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WO2010042743A2 - Multiplexes chimères, compositions et procédés permettant de les utiliser - Google Patents

Multiplexes chimères, compositions et procédés permettant de les utiliser Download PDF

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Publication number
WO2010042743A2
WO2010042743A2 PCT/US2009/060040 US2009060040W WO2010042743A2 WO 2010042743 A2 WO2010042743 A2 WO 2010042743A2 US 2009060040 W US2009060040 W US 2009060040W WO 2010042743 A2 WO2010042743 A2 WO 2010042743A2
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WO2010042743A3 (fr
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Miguel De Los Rios
Timothy L. Bullock
Kenneth J. Oh
Patrick T. Johnson
Jacek Ostrowski
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Chimeros Inc
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Chimeros Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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
    • A61K47/54—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 organic compound
    • A61K47/543—Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6919—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a ribbon or a tubule cochleate
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10141—Use of virus, viral particle or viral elements as a vector
    • C12N2730/10142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule

Definitions

  • Effective therapeutics that include nucleic acids such as interfering RNA may require stability in order to reach target cells or targets before the therapeutic is degraded or excreted. For example, such therapeutics must get into the target cells, link up with a intracellular target, and exert a therapeutic effect. In order to be effective, such therapeutics should exert enough of an effect to improve the health of the person taking it. Further, therapeutics that include nucleic acid should be effective without causing significant toxic effects in either target or nontarget tissues. [0005] Many proposed therapeutic agents have not been successful because of a limited ability to reach the target tissue. Further, some agents can be taken up by cells, and therefore may not allow efficient drug accumulation at a target site in a patient.
  • nucleic acid based therapeutics are typically administered intravenously due to their instability at a high pH in e.g. the stomach after oral administration.
  • the present disclosure is generally directed, at least in part, to therapeutic chimeric multiplexes, e.g. a multiplex that includes two or more capsids formed from chimeric proteins that may include a protein and a nucleic acid, and/or compositions that include disclosed therapeutic chimeric multiplexes.
  • therapeutic chimeric multiplexes e.g. a multiplex that includes two or more capsids formed from chimeric proteins that may include a protein and a nucleic acid, and/or compositions that include disclosed therapeutic chimeric multiplexes.
  • a therapeutic multiplex comprises two or more capsids, e.g. plurality of capsids, for example, about 3 to about 12, or about 6 capsids, each capsid comprising a viral core protein having a modified structural core portion and a modified tail portion, and a nucleic acid bound to said tail portion; and a coating at least partially surrounding, or for example, substantially surrounding, the two or more capsids.
  • the tail portion is capable of releasing said nucleic acid when said therapeutic multiplex is administered in vivo.
  • at least two of the capsids are associated with each other through a disulfide interaction and/or a hydrophobic interaction.
  • a structural core portion comprises a conjugation site allowing attachment of a chemical linker moiety.
  • Contemplated coatings may further comprise a chemical or targeting moiety bonded to said coating.
  • a coating may comprise one or more lipids.
  • at least one lipid molecule may be covalently bound through lipid linker moiety to one of the viral core proteins that form e.g., a capsid.
  • Disclosed coatings may include, cholesterol or one or more neutral lipids.
  • the coating comprises HSPC and/or POPG.
  • the disclosed multiplexes may be substantially free of nuclease and/or substantially free of endogenous nucleic acids.
  • a disclosed multiplex or capsid having a nucleic acid bound to a viral core protein may be substantially protected from serum degradation when administered in vivo, for example, a nucleic acid bound to a viral core protein may be substantially protected from serum degradation for at least two weeks when a disclosed therapeutic chimeric and/or particle and/or composition is exposed at 37 0 C to a composition comprising a 1 : 1 weight ratio of human serum to water.
  • compositions that include disclosed multiplexes and a pharmaceutically acceptable carrier.
  • a therapeutic composition includes a) a multiplex comprising: i) a plurality of capsids, each capsid comprising a first discrete number of modified viral core proteins; and a second discrete number of nucleic acids each bound to one of said modified viral core proteins; and ii) a coating substantially surrounding said capsids; and b) a pharmaceutically acceptable excipient.
  • FIGURE 1 is a computational reconstruction depicting wild-type Hepatitis B Virus
  • HBV capsid reconstructed from electron density maps of the full size HBV dimer from the perspective of looking down at the 6-fold axis.
