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the target mRNA sequence and accordingly is capable of hydrogen bonding to the mRNA.
  • Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5' or 3' untranslated region of the mRNA or a region bridging the coding region and an untranslated region (e.g., at the junction of the 5' untranslated region and the coding region).
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon in the 3' untranslated region of an mRNA.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of an mRNA, but more preferably is antisense to only a portion of the coding or noncoding region of an mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of a target mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 100, 500, 1000 nucleotides or more in length. In some embodiments, the antisense oligonucleotide may be as long as, or longer than, the length of the mRNA that is targeted.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5 -carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, S'-
  • an antisense nucleic acid can be produced biologically using an expression vector into which all or a portion of a cDNA has been subcloned in an antisense orientation (i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • the antisense expression vector is prepared according to standard recombinant DNA methods for constructing recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense oligonucleotides may be employed which are complementary to one or more of the RNA silencing agents (e.g., miRNA molecules) described supra.
  • Said anti-miRNA oligonucleotides may be DNA or RNA oligonucleotides, or they may be comprised of both ribonucleotide and deoxyribonucleotides or analogs thereof.
  • said anti-miRNA oligonucleotides comprise one or more (e.g., substantially all) 2'0-methyl ribonucleotides.
  • Such molecules are potent and irreversible inhibitors of miRNA- mediated silencing and are therefore useful for modulating RNA silencing both in vitro and in vivo.
  • In vivo methodologies are useful for both general RNA silencing modulatory purposes as well as in therapeutic applications in which RNA silencing modulation (e.g., inhibition) is desirable.
  • insulin secretion has been shown to be regulated by at least one miRNA (Poy et al. 2004), and a role for miRNAs has also been implicated in spinal muscular atrophy (SMA; Mourelatos et al. 2002).
  • a nucleic acid molecule employed in the invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).
  • Such a nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
  • rasiRNA repeat-associated siRNA
  • rasiRNAs arise mainly from the antisense strand. rasiRNAs are involved in the shutdown of expression of transpo sable elements, histone modification and DNA methylation modification. They function via partly overlapping components of the siRNA pathway (See, e.g., Vagin,V.V. et al.,(2006) A distinct small RNA pathway silences selfish genetic elements in the germline. Science 313:320-324).
  • tncRNA tiny non-coding RNA
  • RNA interference An emerging generation of biologicals. BiotechnoL J. 3:339-353).
  • piRNA v. piwi-interacting RNA
  • piRNA is a class of small RNA molecules that is expressed uniquely in mammalian testes and forms RNA-protein complexes with Pi wi proteins. These piRNA complexes have been linked to transcriptional gene silencing of retrotransposons and other genetic elements in germ-line cells, particularly those in spermatogenesis. Purification of these complexes has revealed that these oligonucleotides are approximately 29-30 nt long. They are distinct in size from miRNA and are associated with distinct protein complexes. It remains unclear how piRNAs are generated, but their biogenesis pathway is distinct from miRNA and siRNA (See, e.g., Carthew R.W. (2006). A New RNA Dimension to Genome Control. Science 313:305-306).
  • HCV Hepatitis C virus
  • HCV infection is the second leading cause of chronic hepatits, liver cirrhosis and hepatocellular carcinoma, affecting more than 170 million people worldwide.
  • HCV contains a single positive- strand RNA genome that consists of about 9,600 nucleotides that encode a single polyprotein composed of 3,010 amino acid residues.
  • the present invention includes a method of using a complex or a molecule comprising a targeting moiety to deliver therapeutic RNA molecules effectively to Hepatitis C virus or to a cell being infected or having the potential to be infected by Hepatitis C virus, resulting in the prevention, immunization, inhibition, cure, or other beneficial effects towards Hepatitis C virus infection.
  • the therapeutic RNA molecules are RNA interference agents ⁇ e.g., siRNAs, shRNAs, miRNA, smRNA, rasiRNA, tncRNA, piRNA, etc.), with or without modifications.
  • the therapeutic RNA molecules are designed to antagonize one or more elements promoting or positively affecting the infection cycle of Hepatitis C virus, which includes, but is not limited to, the stability and activity of Hepatitis C virus itself, the virus entry into a cell, the replication and translation of viral RNAs and proteins in a cell, the up-regulation of host proteins in the infection cycle of HCV, the virus packaging and exit from a cell, the transport and spread of Hepatitis C virus in a mammal, etc.
  • the therapeutic RNA molecules are designed to promote or positively affect one or more elements inhibiting or negatively affecting the infection cycle of Hepatitis C virus.
  • the invention provides a method of RNA interference in a cell, which is or is not infected by Hepatitis C virus.
  • the RNA interference is performed in an animal, preferably a mammal, most preferably a human, which is or is not infected by Hepatitis C virus.
  • the RNA interference agents are designed to recognize the genomic RNA sequence, or a RNA sequence complementary to the genomic RNA sequence, of Hepatitis C virus.
  • the RNA interference agent is designed to recognize the mRNA sequence, or a RNA sequence complementary to the mRNA sequence, encoding each of viral proteins, i.e., C (a.k.a. core protein), El, E2, NSl (a.k.a. P7), NS2, NS3, NS4A, NS4B, NS5A and NS5B.
  • the RNA interference agent is designed to recognize the mRNA sequences, or RNA sequences complementary to the mRNA sequences, encoding multiple viral proteins selected from the above groups.
  • the RNA interference agents are designed to recognize the mRNA sequences, or RNA sequences complementary to the mRNA sequences, encoding the borders or junctions on the various viral proteins, selected from the above groups, after their cleavage from the single 3010 amino acid- containing viral polyprotein.
  • the RNA interference agents are designed to recognize the mRNA sequences, or RNA sequences complementary to the mRNA sequences, in the overlapping reading frames, e.g., +1, +2, +3, -1, -2, or -3.
  • ARFP alternative reading frame protein
  • F frameshift
  • the RNA interference agents recognize each or multiple of untranslated regions (UTR) at the 5' and 3' ends of the HCV genomic RNA, which may fine -regulate expression of the virus genes.
  • the RNA interference agents recognize a RNA sequence complementary to each or multiple of untranslated regions (UTR) at the 5' and 3' ends of the HCV genomic RNA.
  • the RNA intereference agents recognize the mRNA sequence, or a RNA sequences complementary to the mRNA sequence, representing the ribosome binding site in the 5' UTR of Hepatitis C virus.
  • the RNA interference agent recognized the mRNA sequence, or a RNA sequence complementary to the mRNA sequence, of viral proteases, e.g., NS2-3 and NS3-4A, which are able to cleave the single viral polyprotein into individual proteins.
  • the RNA interference agent recognized the mRNA sequence, or a RNA sequence complementary to the mRNA sequence of host endoplasmic reticulum (ER) signal peptidase(s) (e.g., see Reed K.E. and Rice CM.
  • ER endoplasmic reticulum
  • the RNA interference agents recognize the regions of viral genes that are important for the functional protein-protein interactions between the said viral products and others viral or host proteins. These interactions include, for example, the heterodimers between El and E2 proteins, the hexamers of P7 proteins, NS5A dimers, the interaction between C and E1/E2, etc. In some embodiments, the RNA interference agents are designed to antagonize the virus entry process.
  • the RNA interference agents recognize the mRNA sequence, or a RNA sequence complimentary to the mRNA sequence, of HCV envelope glycoproteins El and E2. In one preferred embodiment, the RNA interference agents recognize the hypervariable regions (HVR) in El and/or E2. In one embodiment, the RNA interference agents recognize the receptors for HCV particles on the surfaces of host cells, e.g., hepatocytes, lymphocytes, monocytes, etc.
  • RNA interference agents are designed to down-regulate the expressions of ubiquitous or specific receptors/co-f actors.
  • RNA interference agents antagonize the mechanisms (e.g., host tropism) of HCV infection and entry of host cells, including binding of Hepatitis C virus to cellular receptors, the following endocytosis and then the release of viral nucleocapsid or other proteins into the cytoplasm of the cell.
  • the RNA interference agents recognize members of the cellular endocytosis machinery, e.g., the clathrin network, which facilitates the Hepatitis C virus entry into the cell.
  • the RNA interference agents antagonize the release of single virus particles from endosomal compartments into the cytoplasm, which is associated with membrane fusion and virus uncoating.
  • Hepatitis C virus have developed mechanisms to utilize certain eukaryotic translation initiation factors (elFs), and thus hijack the translational machinery for their own profit.
  • This invention also includes methods to design RNA interference agents to antagonize the virus replication and translation process in infected cells.
  • the RNA interference agent is designed to recognize the 5' UTR of the HCV genome, preferably one or multiple domain sequences of domains I-IV in the 5'UTR.
  • the RNA interference agent recognizes the IRE sequence on the HCV genome.
  • the IRE sequence comprises nearly the entire 5' UTR of the HCV genome and can also include part of the first coding sequence, e.g., the first 12 to 30 nt.
  • the RNA interference agents are designed to recognize members of the host replication machinery facilitating the replication of HCV.
  • the RNA interference agent down-regulates the expression of these host proteins.
  • the RNA interference agent antagonizes the interaction of these host proteins with HCV viral genome, preferably the viral IRE sequence.
  • host proteins include, for example, the 43S ribosomal complex, containing a small 4OS ribosomal subunit, eukaryotic initiation factor (elF) 3, and a tRNA-eIF2-GTP ternary complex.
  • elF eukaryotic initiation factor
  • Other cellular factors such as La autoantigen, heterogeneous ribonucleoprotein L, poly-C binding protein, and pyrimidine tract-binding protein, also bind to the IRES element and modulate viral translation.
  • the RNA interference agent recognizes the 3' UTR of the HCV genome, preferably one or multiple domains of a variable region of about 40 nt, a variable length poly(U/UC) tract, and a highly conserved, 98-nt 3' terminal segment (3 'X) that putatively forms three stem-loop structures.
  • the RNA interference agent is designed to recognize the host signal peptidases that process the viral polyprotein. In one preferred embodiment, the RNA interference agent down- regulates the expression of these host signal peptidases.
  • the RNA interference agent is designed to recognize the viral proteases that process the viral polyprotein, e.g., the NS2-3 protease, which spans NS2 and the N-terminal domain of NS3, and the NS3-4A protease, which contains NS4A as a cofactor of the NS3 serine protease.
  • the RNA interference agent down-regulates the expression of these viral proteases.
  • the RNA interference agent is designed to recognize the sequence of these host and/or viral proteases to antagonize their interaction with HCV genome.
  • the RNA interference agent is designed to antagonize the maturation and/or post-translational regulation of viral proteins.
  • the RNA interference agent is designed to antagonize the expression or activity of host machinery responsible for HCV protein maturation and/pr post-translational modifications.
  • the RNA interference agent is designed to antagonize the expression or activity of host proteins in ubiquitin-proteasome pathway. These host proteins may include, for example, E6-AP and other HECT-domain E3 ligases, the proteasome activator PA28 ⁇ , which binds to HCV C protein, etc.
  • the RNA interference agents are designed to antagonize the HCV RNA replication.
  • HCV is assumed to replicate its genome through the synthesis of a full-length negative- strand RNA. Positive-strand RNA is then produced from the negative- strand template; it is several-fold more abundant than the negative-stranded RNA and is utilized for translation, replication, and packaging into progeny viruses.
  • the RNA interference agent is complementary to and recognizes the positive- strand RNA of HCV.
  • the RNA interference agent is complementary to and recognizes the negative- strand RNA of HCV.
  • the RNA interference agents recognize and antagonize viral or host factors important for conferring proper RNA replication.
  • viral or host factors may include, for example, the NS5B, NS3, NS4B, NS5A viral proteins, host proteins interacting with these viral proteins, e.g., human vesicle-associated membrane protein associated proteins (h V AP-A and -B), FB L2, cyclophylin B and other components in the replication complexes (RCs).
  • NS5B NS3, NS4B
  • NS5A viral proteins e.g., human vesicle-associated membrane protein associated proteins (h V AP-A and -B), FB L2, cyclophylin B and other components in the replication complexes (RCs).
  • the RNA interference agents antagonize the localization of the viral RNA or proteins within cellular structures. In one preferred embodiment, the RNA interference agent antagonizes the transportation process and host transportation machinery for transporting HCV RNA or proteins into the ER system. In another embodiment, the RNA interference agent antagonizes the transportation process and host transportation machinery for transporting HCV RNA or proteins out of the ER system and into the Golgi fraction. In other embodiments, the RNA interference agents antagonize the transportation process and host transportation machinery for transporting HCV RNA or proteins out of the Golgi fraction.
  • the present invention includes methods to design RNA interference agents to antagonize the assembly and budding process to release mature Hepatitis C virus out of the infected cell.