  • FIGURE 2 is a schematic depicting phosphatidyl ethanolamine (PE) conjugation to an exemplary lipid linker moiety.
  • PE phosphatidyl ethanolamine
  • FIGURE 3 is a schematic depicting conjugating a maleimide-containing linker, with a lipid, to a sulfhydryl-containing protein.
  • FIGURE 4 is a pictorial representation of a therapeutic multiplex.
  • FIGURE 5 depicts a photograph of a gel showing K9 protein-RNA complex.
  • FIGURE 6 depicts results from a capsid stability assay.
  • FIGURE 7 is a quantitative representation of the results of a nuclease protection assay.
  • FIGURE 8 is a quantitative representation of the results of a serum stability assay.
  • FIGURE 9 is a line graph depicting the binding curve for K9 mutants.
  • FIGURE 10 is a line graph depicting the binding curves for K7 and KI l mutants.
  • FIGURE 11 depicts a dose response mRNA knockdown effect when multiplex particles loaded with modified Factor VII inhibitory dsRNA are incubated on primary mouse hepatocytes for 72 hours.
  • Figure 12 depicts a chromatogram obtained by a purification method.
  • Figure 13 depicts particle size measurement. DETAILED DESCRIPTION
  • the present disclosure is generally directed, at least in part, to chimeric multiplex therapeutics, e.g. a therapeutic that includes two or more capsids, wherein one or more capsids include a nucleic acid, e.g. an inhibitory nucleic acid, bound to a modified protein, and/or compositions that include such chimeric multiplex therapeutics.
  • chimeric multiplex therapeutics e.g. a therapeutic that includes two or more capsids, wherein one or more capsids include a nucleic acid, e.g. an inhibitory nucleic acid, bound to a modified protein, and/or compositions that include such chimeric multiplex therapeutics.
  • amphipathic lipid refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase.
  • Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).
  • amphipathic compounds include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • phospholipids include, but are not limited to, phosphatidylcholine such as egg phosphatidylcholine or hydrogenated soy phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, pahnitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidyl glycerol, monosialoganlgolioside, spingomyelin, dimyristoylphosphatidylcholine, and dilinoleoy
  • amphipathic lipids Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and ⁇ -acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipid described above can be mixed with other lipids including triglycerides and sterols.
  • anionic lipid refers to any lipid that is negatively charged at physiological pH.
  • lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.
  • phosphatidylglycerols cardiolipins
  • diacylphosphatidylserines diacylphosphatidic acids
  • N-dodecanoyl phosphatidylethanolamines N-succinyl phosphatidylethanolamines
  • N- glutarylphosphatidylethanolamines N- glutarylphosphatidylethanolamines
  • cationic lipid refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0).
  • cationic lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioctadecyldimethylammonium (DODMA), distearyldimethylammonium (DSDMA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)- N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane
  • DODAC N,
  • anionic lipids can be neutral on the surface with an internal negative charge.
  • An "effective amount” or “therapeutically effective amount” of a therapeutic, composition or multiplex contemplated herein is an amount sufficient to produce a desired effect, e.g., inhibition of expression of a target in comparison to the normal expression level detected in the absence of administration.
  • the therapeutically effective amount will vary depending upon the subject and disease condition being treated, the rate of target transcript turnover, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like.
  • certain compositions of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • the term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.
  • the term “inhibitory nucleic acid” refers to a single- stranded or double-stranded
  • an inhibitory nucleic acid comprises or corresponds to at least a portion of a target nucleic acid or gene, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid or gene.
  • An inhibitory nucleic acid typically has substantial or complete identity or homology ⁇ e.g.
  • lipid refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) "simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.
  • modulation is art-recognized and refers to up regulation (i.e., activation or stimulation), down regulation (i.e., inhibition or suppression) of a response, or the two in combination or apart.
  • neutral lipid refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.
  • nucleic acid refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA.
  • DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre- condensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chromosomal DNA, or derivatives and combinations of these groups.
  • RNA may be in the form of inhibitory RNA, mRNA, tRNA, rRNA, tRNA, vRNA, and combinations thereof.
  • Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).