  • the RNA interference agents recognize the mRNA sequence, or a RNA sequence complimentary to the mRNA sequence, of HCV envelope glycoproteins El and E2.
  • the RNA interference agents recognize the hypervariable regions (HVR) in El and/or E2.
  • the RNA interference agent down-regulates the expression of viral proteins facilitating the formation of the nonenveloped nucleocapsids. These viral proteins include, for example, the C proteins.
  • the RNA interference agent is designed to recognize the sequences of viral and host proteins, as exemplified above.
  • the RNA interference agents are designed to antagonize the formation of enveloped virions.
  • the RNA interference agent down-regulates the expression of viral C and/or E1/E2 proteins.
  • the RNA interference agent is designed to the regions of sequences of these proteins for their interactions.
  • the RNA interference agents antagonize the expression of viral proteins or host proteins capable of inducing cellular membrane curvature and budding off virus particles.
  • the RNA interference agents antagonize the induction of cellular gene expression by Hepatitis C virus.
  • the RNA interference agents antagonize the expression of gene products that are specific to hepatocyte or have a selective effect on hepatocyte functions.
  • the gene products include growth factors, for example, FHF-4 (fibroblast growth factor homologous factor 4) and neuroleukin (a neurotropic growth factor).
  • the gene products include cell-cycle-related proteins, for example, Cyclin Dl, CDK4, CCNG2, BZAP45, etc.
  • the gene products include cell proliferation-related proteins, for example, SSRl, ANXA2, SlOOAlO, PTMA, proteins in Mitogen- Activated Protein Kinase (MAPK) and NF-KB signaling pathways, etc.
  • the gene products include CXC (or ⁇ - chemokines) and CC (or ⁇ -chemokines) chemokine ligands.
  • the host gene products include non-coding microRNAs (miRNAs) associated with the carcinogenic process of liver cancers resulted from HCV infection (for example, see Murakami Y. et al., "Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues.
  • RNA interference agents are designed to recognize and antagonize the expression and/or activity of other gene products resulting from Hepatitis C virus infection.
  • HBXIP Hepatitis B X-interacting protein
  • HBXAP Hepatitis B virus X-associated protein
  • c-FLIP FLICE inhibitory protein
  • PPAR- ⁇ Peroxisome proliferators-activated receptor ⁇
  • PPAR- ⁇ RNA polymerase II subunit 5
  • TATA-binding protein TBP
  • TFIIH TATA-binding protein
  • CBP CBP
  • RXR retinoid X receptor
  • ASC-2 Jun activation domain-binding protein 1
  • Jabl Jun activation domain-binding protein 1
  • HVDAC3 proteasome subunits
  • PSMA7/PSMC1, Skp2 Serine/threonine protein phosphatase PP2C-0C
  • lymphotoxin- ⁇ receptor LT- ⁇ R
  • SpIlOb a repressor for
  • RNA interference agents to antagonize Hepatitis C virus as an IFN antagonist.
  • the present invention also includes methods to design RNA interference agents0 to antagonize the anti-apoptotic or pro-apoptotic activity of Hepatitis C virus.
  • the RNA interference agents antagonize the expression of Fas, FasL or other components in the Fas-FasL signaling pathway induced by Hepatitis C virus infection.
  • the RNA interference agents antagonize the expression and/or activity of cellular anti-apoptotic molecules, which include, for example, FADD, procaspase-8, FLIP, BH3-only proteins (such as Bid, tBid, etc.), Bax, Bak, Bcl-2 family proteins, Bcl-xL, cytochrome c, Apaf-1, caspase-9, Smac/DIABLO, inhibitors of apoptotic proteins (IAPs) proteins, procaspase-3, ICAD, etc.
  • HCV may circulate in various forms in the sera of infected hosts, for example, as
  • the present invention includes methods to design RNA interference agents to antagonize the transport and spread process of Hepatitis C virus in vivo.
  • the RNA interference agents down-regulated the expression of the host proteins facilitating the circulation and spread of HCV. These host proteins include, but are not limited to, low-density lipoproteins, very low density lipoproteins, immunoglobulins, etc.
  • the RNA interference agents antagonize viral proteins to disrupt the balance of replication and transcription of Hepatitis C virus. In another embodiment, the RNA interference agents antagonize the cell mobility stimulated by viral infection, which helps to spread Hepatitis C virus.
  • the invention relates RNA silencing agent (e.g., an siRNA or other dsRNA agents) which is complementary to at least a portion of a 5'- untranslated region (5'-UTR) of HCV.
  • RNA silencing agent e.g., an siRNA or other dsRNA agents
  • the nucleotide sequence is within a highly conserved region of the 5'-UTR.
  • an RNA silencing agent is complementary to at least a portion of a 3'- untranslated region (3'-UTR) of HCV.
  • the nucleotide sequence is within a highly conserved region of the 3'-UTR.
  • the complementary RNA strand comprises the nucleotide sequence of ggaccuuucacagcuagccg uga and the sense RNA strand comprises the nucleotide sequence of acggcuagcugugaaagguccgu.
  • Additional exemplary anti-HCV RNA silencing agents are described in US Patent No. 7,348,314, filed March 7, 2003, International PCT Publication No. WO 03/070750, filed February 20, 2003; International PCT Publication No. WO 05/028650, filed September 15, 2004; International PCT Publication No. WO 05/107816, filed November 17, 2005; International PCT Publication No. WO 06/031901, filed September 12, 2005; International PCT Publication No. WO 06/039656, filed September 30, 2005; and International PCT Publication No. WO 07/076328, filed December 18, 2006; each of which is incorporated by reference herein in its entirety.
  • the antisense oligonucleotide targets miR-122 such that hepatitis C virus (HCV) infection is inhibited.
  • miR-122 miR-122 is specifically expressed in liver cells and has been identified as an endogenous host gene or host factor required for viral infection by HCV. miR-122 interacts directly with a specific sequence in the 5' non-coding sequence region of the HCV genome leading to increased abundance of autonomously replicating Hepatitis C viral RNAs (see Jopling et al., Science, (2005), 309(5740):1577-81).
  • the antisense oligonucleotide (e.g., an "AntagomiR") is sufficiently complementary to miR-122 to inhibit the viral replication function of miR-122.
  • the antisense oligonucleotide comprises the sequence
  • certain embodiments of the invention feature a targeting moiety that can deliver a therapeutic payload to a virus (e.g., an HCV or HCV-like virus) or a cell (e.g., a virally infected cell or a cell that has the capacity to be infected by a virus).
  • a virus e.g., an HCV or HCV-like virus
  • a cell e.g., a virally infected cell or a cell that has the capacity to be infected by a virus.
  • binding proteins including antibodies, antibody fragments, molecules with regions, domains or portions derived from antibodies, and non-antibody binding proteins, that can function as a targeting moiety.
  • the instant invention is not limited to traditional antibodies and may be practiced through the use of binding proteins or polypeptides, antibody fragments and antibody mimetics.
  • binding polypeptide, antibody fragment and antibody mimetic technologies have now been developed and are widely known in the art. While a number of these technologies, such as domain antibodies, Nanobodies, and UniBodies make use of fragments of, or other modifications to, traditional antibody structures, there are also alternative technologies, such as Affibodies, DARPins, Anticalins, Avimers, and Versabodies that employ binding structures that, while they mimic traditional antibody binding, are generated from and function via distinct mechanisms.
  • Domain Antibodies are the smallest functional binding units of antibodies, corresponding to the variable regions of either the heavy (VH) or light (VL) chains of human antibodies. Domain Antibodies have a molecular weight of approximately 13 kDa. Domantis has developed a series of large and highly functional libraries of fully human VH and VL dAbs (more than ten billion different sequences in each library), and uses these libraries to select dAbs that are specific to therapeutic targets. In contrast to many conventional antibodies, Domain Antibodies are well expressed in bacterial, yeast, and mammalian cell systems.
  • Nanobodies are antibody-derived therapeutic proteins that contain the unique structural and functional properties of naturally-occurring heavy-chain antibodies. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a perfectly stable polypeptide harbouring the full antigen-binding capacity of the original heavy-chain antibody. Nanobodies have a high homology with the VH domains of human antibodies and can be further humanized without any loss of activity. Importantly, Nanobodies have a low immunogenic potential, which has been confirmed in primate studies with Nanobody lead compounds. Nanobodies combine the advantages of conventional antibodies with important features of small molecule drugs.
  • Nanobodies Like conventional antibodies, Nanobodies show high target specificity, high affinity for their target and low inherent toxicity. However, like small molecule drugs they can inhibit enzymes and readily access receptor clefts. Furthermore, Nanobodies are extremely stable, can be administered by means other than injection (see e.g. WO 04/041867, which is herein incorporated by reference in its entirety) and are easy to manufacture. Other advantages of Nanobodies include recognizing uncommon or hidden epitopes as a result of their small size, binding into cavities or active sites of protein targets with high affinity and selectivity due to their unique 3-dimensional, drug format flexibility, tailoring of half-life and ease and speed of drug discovery.
  • Nanobodies are encoded by single genes and are efficiently produced in almost all prokaryotic and eukaryotic hosts e.g. E. coli (see e.g. US 6,765,087, which is herein incorporated by reference in its entirety), molds (for example Aspergillus or
  • Nanobodies exhibit a superior stability compared with conventional antibodies, they can be formulated as a long shelf-life, ready-to-use solution.
  • the Nanoclone method (see e.g. WO 06/079372, which is herein incorporated by reference in its entirety) is a proprietary method for generating Nanobodies against a desired target, based on automated high-throughout selection of B -cells and could be used in the context of the instant invention.
  • UniBodies are another antibody fragment technology, however this one is based upon the removal of the hinge region of IgG4 antibodies. The deletion of the hinge region results in a molecule that is essentially half the size of traditional IgG4 antibodies and has a univalent binding region rather than the bivalent binding region of IgG4 antibodies. It is also well known that IgG4 antibodies are inert and thus do not interact with the immune system, which may be advantageous for the treatment of diseases where an immune response is not desired, and this advantage is passed onto UniBodies. For example, UniBodies may function to inhibit or silence, but not kill, the cells to which they are bound. Additionally, UniBody binding to cancer cells do not stimulate them to proliferate.
  • UniBodies are about half the size of traditional IgG4 antibodies, they may show better distribution over larger solid tumors with potentially advantageous efficacy. UniBodies are cleared from the body at a similar rate to whole IgG4 antibodies and are able to bind with a similar affinity for their antigens as whole antibodies. Further details of UniBodies may be obtained by reference to patent WO2007/059782, which is herein incorporated by reference in its entirety.
  • Diabodies are bivalent, bispecific molecules in which V H and V L domains are expressed on a single polypeptide chain, connected by a linker that is too short to allow for pairing between the two domains on the same chain.
  • the V H and V L domains pair with complementary domains of another chain, thereby creating two antigen binding sites (see e.g., Holliger et al, 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al, 1994 Structure 2:1121-1123).
  • Diabodies can be produced by expressing two polypeptide chains with either the structure V HA - V LB and V HB -V LA (V H -V L configuration), or V LA -V HB and V LB -V HA (V L -V H configuration) within the same cell. Most of them can be expressed in soluble form in bacteria.
  • Single chain diabodies are produced by connecting the two diabody- forming polypeptide chains with linker of approximately 15 amino acid residues (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(3-4): 128-30; Wu et al,
  • scDb can be expressed in bacteria in soluble, active monomeric forms (see Holliger and Winter, 1997 Cancer Immunol. Immunother., 45(34): 128-30; Wu et al, 1996 Immunotechnology, 2(l):21-36; Pluckthun and Pack,
  • a diabody can be fused to Fc to generate a "di-diabody" (see Lu et al, 2004 J. Biol. Chem., 279(4):2856-65).
  • the invention further provides binding molecules that exhibit functional properties of antibodies but derive their framework and antigen binding portions from other polypeptides ⁇ e.g., polypeptides other than those encoded by antibody genes or generated by the recombination of antibody genes in vivo).
  • the antigen binding domains ⁇ e.g., anti-HCV epitope binding domains
  • Molecules that have an overall fold similar to that of a variable domain of an antibody are appropriate scaffold proteins.
  • Scaffold proteins suitable for deriving antigen binding molecules include fibronectin or a fibronectin dimer, tenascin, N-cadherin, E-cadherin, ICAM, titin, GCSF-receptor, cytokine receptor, glycosidase inhibitor, antibiotic chromoprotein, myelin membrane adhesion molecule PO, CD8, CD4, CD2, class I MHC, T-cell antigen receptor, CDl, C2 and I-set domains of VCAM-I, I-set immunoglobulin domain of myo sin-binding protein C, I-set immunoglobulin domain of myosin-binding protein H, I-set immunoglobulin domain of telokin, NCAM, twitchin, 5 neuroglian, growth hormone receptor, erythropoietin receptor, prolactin receptor, interferon-gamma receptor, ⁇ -galactosidase/glucuronidase, ⁇ -glucuronidase, transgluta
  • the antigen binding domain (e.g., the immunoglobulin-like fold) of the non- o antibody binding molecule can have a molecular mass less than 10 kD or greater than 7.5 kD (e.g., a molecular mass between 7.5-10 kD).
  • the protein used to derive the antigen binding domain is a naturally occurring mammalian protein (e.g., a human protein), and the antigen binding domain includes up to 50% (e.g., up to 34%, 25%, 20%, or 15%), mutated amino acids as compared to the immunoglobulin-like fold of the 5 protein from which it is derived.
  • the domain having the immunoglobulin-like fold generally consists of 50-150 amino acids (e.g., 40-60 amino acids).
  • a library of clones is created in which sequences in regions of the scaffold protein that form antigen binding surfaces (e.g., regions analogous in position and structure to CDRs of an antibody variable0 domain immunoglobulin fold) are randomized.
  • Library clones are tested for specific binding to the antigen of interest (e.g., an epitope or antigen from HCV) and for other functions (e.g., inhibition of biological activity of HCV). Selected clones can be used as the basis for further randomization and selection to produce derivatives of higher affinity for the antigen.
  • 5 High affinity binding molecules are generated, for example, using the tenth module of fibronectin III ( 10 Fn3) as the scaffold.
  • a library is constructed for each of three CDR-like loops of 10 FN3 at residues 23-29, 52-55, and 78-87.
  • DNA segments encoding sequence overlapping each CDR-like region are randomized by oligonucleotide synthesis.
  • Techniques for producing selectable 10 Fn30 libraries are described in U.S. Pat. Nos. 6,818,418 and 7,115,396; Roberts and Szostak, 1997 Proc. Natl. Acad. Sci USA 94:12297; U.S. Pat. No. 6,261,804; U.S. Pat. No. 6,258,558; and Szostak et al. WO98/31700.
  • Non-antibody binding molecules can be produces as dimers or multimers to increase avidity for the target antigen.
  • the antigen binding domain is expressed as a fusion with a constant region (Fc) of an antibody that forms Fc-Fc dimers.
  • Fc constant region
  • Affibody molecules represent a new class of affinity proteins based on a 58- amino acid residue protein domain, derived from one of the IgG-binding domains of staphylococcal protein A.
  • This three helix bundle domain has been used as a scaffold for the construction of combinatorial phagemid libraries, from which Affibody variants that target the desired molecules can be selected using phage display technology (Nord K, Gunneriusson E, Ringdahl J, Stahl S, Uhlen M, Nygren PA, Binding proteins selected from combinatorial libraries of an ⁇ -helical bacterial receptor domain, Nat Biotechnol 1997; 15:772-7. Ronmark J, Gronlund H, Uhlen M, Nygren PA, Human immunoglobulin A (IgA) -specific ligands from combinatorial engineering of protein A, Eur J Biochem 2002;269:2647-55.).
  • Affibody molecules in combination with their low molecular weight (6 kDa), make them suitable for a wide variety of applications, for instance, as detection reagents (Ronmark J, Hansson M, Nguyen T, et al, Construction and characterization of affibody- Fc chimeras produced in Escherichia coli, J Immunol Methods 2002;261: 199-211) and to inhibit receptor interactions (Sandstorm K, Xu Z, Forsberg G, Nygren PA, Inhibition of the CD28-CD80 co- stimulation signal by a CD28-binding Affibody ligand developed by combinatorial protein engineering, Protein Eng 2003;16:691-7).
  • Affibodies and methods of production thereof may be obtained by reference to US Patent No 5831012 which is herein incorporated by reference in its entirety. Labelled Affibodies may also be useful in imaging applications for determining abundance of isoforms.
  • DARPins Designed Ankyrin Repeat Proteins
  • Repeat proteins such as ankyrin or leucine-rich repeat proteins, are ubiquitous binding molecules, which occur, unlike antibodies, intra- and extracellularly.
  • Their unique modular architecture features repeating structural units (repeats), which stack together to form elongated repeat domains displaying variable and modular target-binding surfaces. Based on this modularity, combinatorial libraries of polypeptides with highly diversified binding specificities can be generated. This strategy includes the consensus design of self- compatible repeats displaying variable surface residues and their random assembly into repeat domains.
  • DARPins can be produced in bacterial expression systems at very high yields and they belong to the most stable proteins known. Highly specific, high-affinity DARPins to a broad range of target proteins, including human receptors, cytokines, kinases, human proteases, viruses and membrane proteins, have been selected.
  • DARPins having affinities in the single-digit nanomolar to picomolar range can be obtained.
  • DARPins have been used in a wide range of applications, including ELISA, sandwich ELISA, flow cytometric analysis (FACS), immunohistochemistry (IHC), chip applications, affinity purification or Western blotting. DARPins also proved to be highly active in the intracellular compartment for example as intracellular marker proteins fused to green fluorescent protein (GFP). DARPins were further used to inhibit viral entry with IC50 in the pM range. DARPins are not only ideal to block protein- protein interactions, but also to inhibit enzymes. Proteases, kinases and transporters have been successfully inhibited, most often an allosteric inhibition mode. Very fast and specific enrichments on the tumor and very favorable tumor to blood ratios make
  • DARPins well suited for in vivo diagnostics or therapeutic approaches.
  • Anticalins are an additional antibody mimetic technology, however in this case the binding specificity is derived from lipocalins, a family of low molecular weight proteins that are naturally and abundantly expressed in human tissues and body fluids.
  • Lipocalins have evolved to perform a range of functions in vivo associated with the physiological transport and storage of chemically sensitive or insoluble compounds.
  • Lipocalins have a robust intrinsic structure comprising a highly conserved ⁇ -barrel which supports four loops at one terminus of the protein. These loops form the entrance to a binding pocket and conformational differences in this part of the molecule account for the variation in binding specificity between individual lipocalins.
  • lipocalins differ considerably from antibodies in terms of size, being composed of a single polypeptide chain of 160-180 amino acids which is marginally larger than a single immunoglobulin domain.
  • Lipocalins are cloned and their loops are subjected to engineering in order to create Anticalins. Libraries of structurally diverse Anticalins have been generated and Anticalin display allows the selection and screening of binding function, followed by the expression and production of soluble protein for further analysis in prokaryotic or eukaryotic systems. Studies have successfully demonstrated that Anticalins can be developed that are specific for virtually any human target protein can be isolated and binding affinities in the nanomolar or higher range can be obtained. Anticalins can also be formatted as dual targeting proteins, so-called Duocalins.
  • a Duocalin binds two separate therapeutic targets in one easily produced monomeric protein using standard manufacturing processes while retaining target specificity and affinity regardless of the structural orientation of its two binding domains.
  • Modulation of multiple targets through a single molecule is particularly advantageous in diseases known to involve more than a single causative factor.
  • bi- or multivalent binding formats such as Duocalins have significant potential in targeting cell surface molecules in disease, mediating agonistic effects on signal transduction pathways or inducing enhanced internalization effects via binding and clustering of cell surface receptors.
  • the high intrinsic stability of Duocalins is comparable to monomeric Anticalins, offering flexible formulation and delivery potential for Duocalins.
  • Anticalins can be found in US Patent No. 7,250,297 and International Patent Application Publication No. WO 99/16873, both of which are hereby incorporated by reference in their entirety.
  • Another antibody mimetic technology useful in the context of the instant invention are Avimers. Avimers are evolved from a large family of human extracellular receptor domains by in vitro exon shuffling and phage display, generating multidomain proteins with binding and inhibitory properties. Linking multiple independent binding domains has been shown to create avidity and results in improved affinity and specificity compared with conventional single-epitope binding proteins. Other potential advantages include simple and efficient production of multitarget- specific molecules in Escherichia coli, improved thermostability and resistance to proteases.
  • Avimers with sub-nanomolar affinities have been obtained against a variety of targets. Additional information regarding Avimers can be found in US Patent Application Publication Nos. 2006/0286603, 2006/0234299, 2006/0223114, 2006/0177831, 2006/0008844, 2005/0221384, 2005/0164301, 2005/0089932, 2005/0053973, 2005/0048512, 2004/0175756, all of which are hereby incorporated by reference in their entirety.
  • Versabodies are another antibody mimetic technology that could be used in the context of the instant invention. Versabodies are small proteins of 3-5 kDa with >15% cysteines, which form a high disulfide density scaffold, replacing the hydrophobic core that typical proteins have.
  • Versabodies Given the structure of Versabodies, these antibody mimetics offer a versatile format that includes multi-valency, multi- specificity, a diversity of half-life mechanisms, tissue targeting modules and the absence of the antibody Fc region. Furthermore, Versabodies are manufactured in E. coli at high yields, and because of their hydrophilicity and small size, Versabodies are highly soluble and can be formulated to high concentrations. Versabodies are exceptionally heat stable (they can be boiled) and offer extended shelf-life.
  • antibody fragment and antibody mimetic technologies are not intended to be a comprehensive list of all technologies that could be used in the context of the instant specification.
  • additional technologies including alternative polypeptide-based technologies, such as fusions of complimentary determining regions as outlined in Qui et al., Nature Biotechnology, 25(8) 921-929 (2007), which is hereby incorporated by reference in its entirety, as well as nucleic acid- based technologies, such as the RNA aptamer technologies described in US Patent Nos.
  • Embodiments of the invention include conjugates formed from monoclonal and polyclonal antibodies raised to antigens and epitopes of HCV. A variety of techniques known in the art may be used to generate antibodies for use with embodiments of the invention.
  • Monoclonal antibodies can be produced in a manner not possible with polyclonal antibodies.
  • Polyclonal antisera vary from animal to animal, whereas monoclonal preparations exhibit a uniform antigenic specificity.
  • Murine animal systems are useful to generate monoclonal antibodies, and immunization protocols, techniques for isolating and fusing splenocytes, and methods and reagents for producing hybridomas are well known.
  • Monoclonal antibodies can be produced by a variety of techniques, including conventional monoclonal antibody methodology, e.g., the standard somatic cell hybridization technique of Kohler and Milstein, Nature, 256: 495, 1975. See generally, Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988.
  • humanized or human antibodies rather than murine antibodies to treat human subjects, because humans mount an immune response to antibodies from mice and other species.
  • the immune response to murine antibodies is called a human anti-mouse antibody or
  • HAMA response (Schroff, R. et al, Cancer Res., 45, 879-885, 1985) and is a condition that causes serum sickness in humans and results in rapid clearance of the murine antibodies from an individual's circulation.
  • the immune response in humans has been shown to be against both the variable and the constant regions of murine immunoglobulins.
  • Human monoclonal antibodies are safer for administration to humans than antibodies derived from other animals and human polyclonal antibodies.
  • One useful type of animal in which to generate human monoclonal antibodies is a transgenic mouse that expresses human immunoglobulin genes rather than its own mouse immunoglobulin genes.
  • Such transgenic mice e.g., "HuMAbTM” mice, contain human immunoglobulin gene miniloci that encode unrearranged human heavy ( ⁇ and ⁇ ) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous ⁇ and K chain loci (see e.g., Lonberg, N. et al., Nature 368(6474): 856-859, 1994, and U.S. Pat. 5,770,429). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgG ⁇ monoclonal antibodies (Lonberg, N.
  • HuMAb mice can be immunized with an immunogen, as described by Lonberg, N. et al. Nature, 368(6474): 856-859, 1994; Fishwild, D. et al ., Nature Biotechnology, 14: 845-851, 1996 and WO 98/24884.
  • the mice are 6-16 weeks of age upon the first immunization.
  • a purified preparation of inactivated HCV can be used to immunize the HuMAb mice intraperitonealy.
  • mice can be immunized with live, killed or nonviable inactivated and/or lyophilized HCV.
  • HCV envelope glycoproteins El and/or E2 can be used as an immunogen.
  • HuMAb transgenic mice respond best when initially immunized intraperitoneally (IP) with antigen in complete Freund's adjuvant, followed by IP immunizations every other week (up to a total of 6) with antigen in incomplete Freund' s adjuvant.
  • IP intraperitoneally
  • the immune response can be monitored over the course of the immunization protocol with plasma samples being obtained by retroorbital bleeds.
  • the plasma can be screened, for example by ELISA or flow cytometry, and mice with sufficient titers of anti-HCV human immunoglobulin can be used for fusions.
  • mice can be boosted intravenously with antigen 3 days before sacrifice and removal of the spleen. It is expected that multiple fusions for each antigen may need to be performed.
  • Several mice are typically immunized for each antigen.
  • the mouse splenocytes can be isolated and fused with PEG to a mouse myeloma cell line based upon standard protocols.