  • Nucleotides contain a deoxyribose (DNA) or ribose (RNA), sugar, a nitrogenous base, and a phosphate group or analog thereof. Nucleotides are linked together through the phosphate groups.
  • Bases include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.
  • a "patient,” “subject” or “host” to be treated by a disclosed method may mean either a human or non-human animal.
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent, involved in carrying or transporting any subject composition or component thereof from one organ, or portion of the body, to another organ, or portion of the body.
  • excipient must be "acceptable” in the sense of being compatible with the subject composition and its components and not injurious to the patient.
  • materials which may serve as pharmaceutically acceptable excipients include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydro
  • Target refers to a nucleic acid or variants thereof required for expression of a polypeptide that is the site or potential site of therapeutic intervention by a therapeutic agent; or a non-peptide entity including a microorganism, virus, bacterium, or single cell parasite (wherein the entire genome of a virus may be regarded as a target); and/or a naturally occurring interfering RNA or microRNA or precursor thereof.
  • target may refer to the sequence of nucleotides corresponding to the portion of a gene's coding mRNA.
  • serum- stable in relation to a nucleic acid- multiplex means that the particle is not significantly degraded after exposure to a serum or nuclease assay that would significantly degrade free DNA or RNA.
  • Suitable assays include, for example, a standard serum assay, a DNase assay, or an RNase assay.
  • the therapeutic chimeric multiplexes disclosed herein may be, e.g., formed from particles , which may be a nanoparticles, for example, may include two or more capsids formed, at least in part, from disclosed viral core proteins and/or disclosed chimeric therapeutics.
  • a plurality of disclosed chimeric therapeutics may self- assemble in to a particle or capsid, several of which may form part of a disclosed multiplex.
  • Such therapeutic chimerics may include a modified viral core protein with a nucleic acid associated with, e.g. bound to, the modified viral core protein.
  • the nucleic acid may be bound to the modified viral core protein by substantially Coulombic forces.
  • the nucleic acid may be bound to the modified viral core protein by Coulombic and/or hydrogen bonding.
  • Nucleic acids associated with a disclosed viral core protein may be, e.g., substantially homologous to a target, e.g. a target gene.
  • the nucleic acid when bound to the modified viral core protein, may be substantially non-immunogenic.
  • a nucleic acid bound to a modified viral core protein may be substantially less immunogenic as compared to an identical unbound nucleic acid.
  • a chimeric therapeutic in another embodiment, includes an e.g. modified viral core protein and a nucleic acid bound to the modified viral core protein (e.g. to a modified tail portion of the viral core protein) with a binding affinity that allows release of the nucleic acid when the chimeric therapeutic is administered in vivo.
  • the nucleic acid of a chimeric therapeutic, wherein bound to a disclosed modified viral core protein may be resistant in an aqueous solution to degradation with a nuclease, e.g. benzonase.
  • a nucleic acid bound to a disclosed modified viral core protein at 1.9 units/nmole, 100 unit/nmole, 500 units/nmole or 945 units/nmole and incubated for 1 hour at room temperature does not substantially degrade as compared to an identical, but unbound, nucleic acid.
  • a nucleic acid bound to a disclosed modified viral core protein is substantially protected from serum degradation in vivo or in vitro, for example, when a chimeric therapeutic is exposed at 37 0 C to a composition comprising a 1:1 weight ratio of human serum to water.
  • Disclosed chimeric therapeutics may be substantially free of nuclease and/or may be substantially free of endogenous nucleic acids.
  • compositions and Particles are Compositions and Particles
  • therapeutic chimeric multiplexes that include particles or capsids formed from a plurality of chimeric therapeutics as described above.
  • Disclosed therapeutic chimeric multiplexes may include a coating at least partially surrounding the two or more capsids.
  • Therapeutic compositions that may include disclosed therapeutic chimeric multiplexes may also include a pharmaceutically acceptable excipient.
  • Figure 1 is representative of how a naked (comprised solely of e.g. capsid proteins and/or chimeric therapeutics) particle or caspid may appear before associating with other capsids and being coated.
  • a disclosed multiplex comprising two or more capsids, e.g. a plurality of capsids, and a coating at least partially surrounding the capsids.
  • multiplex refers to an entity having at least two disclosed particles or capsids.