  • the resulting hybridomas are then screened for the production of antigen- specific antibodies. For example, single cell suspensions of spleenic lymphocytes from immunized mice are fused to one-sixth the number of P3X63-Ag8.653 nonsecreting mouse myeloma cells (ATCC, CRL 1580) with 50% PEG.
  • Cells are plated at approximately 2xlO 5 in flat bottom microtiter plate, followed by a two week incubation in selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES, 0.055 mM 2-mercaptoethanol, 50 units/ml penicillin, 50 mg/ml streptomycin, 50 mg/ml gentamycin and Ix HAT (Sigma; the HAT is added 24 hours after the fusion). After two weeks, cells are cultured in medium in which the HAT is replaced with HT.
  • selective medium containing 20% fetal Clone Serum, 18% "653" conditioned media, 5% origen (IGEN), 4 mM L-glutamine, 1 mM L-glutamine, 1 mM sodium pyruvate, 5 mM HEPES,
  • Supernatants from individual wells are then screened by ELISA for human anti-HCV monoclonal IgM and IgG antibodies.
  • the antibody secreting hybridomas are replated, screened again, and if still positive for human IgG, anti-HCV monoclonal antibodies, can be subcloned at least twice by limiting dilution.
  • the stable subclones are then cultured in vitro to generate small amounts of antibody in tissue culture medium for characterization.
  • the transgenic animal used to generate human antibodies to HCV contains at least one, typically 2-10, and sometimes 25-50 or more copies of the transgene described in Example 12 of WO 98/24884 (e.g., pHCl or pHC2) bred with an animal containing a single copy of a light chain transgene described in Examples 5, 6, 8, or 14 of WO 98/24884, and the offspring bred with the J H deleted animal described in Example 10 of WO 98/24884, the contents of which are hereby expressly incorporated by reference. Animals are bred to homozygosity for each of these three traits.
  • WO 98/24884 e.g., pHCl or pHC2
  • Such animals have the following genotype: a single copy (per haploid set of chromosomes) of a human heavy chain unrearranged mini-locus (described in Example 12 of WO 98/24884), a single copy (per haploid set of chromosomes) of a rearranged human K light chain construct (described in Example 14 of WO 98/24884), and a deletion at each endogenous mouse heavy chain locus that removes all of the functional J H segments (described in Example 10 of WO 98/24884).
  • Such animals are bred with mice that are homozygous for the deletion of the J H segments (Examples 10 of WO 98/24884) to produce offspring that are homozygous for the J H deletion and hemizygous for the human heavy and light chain constructs.
  • the resultant animals are injected with antigens and used for production of human monoclonal antibodies against these antigens.
  • the B cells isolated from such an animal are monospecific with regard to the human heavy and light chains because they contain only a single copy of each gene. Furthermore, they will be monospecific with regard to human or mouse heavy chains because both endogenous mouse heavy chain gene copies are nonfunctional by virtue of the deletion spanning the J H region introduced as described in Examples 9 and 12 of WO 98/24884. Furthermore, a substantial fraction of the B cells will be monospecific with regards to the human or mouse light chains, because expression of the single copy of the rearranged human kappa light chain gene will allelically and isotypically exclude the rearrangement of the endogenous mouse kappa and lambda chain genes in a significant fraction of B-cells.
  • the transgenic mouse will exhibit immunoglobulin production with a significant repertoire, ideally substantially similar to that of a native mouse.
  • the total immunoglobulin levels will range from about 0.1 to 10 mg/ml of serum, e.g., 0.5 to 5 mg/ml, or at least about 1.0 mg/ml.
  • the adult mouse ratio of serum IgG to IgM is preferably about 10:1.
  • the IgG to IgM ratio will be much lower in the immature mouse. In general, greater than about 10%, e.g., about 40 to 80% of the spleen and lymph node B cells will express exclusively human IgG protein.
  • the repertoire in the transgenic mouse will ideally approximate that shown in a non-transgenic mouse, usually at least about 10% as high, preferably 25 to 50% or more as high.
  • at least about a thousand different immunoglobulins ideally IgG
  • the immunoglobulins will exhibit an affinity for preselected antigens of at least about 10 "6 M, 10 "7 M , 10 "8 M , 10 "9 M , 10 "10 M , 10 "11 M , 10 "12 M , 10 "13 M , 10 "14 M , or greater, e.g., up to 10 "15 M or more.
  • HuMAb mice can produce B cells that undergo class-switching via intratransgene switch recombination (cis-switching) and express immunoglobulins reactive with HCV.
  • the immunoglobulins can be human sequence antibodies, wherein the heavy and light chain polypeptides are encoded by human transgene sequences, which may include sequences derived by somatic mutation and V region recombinatorial joints, as well as germline-encoded sequences.
  • human sequence immunoglobulins can be referred to as being substantially identical to a polypeptide sequence encoded by a human VL or VH gene segment and a human JL or JL segment, even though other non-germline sequences may be present as a result of somatic mutation and differential V-J and V-D-J recombination joints.
  • the variable regions of each chain are typically at least 80 percent encoded by human germline V, J, and, in the case of heavy chains, D, gene segments. Frequently at least 85 percent of the variable regions are encoded by human germline sequences present on the transgene. Often 90 or 95 percent or more of the variable region sequences are encoded by human germline sequences present on the transgene.
  • the human sequence antibodies will frequently have some variable region sequences (and less frequently constant region sequences) that are not encoded by human V, D, or J gene segments as found in the human transgene(s) in the germline of the mice.
  • such non-germline sequences or individual nucleotide positions
  • the human sequence antibodies that bind to HCV can result from isotype switching, such that human antibodies comprising a human sequence gamma chain (such as gamma 1, gamma 2, or gamma 3) and a human sequence light chain (such as K) are produced.
  • Such isotype- switched human sequence antibodies often contain one or more somatic mutation(s), typically in the variable region and often in or within about 10 residues of a CDR) as a result of affinity maturation and selection of B cells by antigen, particularly subsequent to secondary (or subsequent) antigen challenge.
  • These high affinity human sequence antibodies have binding affinities of at least about Ix 10 ⁇ 9 M, typically at least 5xlO ⁇ 9 M, frequently more than IxIO "10 M, and sometimes 5xlO ⁇ 10 M to 1x10 " ⁇ M or greater.
  • Anti-HCV antibodies can also be raised in other mammals, including non- transgenic mice, humans, rabbits, and goats.
  • Anti-HCV Antibodies Human monoclonal antibodies that specifically bind to HCVinclude antibodies produced by the clones 95-2, 95-14, 95-38, 83-128 and 073-1 described herein (referred to as, respectively, antibody clones 95-2, 95-14, 95-38, 83-128 and 073-1). Antibodies with variable heavy chain and variable light chain regions that are at least 80% identical to the variable heavy and light chain regions of 95-2, 95-14, 95-38, 83-128 and 073- lean also bind to HCV.
  • anti-HCV antibodies include, for example, complementarity determining regions (CDRs) that are at least 80% identical to the CDRs of the variable heavy chains and/or variable light chains of 95-2, 95-14, 95- 38, 83-128 and 073-1.
  • CDRs complementarity determining regions
  • SEQ ID NOs. for the amino acid and nucleotide sequences of the variable light and heavy chains of these antibodies, as well as their CDRs, are given in Table 1 below.
  • CDRs are the portions of immunoglobulins that determine specificity for a particular antigen.
  • CDRs corresponding to the CDRs in Table 1 having sequence variations may bind to HCV.
  • CDRs, in which 1, 2, 3, 4, or 5 residues, or less than 20% of total residues in the CDR, are substituted or deleted can be present in an antibody (or antigen binding portion thereof) that binds HCV.
  • anti-HCV antibodies can have CDRs containing a consensus sequence, as sequence motifs conserved amongst multiple antibodies can be important for binding activity.
  • the invention provides for the use of one or more CDR regions or derivatives of the disclosed CDRs.
  • Such derivative CDRs are derived from a disclosed CDR or portion thereof and, optionally, altered at one more amino acid positions.
  • Alterations include one or more amino acid additions, deletions, or substitutions as described herein.
  • Exemplary residue positions for altering include those amino acid positions identified as subject to more variance than other amino acid positions, for example, positions subject to somatic mutations as known in the art.
  • positions can be identified by comparing two or more sequences known to have a desired binding activity and identifying CDR residues that vary and CDR residues that are constant.
  • variable regions of heavy and light chains are presented in Figures 37 and 38 and the CDR derivative or consensus sequences that can be determined therefrom are found in SEQ ID NO: 87, 88, 89 (VH consensus sequences for CDRl, CDR2, and CDR3, respectively), 90, 91 and 92 (VL consensus sequences for CDRl, CDR2, and CDR3, respectively). It is also understood that one more of the CDRs disclosed herein (including
  • CDR derivative or consensus sequences can be used for identifying naturally occurring CDRs that are suitable for binding to a HCV epitope.
  • the CDRs can also be combined or cross-cloned between variable regions, for example, light chain CDRs can be introduced into heavy chain variable regions and heavy chain CDRs can be introduced into light chain variable regions and screened to ensure that specific binding is retained.
  • Human anti-HCV antibodies can include variable regions that are the product of, or derived from, specific human immunoglobulin genes.
  • the antibodies can include a variable heavy chain region that is the product of, or derived from, a human VH 3-30-3 or VH3-33 gene (see, e.g., Ace. No.: AJ555951, GI No.: 29836865; Ace.
  • the antibodies can also, or alternatively, include a light chain variable region that is the product of, or derived from a human VK L6, VK Ll 1, VK L13, VK L15, or VK L19 gene (see, e.g., GenBank® Ace. No.: AJ556049, GI No.: 29837033 for a partial sequence of a rearranged human VK Ll 9 gene segment).
  • variable immunoglobulin regions of recombined antibodies are derived by a process of recombination in vivo in which variability is introduced to genomic segments encoding the regions.
  • variable regions derived from a human VH or VL gene can include nucleotides that are different that those in the gene found in non-lymphoid tissues. These nucleotide differences are typically concentrated in the CDRs.
  • the above antibodies exhibit binding activity to HCV and, in particular, to one or more HCV glycoprotein epitopes ⁇ e.g., El or E2 envelope glycoproteins). Such antibodies further exhibit HCV neutralization activity in vitro and protective efficacy against hepatitis sequelae in vivo.
  • Additional anti-HCV antibodies for use in embodiments of the invention include GNI- 104 (monoclonal antibodies to El, from GENImmune) and HuMax-HepC (monoclonal antibody to E2, broadly cross-reactive across multiple HC genotypes, from Genmab A/S) (see http://www.hcvdrugs.com).
  • Additional embodiments of the invention feature a variety of antibodies.
  • Table 2 lists additional human anti-HCV antibodies known in the art that can be used in embodiments of the invention. Further additional antibodies for use with embodiments of the invention can be found in U.S. Patent No. 6,171,782, issued January 9, 2001; European Patent No. 0 569 537 Bl, published June 17, 1998; U.S. Patent No. 5,753,430, issued May 19, 1998; U.S. Patent No.
  • the antibodies can be of the various isotypes, including: IgG (e.g., IgGl, IgG2, IgG3, IgG4), IgM, IgAl, IgA2, IgD, or IgE.
  • the antibody is an IgG isotype, e.g., IgGl.
  • the antibody molecules can be full-length (e.g., an IgGl, IgG2, IgG3, or IgG4 antibody) or can include only an antigen-binding fragment (e.g., a Fab, F(ab')2 > Fv or a single chain Fv fragment).
  • monoclonal antibodies e.g., human monoclonal antibodies
  • recombinant antibodies e.g., chimeric antibodies, and humanized antibodies, as well as antigen-binding portions of the foregoing.
  • Anti-HCV antibodies or portions thereof useful in the present invention can also be recombinant antibodies produced by host cells transformed with DNA encoding immunoglobulin light and heavy chains of a desired antibody.
  • Recombinant antibodies may be produced by known genetic engineering techniques.
  • recombinant antibodies can be produced by cloning a nucleotide sequence, e.g., a cDNA or genomic DNA, encoding the immunoglobulin light and heavy chains of the desired antibody.
  • the nucleotide sequence encoding those polypeptides is then inserted into an expression vector so that both genes are operatively linked to their own transcriptional and translational expression control sequences.
  • the expression vector and expression control sequences are chosen to be compatible with the expression host cell used. Typically, both genes are inserted into the same expression vector.
  • Prokaryotic or eukaryotic host cells may be used.
  • eukaryotic host cells are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.
  • any antibody produced that is inactive due to improper folding can be renatured according to well known methods (Kim and Baldwin, Ann. Rev. Biochem., 51:459-89, 1982). It is possible that the host cells will produce portions of intact antibodies, such as light chain dimers or heavy chain dimers, which also are antibody homologs according to the present invention.
  • the antibodies described herein also can be produced in a host cell transfectoma using, for example, a combination of recombinant DNA techniques and gene transfection methods as is well known in the art (Morrison, S., Science, 229:1202,
  • the gene(s) of interest e.g., human antibody genes
  • an expression vector such as a eukaryotic expression plasmid such as used in a GS gene expression system disclosed in WO 87/04462, WO 89/01036 and EP 338 841, or in other expression systems well known in the art.
  • the purified plasmid with the cloned antibody genes can be introduced in eukaryotic host cells such as CHO-cells or NSO-cells or alternatively other eukaryotic cells like a plant derived cells, fungi, or yeast cells.
  • the method used to introduce these genes can be any method described in the art, such as electroporation, lipofectine, Lipofectamine, transfection (e.g., calcium chloride-mediated), or ballistic transfection, in which cells are bombarded with microparticles carrying the DNA of interest (Rodin, et ⁇ l. Immunol. Lett.,
  • antibodies in which specific amino acids have been substituted, deleted, or added have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • preferred antibodies have amino acid substitutions in the framework region, such as to improve binding to the antigen.
  • a selected, small number of acceptor framework residues of the immunoglobulin chain can be replaced by the corresponding donor amino acids.
  • Preferred locations of the substitutions include amino acid residues adjacent to the CDR, or which are capable of interacting with a CDR (see e.g., U.S. Pat. 5,585,089). Criteria for selecting amino acids from the donor are described in U.S. Pat. 5,585,089 (e.g., columns 12-16), the contents of which are hereby incorporated by reference.
  • the acceptor framework can be a mature human antibody framework sequence or a consensus sequence.
  • the Fc region of antibodies of the invention can be altered to modulate effector function(s) such as, for example, complement binding and/or Fc receptor binding. Criteria and subsets of framework alterations and/or constant regions suitable for alteration (by, e.g., substitution, deletion, or insertion) are described in U.S. Patent Nos. 6,548,640; 5,859,205; 6,632,927; 6,407,213; 6,054,297; 6,639,055; 6,737,056; and 6,673,580.
  • a "consensus sequence” is a sequence formed from the most frequently occurring amino acids (or nucleotides) in a family of related sequences (See e.g.,
  • a "consensus framework" of an immunoglobulin refers to a framework region in the consensus immunoglobulin sequence.
  • an anti-HCV antibody, or antigen-binding portion thereof can be derivatized or linked to another functional molecule (e.g., another peptide or protein).
  • another functional molecule e.g., another peptide or protein
  • an antibody can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody, a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a protein or peptide that can mediate association with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • One type of derivatized antibody is produced by crosslinking two or more of such proteins (of the same type or of different types).
  • Suitable crosslinkers include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (e.g., disuccinimidyl suberate).
  • spacer e.g., m-maleimidobenzoyl-N- hydroxysuccinimide ester
  • homobifunctional e.g., disuccinimidyl suberate
  • Useful detectable agents with which an antibody (or fragment thereof) can be derivatized (or labeled) include fluorescent compounds, various enzymes, prosthetic groups, luminescent materials, bioluminescent materials, and radioactive materials.
  • Exemplary fluorescent detectable agents include fluorescein, fluorescein isothiocyanate, rhodamine, and phycoerythrin.
  • a protein or antibody can also be derivatized with detectable enzymes, such as alkaline phosphatase, horseradish peroxidase, ⁇ - galactosidase, acetylcholinesterase, glucose oxidase and the like.
  • a protein When a protein is derivatized with a detectable enzyme, it is detected by adding additional reagents that the enzyme uses to produce a detectable reaction product. For example, when the detectable agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is detectable.
  • a protein can also be derivatized with a prosthetic group (e.g., streptavidin/biotin and avidin/biotin).
  • a prosthetic group e.g., streptavidin/biotin and avidin/biotin.
  • an antibody can be derivatized with biotin, and detected through indirect measurement of avidin or streptavidin binding.
  • Labeled proteins and antibodies can be used, for example, diagnostically and/or experimentally in a number of contexts, including (i) to isolate a predetermined antigen by standard techniques, such as affinity chromatography or immunoprecipitation; and (ii) to detect a predetermined antigen (e.g., HCV, or a HCV protein, carbohydrate, or lipid, or combination thereof, e.g., in a cellular Iy sate or a patient sample) in order to monitor virus and/or protein levels in tissue as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.
  • a predetermined antigen e.g., HCV, or a HCV protein, carbohydrate, or lipid, or combination thereof, e.g., in a cellular Iy sate or a patient sample
  • HCV proteins such as an El or E2 glycoprotein or fragment(s) thereof.
  • Anti-HCV antibodies can be characterized for binding to HCV by a variety of known techniques. Antibodies are typically characterized by ELISA first. Briefly, o microtiter plates can be coated with the target antigen in PBS, for example, HCV or the El and/or E2 glycoprotein or portion thereof, and then blocked with irrelevant proteins such as bovine serum albumin (BSA) diluted in PBS. Dilutions of plasma from mice immunized with the target antigen, for example, a HCV vaccine, are added to each well and incubated for 1-2 hours at 37 0 C.
  • BSA bovine serum albumin
  • mice which develop the highest titers will be used for fusions.
  • An ELISA assay as described above can be used to screen for antibodies and,0 thus, hybridomas that produce antibodies that show positive reactivity with HCV.
  • Hybridomas that produce antibodies that bind, preferably with high affinity, to HCV can than be subcloned and further characterized.
  • One clone from each hybridoma, which retains the reactivity of the parent cells (by ELISA), can then be chosen for making a cell bank, and for antibody purification.
  • selected hybridomas can be grown in roller bottles, two-liter spinner- flasks or other culture systems. Supernatants can be filtered and concentrated before affinity chromatography with protein A-Sepharose (Pharmacia, Piscataway, NJ) to purify the protein. After buffer exchange to PBS, the concentration can be determined by spectrophotometric methods.
  • each antibody can be biotinylated using commercially available reagents (Pierce, Rockford, 111.). Biotinylated MAb binding can be detected with a streptavidin labeled probe.
  • Anti- HCV antibodies can be further tested for reactivity with HCV or one or more HCV proteins by immunoprecipitation or immunoblot.
  • HCV El or E2 glycoprotein epitopes can be linear epitopes, conformational epitopes, discontinuous epitopes, or combinations of such epitopes.
  • the epitope of HCV El glycoprotein is found within amino acid residues 190-330. In some embodiments, the HCV El glycoprotein is found within amino acid residues 315-328. Without wishing to be bound by any particular theory, in some embodiments, the epitope of HCV E2 glycoprotein is found within amino acid residues 412-464. In some embodiments, the HCV E2 glycoprotein is found within amino acid residues 412-423.
  • the HCV E2 glycoprotein is found within residues 413, 418, 420.
  • an epitope of a HCV glycoprotein comprises alterations to the canonical or published sequences of HCV, such as substitutions or deletions.
  • Other assays to measure activity of the anti- HCV antibodies include neutralization assays. In vitro neutralization assays can measure the ability of an antibody to inhibit a cytopathic effect, infectivity, or presence of a virus on or in cells in culture (see Example 1, below). In vivo neutralization or survival assays can be used to measure HCV neutralization as a function of reduced morbidity and/or mortality in an appropriate animal model (see Example 5 of PCT/US08/01922, filed February 13, 2008, hereby incorporated by reference).
  • An antibody of the invention can be prepared using an antibody having one or more V H and/or V L sequences as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody.
  • An antibody can be engineered by modifying one or more residues within one or both variable regions ⁇ i.e., V H and/or V L ), for example within one or more CDR regions and/or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody.
  • variable region engineering One type of variable region engineering that can be performed is CDR grafting.
  • Antibodies interact with target antigens predominantly through amino acid residues that are located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within CDRs are more diverse between individual antibodies than sequences outside of CDRs. Because CDR sequences are responsible for most antibody- antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific naturally occurring antibodies by constructing expression vectors that include CDR sequences from the specific naturally occurring antibody grafted onto framework sequences from a different antibody with different properties (see, e.g., Riechmann et al, 1998 Nature 332:323-327; Jones et al, 1986 Nature 321:522-525; Queen et al,
  • Framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • germline DNA sequences for human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrc- cpe.cam.ac.uk/vbase), as well as in Kabat et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson et al., 1992 J. MoI. Biol. 227:776-798; and Cox et al., 1994 Eur. J. Immunol. 24:827-836; the contents of each of which are expressly incorporated herein by reference.
  • V H CDRl, 2 and 3 sequences and the V L CDRl, 2 and 3 sequences can be grafted onto framework regions that have the identical sequence as that found in the germline immunoglobulin gene from which the framework sequence is derived, or the CDR sequences can be grafted onto framework regions that contain one or more mutations as compared to the germline sequences. For example, it has been found that in certain instances it is beneficial to mutate residues within the framework regions to maintain or enhance the antigen binding ability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370). CDRs can also be grafted into framework regions of polypeptides other than immunoglobulin domains.
  • Appropriate scaffolds form a conformationally stable framework that displays the grafted residues such that they form a localized surface and bind the target of interest (e.g., an epitope of a HCV protein).
  • CDRs can be grafted onto a scaffold in which the framework regions are based on fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CPl zinc finger, PSTl, coiled coil, LACI-Dl, Z domain or tendramisat (See e.g., Nygren and Uhlen, 1997 Current Opinion in Structural Biology, 7, 463-469).
  • variable region modification is mutation of amino acid residues within the V H and/or V L CDRl, CDR2 and/or CDR3 regions to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as "affinity maturation.”
  • Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce the mutation(s), and the effect on antibody binding, or other functional property of interest, can be evaluated in in vitro or in vivo assays as described herein.
  • Conservative modifications can be introduced.
  • the mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within a CDR region are altered.
  • Engineered antibodies of the invention include those in which modifications have been made to framework residues within V H and/or V L , e.g., to improve the properties of the antibody. Typically such framework modifications are made to decrease the immunogenicity of the antibody. For example, one approach is to "backmutate" one or more framework residues to the corresponding germline sequence. More specifically, an antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequences to the germline sequences from which the antibody is derived.
  • somatic mutations can be "backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.
  • site-directed mutagenesis or PCR-mediated mutagenesis.
  • Such "backmutated” antibodies are also intended to be encompassed by the invention.
  • Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell -epitopes to thereby reduce the potential immunogenicity of the antibody. This approach is also referred to as "deimmunization" and is described in further detail in U.S. Pat. Pub. No. 20030153043 by Carr et al.
  • antibodies of the invention may be engineered to include modifications within the Fc region, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • modifications within the Fc region typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity.
  • an antibody of the invention may be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the hinge region of CHl is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased.
  • This approach is described further in U.S. Pat. No. 5,677,425 by Bodmer et al.
  • the number of cysteine residues in the hinge region of CHl is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half- life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody has impaired Staphylococcyl protein A (SpA) binding relative to native Fc-hinge domain SpA binding.
  • SpA Staphylococcyl protein A
  • the antibody is modified to increase its biological half- life.
  • U.S. Pat. No. 6,277,375 describes the following mutations in an IgG that increase its half-life in vivo: T252L, T254S, T256F.
  • the antibody can be altered within the CHl or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector functions of the antibody.
  • one or more amino acids can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an Fc receptor or the Cl component of complement. This approach is described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.
  • one or more amino acids selected from amino acid residues can be replaced with a different amino acid residue such that the antibody has altered CIq binding and/or reduced or abolished complement dependent cytotoxicity (CDC).
  • CDC complement dependent cytotoxicity
  • one or more amino acid residues are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in WO 94/29351 by Bodmer et al.
  • the Fc region is modified to increase the ability of the antibody to mediate antibody dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for an Fc ⁇ receptor by modifying one or more amino acids.
  • ADCC antibody dependent cellular cytotoxicity
  • This approach is described further in WO 00/42072 by Presta.
  • the binding sites on human IgGl for Fc ⁇ Rl, Fc ⁇ RII, Fc ⁇ RIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R.L. et al., 2001 J. Biol. Chem. 276:6591-6604).
  • the glycosylation of an antibody is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation.
  • glycoprotein-modifying glycosyl transferases e.g., beta(l,4)-N acetylglucosaminyltransferase III (GnTIII)
  • GnTIII glycoprotein-modifying glycosyl transferases
  • An antibody can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody.
  • the antibody, or fragment thereof typically is reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG moieties become attached to the antibody or antibody fragment.
  • PEG polyethylene glycol
  • the pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer).
  • polyethylene glycol is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl-ClO) alkoxy- or aryloxy- polyethylene glycol or polyethylene glycol-maleimide.