  • a disclosed multiplex has about 3 to about 12 capsids, or about 4 to about 8 capsids.
  • a disclosed multiplex has about 6 capsids, e.g. associated with each other, and a coating at least partially, or substantially, surrounding e.g. the 6 capsids.
  • a therapeutic composition comprising a multiplex disclosed herein, and optionally a pharmaceutically acceptable excipient.
  • a disclosed multiplex comprises two or more capsid particles, e.g. about 3 to about 10 capsids, each formed from at least: i) a first discrete number of modified viral core proteins; and ii) a second discrete number of nucleic acids each bound to one of said modified viral core proteins.
  • capsid particles e.g. about 3 to about 10 capsids, each formed from at least: i) a first discrete number of modified viral core proteins; and ii) a second discrete number of nucleic acids each bound to one of said modified viral core proteins.
  • not all of the first discrete number of modified viral core proteins may be associated or bound to a nucleic acid.
  • only a portion of modified viral core proteins that form part of a particle or capsid are bound to a nucleic acid.
  • a capsid may include different modified viral core proteins, e.g. those with different modified tail portions, or a capsid may be, e.g., formed from all the same modified viral core proteins.
  • a multiplex may be formed with each capsid, or one or more of the capsids, formed from different viral core proteins than other capsids in the multiplex, or a multiplex may have substantially identical capsids.
  • the first discrete number of modified viral core proteins is about 180 to about 250, about 200 to about 245, e.g. about 240 modified viral core proteins.
  • the first discrete number of modified viral core proteins is about 160 to about 250, e.g. about 180 modified viral core proteins.
  • the second discrete number of nucleic acids, wherein each nucleic acid is bound to one of the viral core proteins is about 2 to about 60, about 8 to about 20, or about 14 to about 18, e.g.
  • a disclosed particle is formed from 240 modified viral core proteins, about 14 to about 18 of those modified viral core proteins are bound to a nucleic acid.
  • a given particle can include e.g. about 8 to about 20 of the same nucleic acid, or one or more nucleic acids may be substantially different, e.g. directed to a different area of a gene target or to a different gene target.
  • a multiplex that includes two or more particles or capsids that include a plurality of viral core proteins each comprising a structural core portion and a modified tail portion, wherein said structural core portions form the capsid; and said modified tail portions are substantially disposed within said capsid; and a plurality of nucleic acids, bound to a modified tail portion of one of the viral core proteins.
  • the number of nucleic acids bound to a modified tail portion is less than that number of viral core proteins present in the particle.
  • contemplated particles or capsids formed from e.g. disclosed chimeric therapeutics are about 20 to about 25 nm in diameter, or about 30 to about 35 nm in diameter or more.
  • Particles contemplated herein may be substantially spherical and/or may be icosahedral in form.
  • particles e.g. particles that form part of disclosed multiplexes, contemplated herein may be referred to as “capsids,” “particles,” “therapeutic particles,” and “therapeutic chimeric particles.”
  • Disclosed multiplexes may further, in some embodiments, comprise a coating that partially or substantially or completely coats disposed on the particle that includes one or more lipids.
  • at least one lipid molecule may covalently bound through a chemical linker moiety, e.g. a lipid linker moiety, to a viral core protein, e.g., to a structural core portion of a disclosed viral core protein, on one capsid.
  • More than one capsid within a multiplex may have a lipid molecule covalently bound, e.g. through a linker to e.g. a structural core portion of a viral core protein of the capsid.
  • the lipid may be attached via bond or chemical linker moiety, to an engineered location on the structural core portion of the viral core protein, for example at position 77, 78 or 80 of a hepatitis B structural core portion, as described above.
  • Contemplated lipid linker moieties may include those discussed above.
  • Exemplary lipid linker moieties may be formed from contacting e.g. a succinimidyl derivative such as succinimidyl-4-(p-maleimidophenyl)butyrate (SMPB) or m-maleimidobenzoyl-N- hydroxysuccinimide ester with a modified structural core portion of the viral core protein.
  • SMPB succinimidyl-4-(p-maleimidophenyl)butyrate
  • m-maleimidobenzoyl-N- hydroxysuccinimide ester with a modified structural core portion of the viral core protein.
  • a disclosed multiplex may have a layer or coating comprising one or more lipids, e.g., a neutral lipid, an anionic lipid, and/or a cationic lipid.