  • the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the invention. See for example, EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.
  • pegylation can be achieved in any part of an anti- HCV binding polypeptide of the invention by the introduction of a nonnatural amino acid.
  • Certain nonnatural amino acids can be introduced by the technology described in Deiters et al., J Am Chem Soc 125:11782-11783, 2003; Wang and Schultz, Science 301:964-967, 2003; Wang et al, Science 292:498-500, 2001; Zhang et al, Science 303:371-373, 2004 or in US Patent No. 7,083,970. Briefly, some of these expression systems involve site- directed mutagenesis to introduce a nonsense codon, such as an amber TAG, into the open reading frame encoding a polypeptide of the invention.
  • a nonsense codon such as an amber TAG
  • Such expression vectors are then introduced into a host that can utilize a tRNA specific for the introduced nonsense codon and charged with the nonnatural amino acid of choice.
  • Particular nonnatural amino acids that are beneficial for purpose of conjugating moieties to the polypeptides of the invention include those with acetylene and azido side chains.
  • the polypeptides containing these novel amino acids can then be pegylated at these chosen sites in the protein.
  • compositions e.g., pharmaceutically acceptable compositions, which include conjugates of the present invention, formulated together with a pharmaceutically acceptable carrier, as well as methods of treating subjects with said compositions.
  • the conjugates feature targeting antibodies or antibody variants.
  • the antibodies are therapeutic antibodies, for example neutralizing antibodies.
  • exemplary conjugates of the invention have a "dual modality".
  • exemplary conjugate are capable of neutralizing virus outside the cell (e.g., free virus) to inhibit viral infection and accessing infected cells to prevent viral replication.
  • “Pharmaceutically acceptable carriers” include any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carriers can be suitable for intravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, or epidermal administration (e.g., by injection or infusion).
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes and suppositories.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., dispersions or suspensions
  • liposomes e.g., liposomes and suppositories.
  • useful compositions are in the form of injectable or infusible solutions.
  • a useful mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).
  • conjugates can be administered by intravenous infusion or injection.
  • conjugates can be administered by intramuscular or subcutaneous injection.
  • composition of the invention may be co-administered with a) one or more other antibodies, e.g., anti- HCV antibodies, b) HCV protein(s), e.g., a HCV vaccine, c) toxin(s) d) other therapeutic agent(s) (e.g., antivirals), and/or e) label(s).
  • administration of one or more anti-HCV antibodies can precede administration of one or more anti-HCV antibody-silencing agent conjugates.
  • parenteral administration and “administered parenterally” as used herein mean modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intracranial, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.
  • Therapeutic compositions typically should be sterile and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high antibody concentration.
  • Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the useful methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • conjugates described herein can be administered by a variety of methods known in the art, and for many therapeutic applications. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
  • a conjugate may be orally administered, for example, with an inert diluent or an assimilable edible carrier.
  • the compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet.
  • the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
  • To administer a compound of the invention by other than parenteral administration it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.
  • Therapeutic compositions can be administered with medical devices known in the art.
  • Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of a conjugate of the invention is 0.1-60 mg/kg, e.g., 0.5-25 mg/kg, 1-2 mg/kg, or 0.75-10 mg/kg.
  • the amount of conjugate administered is at or about 0.125 mg/kg, 0.25 mg/kg, 0.5 mg/kg, or at an interval or range thereof. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
  • kits including conjugates of the invention, for example, anti-HCV conjugates.
  • the kits can include one or more other elements including: instructions for use; other reagents, e.g., a label, a therapeutic agent, or an agent useful for chelating, or otherwise coupling, an antibody to a label or therapeutic agent, or other materials for preparing the antibody for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.
  • kits can include anti-HCV conjugates, antibodies that bind to HCV (e.g., antibodies that include the variable heavy and/or light chain regions of 95-2, 95-14, 95-38, 83-128 and 073-1), or a combination thereof.
  • the anti-HCV antibodies are blocking or neutralizing antibodies.
  • kits of the invention may include more than one conjugate, for example, conjugates delivering different silencing agents targeting distinct genes. The compounds can be mixed together, or packaged separately within the kit.
  • Instructions for use can include instructions for therapeutic application including suggested dosages and/or modes of administration, e.g., in a patient with a symptom or indication of a hepatitis virus/HCV infection, or exposure or suspected exposure to a hepatitis virus/HCV.
  • Other instructions can include instructions on coupling of the antibody to a chelator, a label or a therapeutic agent, or for purification of a conjugated antibody, e.g., from unreacted conjugation components.
  • the kit can include a detectable label, a therapeutic agent, and/or a reagent useful for chelating or otherwise coupling a label or therapeutic agent to a conjugate or antibody.
  • Coupling agents include agents such as N-hydroxysuccinimide (NHS).
  • the kit can include one or more of a reaction vessel to carry out the reaction or a separation device, e.g., a chromatographic column, for use in separating the finished product from starting materials or reaction intermediates.
  • the kit can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional anti-HCV antibodies or conjugates (or portions thereof), formulated as appropriate, in one or more separate pharmaceutical preparations.
  • additional reagent such as a diagnostic or therapeutic agent, e.g., a diagnostic or therapeutic agent as described herein, and/or one or more additional anti-HCV antibodies or conjugates (or portions thereof), formulated as appropriate, in one or more separate pharmaceutical preparations.
  • kits can include optimized nucleic acids encoding anti-HCV antibodies, for use as passive immunotherapy, and/or HCV protein(s), or fragments thereof, for use as, e.g., vaccines (active immunotherapy), and instructions for expression of the nucleic acids.
  • conjugates of the present invention can have in vitro and in vivo therapeutic, prophylactic, and diagnostic utilities.
  • conjugates can be administered to cells in culture, e.g., in vitro or ex vivo, or in vivo, to an animal, preferably a human subject, to treat, inhibit, prevent relapse, and/or diagnose HCV and disease associated with HCV.
  • the term "subject” is intended to include human and non-human animals.
  • the term “non-human animals” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, dogs, cats, pigs, cows, and horses.
  • Conjugates of the invention can be used on cells in culture, e.g., in vitro or ex vivo in some embodiments.
  • cells can be cultured in vitro in culture medium and the contacting step can be affected by adding a conjugate to the culture medium.
  • the methods can be performed on virions or cells present in a subject, as part of an in vivo ⁇ e.g., therapeutic or prophylactic) protocol.
  • the contacting step is effected in a subject and includes administering conjugate to the subject under conditions effective to permit binding of the binding protein, antibody, or portion thereof, to HCV or any portion thereof present in the subject, e.g., in or around a wound or on or near cells of neuronal origin.
  • the subjects are subjects having a liver disorder, liver dysfunction, or liver transplant patients. In other embodiments, the subjects suffer from chronic HCV infection. Methods of administering compounds of the invention are described herein.
  • Suitable dosages of the molecules used will depend on the age and weight of the subject and the particular drug used.
  • the compounds can be used as competitive agents for ligand binding to inhibit or reduce an undesirable interaction, e.g., to inhibit binding and/or infection of HCV of cells, e.g., neuronal cells.
  • an anti-HCV conjugate can be administered in combination with other anti-HCV therapies (e.g., other monoclonal antibodies, polyclonal gamma-globulin, e.g., human serum comprising anti-HCV immunoglobulins).
  • Combinations of conjugates and therapies that can be used in some embodiments include an anti-HCV antibody or antigen binding portion thereof and/or an anti-HCV El or E2 protein antibody or antigen binding portion thereof.
  • the anti-HCV or HCV El or E2 protein antibody for use in the formation of a conjugate of the invention can be antibody clone 95-2, 95-14, 95-38, 83-128 and/or 073-1 that includes the variable regions of such an antibody or antibodies, or an antibody with variable regions at least 90% identical to the variable regions of such an antibody or antibodies.
  • conjugates comprising the anti-HCV virus antibody can be 83-128, 95-2, 073-1 or portion thereof or an antibody with variable regions at least 90% identical to the variable regions of the foregoing, e.g., 95-2, 95-14, 95-38, 83-128 and/or 073-1.
  • Combinations of anti-HCV virus conjugates based on anti-HCV antibodies ⁇ e.g., 95-2, 95-14, 95-38, 83-128 and/or 073-1) can provide potent inhibition of HCV, especially, e.g., particular HCV genotypes and subgenotypes.
  • Characteristic HCV genotypes and subgenotypes for which the conjugates of the invention are suitable for treating, detecting, diagnosing and the like include genotypes 1, 2, 3, 4, 5, and 6, subgenotypes Ia, Ib, Ic, 2a, 2b, 2c, 3a, 3b, 4a, 4b, 4c, 4d, 4e, 5a, 6a and combinations thereof.
  • any of the agents of the invention for example, conjugates comprising anti-HCV antibodies, or fragments thereof, can be combined, for example in different ratios or amounts, for improved therapeutic effect.
  • the agents of the invention can be formulated as a mixture, or chemically or genetically linked using art recognized techniques thereby resulting in covalently linked conjugates having anti- HCV binding properties, for example, multi-epitope binding properties to, for example, HCV El and/or E2 glycoprotein.
  • the combined formulation may be guided by a determination of one or more parameters such as the affinity, avidity, or biological efficacy of the agent alone or in combination with another agent.
  • the agents of the invention can also be administered in combination with other agents that enhance access, half-life, or stability of the therapeutic agent in targeting, clearing, and/or sequestering HCV or an antigen thereof.
  • Such combination therapies are preferably additive and even synergistic in their therapeutic activity, e.g., in the inhibition, prevention, infection, and/or treatment of HCV -related disease or disorders.
  • Administering such combination therapies can decrease the dosage of the therapeutic agent (e.g., a conjugate mixture, with or without an antibody or antibody fragment mixture, or cross-linked or genetically fused bispecific antibody or antibody fragment) needed to achieve the desired effect.
  • the therapeutic agent e.g., a conjugate mixture, with or without an antibody or antibody fragment mixture, or cross-linked or genetically fused bispecific antibody or antibody fragment
  • Immunogenic compositions can be used with the conjugates of the invention in certain embodiments.
  • Immunogenic compositions typically contain an immunogenically effective amount of a HCV component, for example, HCV El and/or E2 glycoprotein, or fragments thereof, and can be used in generating anti-HCV antibodies.
  • a HCV component for example, HCV El and/or E2 glycoprotein, or fragments thereof
  • Immunogenic epitopes in a HCV protein sequence can be identified as described herein (see e.g. Example 4 of PCT/US08/01922, filed February 13, 2008, hereby incorporated by reference) or according to methods known in the art, and proteins, or fragments containing thoseepitopes can be delivered by various means, in a vaccine composition.
  • Suitable compositions can include, for example, lipopeptides (e.g., Vitiello et al, J. Clin.
  • peptide compositions encapsulated in poly (DL-lactide-co-glycolide) ("PLG") microspheres see, e.g., Eldridge et al., Molec. Immunol. 28:287-94 (1991); Alonso et al, Vaccine 12:299-306 (1994); Jones et al., Vaccine 13:675-81 (1995)
  • PLG poly (DL-lactide-co-glycolide)
  • IVS immune stimulating complexes
  • MAPs multiple antigen peptide systems
  • compositions of the invention include, for example, thyroglobulin, albumins such as human serum albumin, tetanus toxoid, polyamino acids such as poly L-lysine, poly L-glutamic acid, influenza, hepatitis B virus core protein, and the like.
  • the compositions can contain a physiologically tolerable (i.e., acceptable) diluent such as water, or saline, typically phosphate buffered saline.
  • the compositions and vaccines also typically include an adjuvant.
  • Adjuvants such as incomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, or alum are examples of materials well known in the art.
  • CTL responses can be primed by conjugating target antigens, for example a HCV protein(s) (or fragments, inactive derivatives or analogs thereof) to lipids, such as tripalmitoyl-S-glycerylcysteinyl-seryl- serine (P 3 CSS).
  • target antigens for example a HCV protein(s) (or fragments, inactive derivatives or analogs thereof) to lipids, such as tripalmitoyl-S-glycerylcysteinyl-seryl- serine (P 3 CSS).
  • the anti-HCV conjugates can be administered in combination with other agents, such as compositions to treat HCV -mediated disease.
  • therapeutics including antiviral agents, serum immunoglobulin, and/or vaccines, can be administered in combination with anti-HCV conjugates for treating, preventing, or inhibiting HCV.
  • the conjugate can be administered before, after, or contemporaneously with a HCV vaccine.
  • siRNAs duplex small interfering RNAs
  • the major hurdle in therapeutic application of siRNA is how to direct the molecule into a desired cell population to achieve maximal therapeutic effect and avoid non-specific silencing or other toxicity in cells other than intended targets. Since duplex siRNAs cannot choose their cellular targets, it is necessary to design a "targeting agent" to deliver these siRNAs.
  • Cell surface receptors have been explored as potential targets for gene delivery. Antibodies specifically binding to these surface receptors can serve as a carrying vector to delivery siRNAs into target cells expressing the corresponding cell surface protein recognized by the antibody.
  • full length or truncated human protamine polypeptides which is positive-charged and interacts with negative-charged RNA non-covalently, has been suggested to fuse to the C-terminus of a human single-chain antibody fragment (ScFv) or of a human heavy chain.
  • ScFv human single-chain antibody fragment
  • fusion to polypeptides may destroy the activity of antibodies.
  • the non-covalent binding between the full-length protamine or its fragment and the siRNA can be disrupted by other charged molecules in vitro or in vivo, in which case it cannot promise the safe delivery of the siRNA into target cells. Therefore, a better way to realize the antibody- siRNA interaction is needed.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in polypeptide preparation.
  • conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in polypeptide preparation See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology), 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., IrI Pr (1996); Antibodies: A Laboratory Manual, Harlow et al, C. S. H. L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al. , John Wiley & Son
  • HCV genotype Ia and Ib - soluble E2 ELISA The reactivity of human antibody 95-2 ( Figure IA) and human antibody 83-128 (Figure IB) to soluble HCV genotype Ia E2-660 (triangles) and soluble HCV genotype Ib E2- 661 (squares) was compared. ELISA plates were coated with 2 ⁇ g/ml of antigen and probed with antibody in two-fold dilutions. Bound antibody was detected with goat- anti-human secondary antibody conjugated to alkaline phosphatase and PNPP substrate. Both human antibodies, 95-2 and 83-128 were found to react equivalently to both genotypes (see Figure 1). These experiments were also performed with the 95-14, 95-38 and 073-1 antibodies and all three recognized genotype Ia and Ib equivalently (data not shown).
  • the capacity of human antibodies 95-2 and 83-128 to neutralize both multiple genotypes of HCV pseudovirus was determined using Hep3B cells. Five-fold dilutions of antibody were incubated with HCV pseudovirus for one hour at room temperature. The virus-antibody mixture was added to Hep3B cells followed by incubation at 37 0 C for 72 hours. Infection was quantitated with Brightglo luciferase assay and read in a Victor3 plate reader for light output. An isotype-matched irrelevant human antibody was used as a negative control.
  • human antibodies 95-2 and 83-128 to soluble E2 protein (E2-661) from HCV genotype Ia and Ib subjected to reducing or non-reducing SDS-PAGE was analyzed.
  • Westerns blots were performed with anti-his tag monoclonal (his), 83-128 and 95-2 using anti-mouse IgG (his) or anti-human IgG (95-2 and 83-128) conjugated to HRP with enhanced chemiluminescent detection reagent.
  • Both human antibodies 95-2 and 83-128 were found to recognize E2-661 subjected to denaturing, reducing gel followed by transfer to PVDF membrane (see Figure 3).
  • Binding affinity of human antibodies 95-2 and 83-128 for E2 412-423 epitope expressed as a bacterial fusion protein was determined and summarized in Figure 4.
  • Goat anti- human IgG Fc was amide coupled to the Biacore chip.
  • Human antibodies 95-2 and 83- 128 were separately captured on the chip and E2-G bacterially expressed protein containing E2 412-423 amino acids was flowed over at varying concentrations.
  • the BIAevaluationTM software was used to fit the curves and calculate the affinity constants.
  • the present application discloses methods to form bio-functional molecules between DNA or RNA oligos and proteins using cross-linking reagents.
  • These reagents can be, for example, heterobifunctional, containing an N-hydroxysuccinimde ester (NHS-ester) and either a hydrazine/hydrazide or an aldehyde group.
  • NHS-ester N-hydroxysuccinimde ester
  • the RNA/DNA oligos containing a 5' amino group were modified with SFB (succinimidyl 4-formylbenzoate, Pierce Cat. #22419) to form an active biomolecule for further conjugation with a protein or polypeptide.
  • SFB succinimidyl 4-formylbenzoate
  • RNA/DNA oligos used in this Example had an amino modification of an N6 linker (Figure 5) for SFB modification.
  • a DNA duplex formed by annealing may have an N6 linker on the 5' terminus of the Sense Strand as 5 Sense 5 ' -N6-CAAGCTGACCCTGAAGTTCTT-3 '
  • Antisense 3'-TTGTTCGACTGGGACTTCAAG-S' Antisense 3'-TTGTTCGACTGGGACTTCAAG-S' .
  • RNA/DNA two complimentary strands of RNA/DNA (e.g., strand A and strand B) were resuspended in RNase-free water with a final concentration of 250 pmol/ul, as confirmed by common spectrophotometry. Solutions of each strand o was mixed together in an equal molar ratio. To create 100 nmol of annealed RNA/DNA duplex, 400 ul strand A (250 pmol/ul) was mixed with 400 ul strand B (250 pmol/ul). 200 ul 5X lysis buffer (Dharmacon's 5X buffer) was then added to this nucleic acid solution mixture. The mixture was incubated at 95°C for 5 minutes and then at 37°C for 1.5 hour.
  • 5X lysis buffer Dharmacon's 5X buffer
  • duplex 1 pmol duplex was analyzed via electrophoresis5 (mini TBE gel). The duplex solution was then dialyzed into modification buffer (100 mM Naphosphate, Pierce, Cat#28372, 150 mM NaCl, PH7.0) for 3 hours before proceeding with the SFB modification step.
  • modification buffer 100 mM Naphosphate, Pierce, Cat#28372, 150 mM NaCl, PH7.0
  • Step 2 Modify Duplex with SFB 0
  • the N6 linker at the 5' terminus of the sense strand of the RNA/DNA duplex is modified by SFB to form an aldehyde group, as illustrated in Figure 5.
  • Modification may also be performed on non-duplexed, N ⁇ -linked oligonucleotides.
  • RNA/DNA duplex RNA/DNA duplex
  • DMF half volume of the duplex volume, added to increase solubility of SFB in the reaction mixture
  • modified duplex molecules were collected by centrifugation, the pellet was further washed with 70% EtOH, followed by resuspension in conjugation buffer (100 rnM Naphosphate, 15OmM NaCl, PH5.0) at a concentration betweem 1-2 nmol/ul.
  • conjugation buffer 100 rnM Naphosphate, 15OmM NaCl, PH5.0
  • concentration betweem 1-2 nmol/ul The final concentration of modified RNA/DNA duplex was confirmed by Quan-it RNA kit (Invitrogen).
  • a small sample of the modified duplex (1 pmol) was analyzed using a 20 cm 15% TBE urea gel, to assess modification efficiency.
  • Figure 6 shows the results of modification of the sense strand of the duplex.
  • lane 1 shows unmodified sense strand RNA
  • lanes 2, 3, 4 and 5 show sense strand RNA oligonucleotides that had been modified via the procedure above, with different amounts of SFB in each reaction (25:1, 35:1, 45:1 and 55:1 molar ratios of SFB :N6 GTP duplex in the reaction mix, respectively). Due to a greater molecular weight, the sense strand RNA oligonucleotides with SFB modification ( Figure 6, lanes 2-5) moved more slowly than those without modification ( Figure 6, lane 1).
  • the modification efficiency was at least 90% and these modifications were seen at the lowest molar ratio of SFB:N6 GTP duplex (25:1) ( Figure 6, lane T). No significant increase in modification was seen with greater SFB:N6 GTP duplex molar ratios (i.e., when the SFB: N6 GTP duplex molar ratio was 35:1, 45:1 or 55:1).
  • the upper band in the duplex oligo lane had the same mobility during gel electrophoresis as the modified sense strand oligo in lane 2; the lower band in the duplex oligo lane had the same mobility during gel electrophoresis as the unmodified anti-sense strand oligo in the last lane on the right hand side of the figure.
  • Example 3 SFB modification of siRNA (N ⁇ -GFPz stable 2) and result detection using 2-HP method
  • a specific siRNA (N6-linked GFPz stable 2) was used for SFB modification as part of a RNA/DNA duplex, as demonstrated in Example 2.
  • the sample was diluted 20 to 50 fold into 2-HP (2- hydrazino pyridine.2HCl) solution (0.5 mM 2-HP in 10OmM MES, pH 5.0 and incubated at 37°C for 1 hour.
  • 2-HP 2- hydrazino pyridine.2HCl
  • the aldehyde-modified siRNA duplex and 2-HP will form a hydrazone structure, which has a spectroscopic UV signature and can be read at 260 nm and 350 nm ( Figure 8).
  • Spectroscopy results can be converted via a Solulink calculator to quantitatively determine the percentage of oligo being modified.
  • 94.3% and 94.5% of the siRNA duplex was modified with SFB in two separate trials (right panel), which suggests that this detection methods is comparable to results visualized on gel (middle panel).
  • the efficiency for SFB modification of the passenger strand (DNA) of the siRNA duplex was quite low, at only 28.8% efficiency (left panel).
  • RNA/DNA duplexes were assessed before further conjugation process with any proteins or polypeptides, by measuring the activity of a specific siRNA (GFPz) before and after SFB modification.
  • GFPz siRNA duplexes were previously modified with various 2' O-Met nucleotides (named Stable 6) and the N6 linker to become N6 GFPz Stable 6 siRNA oligo duplex. (This was done using psiCheck with GFP cloned into the psiCheck vector. 293T cells were transfected with siRNAs and psiCheck vector containing GFP DNA. Then luciferase reduction was measured to assess delivery of the anti-GFP siRNA).
  • siRNA oligos both with and without SFB modification on the N6 linker at the 5' terminus, to knock down targeted genes was measured. As shown in Figure 10, the knockdown efficiency of SFB-N ⁇ -GFPz Stable 6 oligos was similar to that of unmodified GFPz oligos.
  • SANH succinimidyl 4-hydrazinonicotinate acetone hvdrazone
  • the present application includes methods of cross-linking DNA or RNA oligos and proteins or polypeptides to form bio-functional molecules.
  • the proteins include, but are not limited to, antibodies or fragments thereof.
  • SANH succinimidyl 4-hydrazinonicotinate acetone hydrazone
  • the SANH solution was added 25-50 fold in molar excess to 2 mg of the antibody in a volume of 400 ul of modification buffer (e.g., 100 mM Naphosphate, Pierce, Cat. #28372, 150 mM NaCl, pH 7.0) to make final concentration of 5mg/ml.
  • modification buffer e.g., 100 mM Naphosphate, Pierce, Cat. #28372, 150 mM NaCl, pH 7.0
  • Step 4 Removal of excess SANH using Zeba Desalting columns After the SANH-antibody modification step, excess SANH in the reaction mixture was removed using Zeba Desalting columns (Pierce Cat. #89889, 2ml columns). After loosening the cap and snapping off the bottom tip, the Zeba column was placed in a 15 ml tube and centrifuged for 1 minute to remove the storage solution. A mark was placed on the side of the column, indicating where the highest point of the upwardly slanted compacted resin was located. The column was placed in centrifuge with the mark facing outward in all subsequent steps.
  • Zeba Desalting columns Pieris Cat. #89889, 2ml columns
  • SANH reaction mixture was applied slowly to the center of the resin bed (200-700 ul volume capacity).
  • a stacker of pH 5 conjugation buffer (40 ul) was applied to resin bed after sample has been fully absorbed.
  • the sample was then collected using a Mini quick spin oligo column (Roche Cat#l 1814397001) after centrifuging at 1,000 x g for 2 minutes.
  • Step 5 Estimation of the number of SANH linkers per antibody
  • a p-NB (p-nitrobenzaldehyde,Solulink) solution was prepared by dissolving 5 mg p-NB in 100 ul DMF and adding a 75 ul aliquot of this solution to a 50 ml volume of 100 mM MES buffer (Sigma C/N M2933, pH 5.0). The solution was protected from light and kept refrigerated. This solution will remain stable for 30 days at 4° C.
  • MES buffer Sigma C/N M2933, pH 5.0
  • the conjugation mechanism and procedures are illustrated in Figure 11, 12 and 13.
  • the free aldehyde formed by the SFB modification of the oligo reacts with SANH 5 on the modified antibody.
  • the reaction mixture was incubated at room temperature overnight, or for at least 3 hours, and then stored at -80 0 C. 1-2 ug of the conjugates were then analyzed by electrophoresis on a Tris-glycine gel, while 20 ug was analyzed by IEF gel. Additionally, the antigen binding activity of the conjugates was analyzed by ELISA. o Conjugates were assessed for variation in conjugation efficiency.
  • This Example illustrates one of other oligo modification methods for Oligo- protein conjugations disclosed in the present application. Specifically, sodium periodate (NaIO 4 ) was used to treat the siRNA oligos, to oxidize hydroxyls (-OH) at the 2' and 3' positions of the ring to generate aldehydes for subsequent conjugations ( Figure 20).