  • a neutral lipid and/or an amphipathic lipid for example, a phospholipid such as phophatidyl serine, may be covalently bonded to a lipid linker moiety.
  • Such covalently bound lipid molecules may guide the placement of a coating around the particles or capsids, e.g. that may include one more neutral lipids, and/or may include an anionic lipid that is surface neutral, such as POPG.
  • Exemplary phospholipids suitable for use include, but are not limited to, hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), phosphatidyl ethanolamine (PE), phosphatidyl glycerol (PG), phosphatidyl inositol (PI), monosialogangolioside, spingomyelin (SPM), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylcholine (DMPC), or dimyristoylphosphatidylglycerol (DMPG).
  • HSPC hydrogenated soy phosphatidylcholine
  • EPC egg phosphatidylcholine
  • PE phosphatidyl ethanolamine
  • PG phosphatidyl glycerol
  • PI phosphatidyl inositol
  • SPM distearoylphosphatidylcholine
  • DMPC dimy
  • multiplexes contemplated herein include one or more lipids including one, two, or more of lipids such as palmitoyloleoylphosphatidylglycerol (POPG), hydrogenated soy phosphatidylcholine (HSPC).
  • Contemplated lipids include PEG- phospholipids, including poly(ethylene glycol) -derivatized distearoylphosphatidylethanolamine (PEG-DSPE) and/or poly(ethylene glycol)-derivatized ceramides (PEG-CER).
  • multiplexes may include a coating comprising one or more lipids and cholesterol, for example, may include various amounts of cholesterol, HSPC or POPG.
  • the lipid coating may include about 5% to about 40% cholesterol, about 10% to about 80% HSPC and/or about 5% to about 80% POPG, or any specific percentage within said ranges.
  • a coating may comprise, for example, (a) about 20% cholesterol and about 80 % HSPC; (b) about 50% cholesterol and about 50 % HSPC; (c) about 20% cholesterol and about 20 % HSPC and about 60% POPG; (d) about 50% cholesterol and about 50% POPG; (e) 20% cholesterol and 80 % POPG; or (f) about 10 % cholesterol and about 15% HSPC and about 65% POPG.
  • a coating may include about 20% cholesterol, about 20 % HSPC and about 60% POPG.
  • a coating composition may have a mass value of the multiplex of about 10% to about 60%, about 10% to about 50%, about 15 to about 40%, about 20% to about 35% or more of the total protein (w/w), or any specific percentage with the recited ranges.
  • a lipid coating composition may coat a particle at a mass value of about 30% (w/w).
  • a disclosed multiplex that includes a lipid coating may be generally prepared by 1) first mixing a modified viral core protein with an nucleic acid of choice; 2) placing the core protein in a buffered solution, e.g., phosphate, citrate, tris, sodium buffer, causing particles to be formed that substantially encapsulate the nucleic acid and associate with other capsids, e.g. by a disulfide interaction and/or a hydrophobic interaction; 3) adding sonicated phospholipids solution to the mixture which may bind with modified sites on the viral core protein; 4) adding cholesterol or lipid-tagged polyethylene glycol to the mixture; and 5) purifying the system by centrifugation or size exclusion chromatography.
  • a buffered solution e.g., phosphate, citrate, tris, sodium buffer
  • the viral core proteins may be maintained in any suitable chemical denaturant or denaturing agent known in the art (e.g., urea, guanidine hydrochloride (GuHCl), sodium dodecyl sulfate (SDS)) in a concentration of about IM to about 6M, about 1.5M to about 5M, about 1.75M to about 4.5M, or any integer disposed within said ranges.
  • the chemical denaturant may be urea, which may be present in e.g. a concentration of about 2M to about 6M, about 3M to about 5M, about 3.5M to about 4.5M, e.g.
  • the ionic strength of a solution of viral core proteins can be raised to a final concentration of about 50 mM to about 600 mM using e.g. a salt, e.g. NaCl.
  • the final concentration can be about 100 mM to about 550 mM, about 150 mM to about 500 mM, about 200 to about 450 mM, about 250 mM to about 400 mM or about 300 mM to about 350 mM, or any integer disposed within said ranges.
  • the final ionic concentration of the solution may be directly related to the amount of chemical denaturant present in the solution.
  • temperature may facilitate self-assembly of the capsid.
  • a temperature of about 25°C to about 105 0 C, about 40 0 C to about 90 0 C or about 55°C to about 75°C (or any specific temperature within the recited ranges) may trigger self-assembly of the capsid.
  • reducing agents such as DTT or beta-mercaptoethanol may also be used to facilitate self-assembly of a capsid.
  • Particles and/or multiplexes disclosed herein may be substantially non-replicating.
  • the viral core proteins may be designed so that once the multiplex and/or particle starts to disintegrate, they are degraded quickly so as to limit any potential immune response. Disclosed particles do not substantially incorporate any attenuated wild type virus.
  • any viral core protein that is capable, together with other viral core proteins, of self-assembling into a capsid is suitable for use in the disclosed therapeutics.
  • Exemplary viral core proteins include hepatitis core proteins such as human and duck Hepatitis B Virus core protein, Hepatitis C Virus core protein, and may also include Human Papilloma Virus type 6 and 16 Ll and L2 protein, cowpea chlorotic mottle virus coat protein, Norwalk virus protein, simian virus, dragon grouper nervous necrosis virus, and tomato bushy stunt virus.
  • An exemplary viral core protein is Hepatitis B Virus (HBV) core protein (C-protein).
  • HBV-C include SEQ ID NO: 1 and 2, with amino acid sequence 1 to 183 include NCBI Protein Database Accession Number BAD86623 and AY741795.
  • a modified viral core protein contemplated herein may include a structural core protein and a tail portion.
  • a modified viral core protein may include a modified structural core as compared to the wild type structural core, and/or a modified tail portion as compared to the wild type tail portion.
  • a modified viral core protein for use in the disclosed therapeutics may include a modified structural core portion and a tail portion, e.g. a carboxyl terminal tail portion and/or a N-terminal tail portion, or may include a structural core portion and a modified tail portion, or may include a modified structure core portion and a modified tail portion.
  • the structural core portions of modified viral core proteins may form a capsid, and the tail portion of the modified viral core proteins may be substantially disposed within the capsid.
  • a modified viral core protein e.g. a modified HBV C protein may include a modified structural core portion and a modified C-terminal tail portion.
  • an inward facing surface of formed capsid may act as modification location.
  • modifications can include alterations, truncations and/or mutations, etc. to the structural core portion and/or the modified tail portion of the viral core protein.
  • modifications may enhance the structural and functional characteristics of the HBV C-protein and may provide more effective therapeutics, e.g.
  • a modified viral core protein is bound to a nucleic acid, e.g., an inhibitory nucleic acid.
  • a modified e.g. C-terminal tail portion of a viral core protein may provide a therapeutic that is substantially free of endogenous nucleic acids and/or substantially free of nuclease.
  • HBV C-protein can be made or engineered according to any method known in the art, including without limitation genetic engineering, chemical modifications, etc.
  • Modification to the viral core protein for example, a viral core protein with a modified tail portion, may also optimize binding and release of a nucleic acid bound to a viral core protein.
  • the binding affinity of a nucleic acid bound to a disclosed modified viral core protein may be about 50 nM to about 500 nM, or about 55 nM to about 400 nM, at 2OmM NaHCO 3 , and a pH of 9.5, or may be about 50 nM to about 500 nM, or about 55 nM to about 400 nM, at 2OmM (CH 2 OH) 3 CNH 2 , and a pH of 7.7.
  • Disclosed viral core proteins can be expressed and purified using common molecular biology and biochemistry techniques.
  • recombinant expression vectors can be used which can be engineered to carry a viral core protein gene into a host cell to provide for expression of the viral core protein.
  • Such vectors for example, can be introduced into a host cell by transfection means including, but not limited to, heat shock, calcium phosphate, DEAE-dextran, electroporation or liposome-mediated transfer.
  • Recombinant expression vectors include, but are not limited to, Escherichia coli based expression vectors such as BL21 (DE3) pLysS, COS cell-based expression vectors such as CDM8 or pDC201, or CHO cell-based expression vectors such as pED vectors.
  • a C-protein gene coding region can be linked to one of any number of promoters in an expression vector that can be activated in the chosen cell line.
  • a cassette (capsid gene and promoter) is carried by a vector that contains a selectable marker such that cells receiving the vector can be identified.