  • NaIO 4 sodium periodate
  • Step 1 Set up annealing reactions to make duplex siRNA 200 pmoles of passenger strand RNA was 5 '-radiolabeled with 32 P using T4 polynucleotide kinase.
  • 4 uL 5X lysis buffer (Dharmacon's 5X buffer) was mixed with 5 uL of 10 uM passenger strand solution (50 pmol), 5 uL of lOuM guide strand solution (50 pmol) and 6 uL dH 2 O. The mixture was incubated at 95 0 C for 2 minutes and then at 37 0 C for 1 hour for annealing.
  • the resulting siRNA duplex contained 50 pmol of siRNA in a total volume of 20 uL. Therefore the concentration of siRNA in this reaction was 50 pmol/20uL or 2.5pmol/uL (2.5uM).
  • NaIO 4 solution Before siRNA oxidation, fresh 200 mM NaIO 4 solution was prepared by resolving 42.7 mg of NaIO 4 in 1 mL RNAse free water. The NaIO 4 solution was stored in vials wrapped with foil and placed on ice until needed.
  • siRNA oxidation process 1.25 uL of fresh NaIO 4 solution was mixed with 5 uL duplex siRNA (12.5 pmol siRNA), 1 uL PBS (10OmM, pH 7.2, 15OmM NaCl), and 2.75 uL water.
  • a control reaction the same amounts of siRNA duplex, PBS and water were mixed without NaIO 4 .
  • the reaction mixture (or the control) was incubated without light at room temperature for 30 min. Ethanol precipitation according to the protocol above was then performed to create a pellet of oxidized duplex, which was then resuspended in 20 uL RNAse free water.
  • Samples were tested via gel electrophoresis. 10 uL of each sample was mixed with 5 uL water and 15 uL formamide loading dye and stored in freezer. The other 10 uL of each sample was first tested via ⁇ -elimination, in order to ascertain the completeness of the previous oxidation reaction. To perform this test, 10 uL of each sample was mixed with 0.5 uL 1 M NaOH and 4.5 uL water and incubated at 42°C for 90 minutes. The resulting mixture was mixed with 15 uL formamide loading dye and were analyzed along with the samples without ⁇ -elimination on a 15% TBE-Urea gel. The gel was analyzed using a phosphoimager to determine the oxidation efficiency.
  • Step 3 Reaction of oxidized siRNA with the EMCH linker 3,3'-N-[e-Maleimidocaproic acid] hydrazide, trifluoroacetic acid salt (EMCH)
  • the oxidized siRNA oligo was linked with EMCH to form an siRNA-EMCH conjugate for subsequent siRNA-antibody conjugation (Figure 24).
  • the reaction was carried out for different lengths of time and the siRNA-EMCH conjugation was compared after electrophoresis.
  • Figure 25 the conjugation with EMCH led to a band shift of oxidized siRNA (21-mer). This conjugation was complete, since the unconjugated oxidized siRNA band disappeared almost completely ( Figure 25).
  • siRNA-EMCH conjugate was separated from free, unconjugated siRNA oligos in the conjugation reaction.
  • HPLC was used for the separation (0.1 M ammonium acetate solution for Buffer A and 0.1 M ammoniun acetate in 50% acetonitrile for Buffer B) ( Figure 27).
  • the sample reaction mixture was loaded for HPLC and the different components in the mixture were eluted out individually after their characteristic retention time in the column ( Figure 28). After removing the unmodified passenger and 2OMe guide fractions ( Figure 29), the oxidized passenger-EMCH conjugate fraction was concentrated and purified.
  • Traut' s Reagent was used to modify lysine residues on the antibody to generate sulfhydryls (-SH) ( Figure 30), which were used in subsequent conjugations with the siRNA-EMCH conjugates.
  • Antibody solution was concentrated down to ⁇ 5mg/mL in 1 X PBS.
  • a 14 mM stock of Traut's Reagent was prepared by dissolving approximately
  • Ellman's reagent 4 mg was dissolved in 1 mL of 100 mM PBS, pH 8, 1 mM EDTA.
  • a cysteine standard curve was made by serial dilutions of a 1.5 mM cysteine stock using a 1:2 dilution in 100 mM PBS, pH8, 1 mM EDTA.
  • the concentration of sulfhydryl groups in a reaction was determined using the cysteine standard curve.
  • the number of free SH groups was determined by dividing the sulfhydryl concentration in each sample by the corresponding antibody concentration (as determined by Octet).
  • the number of modified lysine residues on the antibody increased with more Traut's reagent in the reaction ( Figure 31).
  • Figure 31 For example, with 50-fold excess Traut's reagent in two side-by-side experiments, an average of 0.8 and 1.6 lysine residues per antibody were accessed and modified by Traut's reagent.
  • the amount of Traut's reagent increased to 200-fold excess, the average number of modified lysine residues on the antibody increased to 6.1 and 6.7.
  • the present invention further includes methods to conjugate the modified antibody with the modified siRNA with a linker.
  • an siRNA-EMCH conjugate was further linked with a human monoclonal antibody modified by Traut' s reagent to form an siRNA-mAb conjugate ( Figure 33).
  • the antibody modified by Traut's was reacted with a 5-fold molar excess of the siRNA-EMCH conjugate.
  • the reaction mixture was incubated at room temperature for 2 hours and the resulting conjugates were resolved on a native 4-20% gradient TRIS-HCL gel. Nucleic acids were analyzed by SYBR-GoId staining. Protein was analyzed by SYPRO-Ruby staining.
  • siRNAs antibodies and linkers used in the conjugation
  • siRNAs duplex small interfering RNAs
  • the major hurdle in therapeutic application of siRNA has been finding a method to direct the molecule into a desired cell population to achieve maximal therapeutic effect and avoid non-specific silencing or toxicity in cells other than intended targets.
  • siRNA-antibody bioconjugates are shown to specifically and effectively deliver siRNAs into cells via cell surface receptors.
  • a heterobifunctional linker is used to link the chosen antibody and the chosen RNA at each of the side of the linker.
  • the list of possible heterobifunctional linkers that could be used includes, but is not limited to: MaleimidoCaproic acid Hydrazide, HCl (EMCH, with the formula as C 10 H 16 N 3 O 3 CI); MaleimidoPropionic acid Hydrazide, HCl (MPH, with the formula as C 8 Hi 2 N 3 O 3 Cl); N- (k-Maleimidoundecanoic acid)hydrazide (KMUH, with the formula as C 15 H 25 N 3 O 3) ; and 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide, HCl (MPBH).
  • the heterobifunctional linker is comprised of a maleimide molecule and a hydrazide molecule linked together.
  • the maleimide side of this linker is further linked to one molecule of thiolated antibodies, through a thioether bond with a thiol group on a -NH 2 side chain of a lysine residue found in the sequence of a heavy chain of the antibody.
  • the thiolation agent for use in this example is 2-Iminothiolane (Traut's Reagent).
  • the hydrazide side of this heterobifunctional linker is further linked through a pH-liable hydrazone bond to the aldehyde on one molecule of alkylated siRNAs.
  • This hydrazone bond in the linker is stable in serum at pH 7.4, but can hydrolyze at pH 5.0 in the endosomes, leading to the release of the siRNA conjugated to the antibody.
  • the linker bond is stable in the endosomes.
  • a stable linker bond in the endosome leads to the guide strand remaining attached to the antibody for some time after the passenger strand separates from the guide strand.
  • bioconjugates are created by using one of the antibodies of the invention, including 95-2, 95-14, 95-38, 83-128 and 073-1 and an anti-HCV siRNA, such as those described above in Section IV, for example.
  • an anti-HCV siRNA such as those described above in Section IV, for example.
  • Example 11 Testing of anti-HCV siRNAs
  • a replicon is a subgenomic RNA that contains all essential elements and genes reuired for replication in the absence of structural genes.
  • the HCVcc system also provides a means to study HCV in vitro (Lindenbach et al., Science 309:623-626 (2005)).
  • the HCVcc system features a genetically engineered, chimeric full-length HCV genome (including structural genes) that replicates and produces virus particles in vitro.
  • the siRNAs and protein conjugates of the invention are tested using a replicon or chimeric version of HCV.
  • Anti-HCV antibodies are conjugates to anti-HCV siRNA molecules and to control siRNA molecules, either with a randomized sequence or with a sequence containing point mutations in an anti-HCV siRNA sequence that render the siRNA ineffective at knocking down HCV RNA, gene expression and/or viral particle production.
  • the antibody in the bioconjugate can be substituted by any antibody or antigen binding portion of antibody recognizing other viral proteins.
  • the siRNA in the bioconjugate can be substituted by any siRNA targeting the genome of the virus, different isolates of HCV or genetic elements of cells that are or may be infected with HCV.
  • the heterobifunctional linker connecting the siRNA and the antibody can comprise of any spacer moiety between a maleimide moiety and a hydrazide moiety.

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Abstract

L’invention concerne un procédé d’utilisation d’un complexe ou d’une molécule comportant un fragment de ciblage pour distribuer efficacement une seconde molécule au virus de l’hépatite C (VHC) ou à une cellule infectée ou présentant le potentiel d’être infectée par le VHC, conduisant à la prévention, à l’immunisation, à l’inhibition ou à la guérison d’une infection par le VHC, ou d’autres effets bénéfiques liés à une infection par le VHC.
PCT/US2009/044840 2008-05-22 2009-05-21 Conjugués agent-protéine désactivant des acides nucléiques et leur utilisation pour traiter des troubles liés au vhc WO2009143345A2 (fr)

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CN109694868A (zh) * 2018-12-27 2019-04-30 合肥中科干细胞再生医学有限公司 一种抑制sFRP1基因表达的siRNA及其应用
CN109959641A (zh) * 2017-11-27 2019-07-02 美天施生物科技有限责任公司 用于光漂白染色细胞的方法
CN112924665A (zh) * 2021-02-19 2021-06-08 山东莱博生物科技有限公司 一种抗体辣根过氧化物酶标记物及其制备与应用
WO2021237160A3 (fr) * 2020-05-22 2022-02-17 City Of Hope Conjugués d'acide nucléique de phosphorothioate comprenant des enzymes d'édition génique
WO2021260061A3 (fr) * 2020-06-24 2022-02-24 Sapreme Technologies B.V. Dérivés de saponine à fenêtre thérapeutique améliorée
WO2021261996A3 (fr) * 2020-06-24 2022-03-10 Sapreme Technologies B.V. Conjugués de saponine à base de nhs
CN116801909A (zh) * 2020-12-02 2023-09-22 S·鲍默 静电纳米颗粒及其用途

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US8202979B2 (en) * 2002-02-20 2012-06-19 Sirna Therapeutics, Inc. RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid
US20040209831A1 (en) * 2002-02-20 2004-10-21 Mcswiggen James RNA interference mediated inhibition of hepatitis C virus (HCV) gene expression using short interfering nucleic acid (siNA)
AU2006325030B2 (en) * 2005-12-16 2012-07-26 Cellectis Cell penetrating peptide conjugates for delivering nucleic acids into cells
ES2456961T3 (es) * 2007-02-21 2014-04-24 University Of Massachusetts Anticuerpos humanos contra el virus de la hepatitis C (VHC), usos de los mismos

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CN109959641A (zh) * 2017-11-27 2019-07-02 美天施生物科技有限责任公司 用于光漂白染色细胞的方法
CN109694868A (zh) * 2018-12-27 2019-04-30 合肥中科干细胞再生医学有限公司 一种抑制sFRP1基因表达的siRNA及其应用
WO2021237160A3 (fr) * 2020-05-22 2022-02-17 City Of Hope Conjugués d'acide nucléique de phosphorothioate comprenant des enzymes d'édition génique
WO2021260061A3 (fr) * 2020-06-24 2022-02-24 Sapreme Technologies B.V. Dérivés de saponine à fenêtre thérapeutique améliorée
WO2021261996A3 (fr) * 2020-06-24 2022-03-10 Sapreme Technologies B.V. Conjugués de saponine à base de nhs
WO2021260054A3 (fr) * 2020-06-24 2022-04-21 Sapreme Technologies B.V. Dérivés de saponine à fenêtre thérapeutique améliorée
CN116801909A (zh) * 2020-12-02 2023-09-22 S·鲍默 静电纳米颗粒及其用途
CN112924665A (zh) * 2021-02-19 2021-06-08 山东莱博生物科技有限公司 一种抗体辣根过氧化物酶标记物及其制备与应用
CN112924665B (zh) * 2021-02-19 2023-10-03 山东莱博生物科技有限公司 一种抗体辣根过氧化物酶标记物及其制备与应用

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