  • promoters to express the capsid proteins within a cell line can be drawn from those that are functionally active within the host cell.
  • Such promoters can include, but are not limited to, a T7 promoter, a CMV promoter, a SV40 early promoter, a herpes TK promoter, and others known in recombinant DNA technology.
  • Inducible promoters can be used, and include promoters such as metallothionine promoter (MT), mouse mammary tumor virus promoter (MMTV), and others known to those skilled in the art.
  • Exemplary selectable markers and their attendant selection agents can be drawn, for example, from the group including, but not limited to, ampicillin, kanamycin, aminoglycoside phosphotransferase/G418, hygromycin-B phosphotransferase/hygromycin-B, and amplifiable selection markers such as dihydrofolate reductase/methotrexate and others known to skilled practitioners.
  • a variety of eukaryotic, prokaryotic, insect, plant and yeast expression vector systems e.g., vectors which contain the necessary elements for directing the replication, transcription, and translation of viral core protein coding sequences
  • vectors which contain the necessary elements for directing the replication, transcription, and translation of viral core protein coding sequences
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the capsid protein coding sequences; yeast transformed with recombinant yeast expression vectors containing the capsid protein coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the capsid protein coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the capsid protein coding sequences.
  • microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the capsid protein coding sequences; yeast transformed with recombinant yeast expression vectors containing the capsid protein
  • the wildtype HBV C protein is 183 amino acids of which the first 149 amino acids form a globular fold followed by a 35 amino acid C-terminal tail.
  • the first 149 amino acids of a hepatitis B core protein e.g. a modified viral core protein
  • the C-terminal tail of a hepatitis B core protein can be engineered to, for example, provide appropriate properties for binding a nucleic acid to the modified viral core protein.
  • a therapeutic chimeric is provided that includes a viral core protein with a modified tail portion and a nucleic acid associated with, e.g. bound to the modified tail portion.
  • the 35 amino C-terminal tail of the wild type HBV-C protein is presumed to hang inside the fully formed viral capsid and bind the viral nucleic acid, and is shown below:
  • SEQ ID NO: 3 C-terminal tail amino acid sequence 150 to 183
  • This wild type tail can be modified, truncated, and/or mutated to provide a modified tail portion, that, together with a structural core portion, provides a complete viral core protein for use in the disclosed therapeutic chimerics, multiplexes, particles, and compositions.
  • a modified tail portion may include a modification that includes one or more poly-lysines.
  • the modified tail portion may include about 4 to about 30 lysines, or about 5 to about 20 lysines, e.g., about 7, 8, 9, or 10 lysines.
  • the modified tail portion may include one or more lysine domains.
  • each poly-lysine domain may comprise about one to about thirty lysine residues.
  • the poly-lysine domain may comprise about 5 lysine residues to about 20 lysine residues.
  • the poly-lysine domains can be separated by about 1 to about 20 amino acid residues.
  • the each poly-lysine domain can comprise about 4 lysine residues to about 20 lysine residues (or any specific amino acid length disposed with the range).
  • polylysines and poly-lysine domains and/or a polyhistidine tag can form part of a modified C-terminal tails separately or in combination.
  • a polyhistidine tag may, in some embodiments, facilitate purification of the proteins.
  • Exemplary C-terminal tail portions include those having e.g., 5 lysines (K5), 7 lysines (Kl), 9 lysines (K9), 10 lysines (KIO), 11 lysines (KU), 13 lysines (K13), 20 lysines (K20).
  • C-terminal tail portions include those with a poly-lysine region with nine lysines alternating with a poly-alanine region with nine alanines (KA9), a poly-lysine region with nine lysines alternating with a poly-glycine region with nine glycines (KG9) and a poly-lysine region with nine lysines interrupted by a sequence of at least four amino acids between the fourth and fifth lysines (K4-5).
  • an about four amino acid stretch between the fourth and fifth lysines of the K4-5 tail may be amino acids Ser-Gln-Ser- Pro.
  • a modified tail portion may be represented by:
  • modified tail portion may form part of a modified viral core protein as shown below together with the corresponding nucleic acid sequences.
  • the viral core proteins are contemplated for use in the therapeutic chimerics, particles, multiplexes, and compositions disclosed herein.
  • modified tail portions (and associated nucleic acids include:
  • a modified tail portion may be formed from alternating lysines.
  • a modified tail portion can be represented by:
  • DKLAA [ AK ] P LE [ H ] SEQ ID NO: 32 wherein p is an integer from 5 to 12, and j is an integer from 0 to 10. For example, p may be 5, 6, 7, 8, 9, 10, 11, or 12; j may be 0, 1, 2, 3, 4, 5 or more.
  • a viral core protein may be represented by a viral core protein selected from:
  • HBV C-protein variant 1 (SEQ ID NO: 1).
  • a modification indentified with K5 and based on a modified structural core of SEQ ID NO: 1 can be represented by (SEQ ID NO: 25)
  • MDIDPYKEFGASVELLSFLPSDFFPS IRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVDKLAAAKKKKKKKKKLEHHHHHH
  • KlO (SEQ ID NO: 28) MDIDPYKEFGASVELLSFLPSDFFPS IRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVDKLAAAKKKKKKKKKKLEHHHHHH
  • MDIDPYKEFGASVELLSFLPSDFFPS IRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVDKLAAAKKKKKKKKKLEHHHHHH
  • MDIDPYKEFGASVELLSFLPSDFFPS IRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVDKLAAAKKKKKKKKKKKLEHHHHHH
  • MDIDPYKEFGASVELLSFLPSDFFPS IRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPAYRPPNAPILSTLPETTVVDKLAAAKGKGKGKGKGKGKGKGKGKGKGKGKGKLEHHHHHH
  • mutations creating e.g., various poly-lysine domains of differing lengths after e.g. first 149 amino acids, or the first 138 amino acids, of HBV core protein can be engineered using any methods known in the art.
  • the core protein gene can be amplified via PCR up to amino acid 149 and various numbers of lysine (or other) residues can be added to amino acids 1-149.
  • a modified tail portion includes one or more poly-arginines.
  • the modified tail portion may include about 4 to about 30 arginines, or about 5 to about 20 arginines, e.g. about 7, 8, 9, or 10 arginines.
  • the modified tail portion may include one or more arginine domains.
  • the poly-arginine domains can be separated by about 1 to about 20 amino acid residues.
  • each poly-arginine domain may comprise about one to about thirty arginine residues.
  • the each poly-arginine domain can comprise about 4 arginine residues to about 20 arginine residues (or any specific amino acid length disposed with the range).
  • a modified C-terminal tail includes at least four or at least five consecutive arginine residues.
  • Poly-arginine domains and/or a poly histidine tag can be added to the C-terminal tails separately or in combination.
  • a poly histidine tag may, in some embodiments, facilitate purification of the proteins.
  • Exemplary C-terminal tail portions may include 5 arginines (R5), 7 arginines (R7), 9 arginines (R9), 11 arginines (RI l), 13 arginines (R13), and 20 arginines (R20).
  • modified tail portions that include poly-arginine domains may be represented by:
  • q is an integer from 4 to 21 or more, and j is an integer from 0 to 10.
  • q may be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; j may be 0, 1, 2, 3, 4, 5 or more.
  • exemplary modified viral core proteins and corresponding nucleic acid that include a arginine modified tail portion include the following (together with associated nucleic acids):
  • R9 (SEQ ID NO: 41) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG
  • R9 (SEQ ID NO: 42) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNL CDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVDKL AAARRRRRRRLEHHHHHH
  • a modified tail portion includes one or more truncation mutations.
  • such modified tail portions may form part of a viral core protein as provided below, together with the corresponding nucleic acids.
  • the modified tail portion is underlined for ease of identification.
  • Some modified tail portions may or may not include a histidine tag.
  • Exemplary truncation mutants include a mutation at CP 155 with the following nucleic acid sequence: (SEQ ID NO: 49)
  • CP 155 has the following amino acid sequence, with the modified tail portion underlined: (SEQ ID NO: 50)
  • modified viral core proteins include: CP162
  • GGT CGC AGC CCG CGC CGT CGT ACC CCG AGC CCG CGT CGT CGT AGC CAG AGC CTC GAG CAC CAC CAC CAC CAC CAC CAC CAC CAC
  • CP170 (SEQ ID NO: 54) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNL CDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVRRR GRSPRRRTPSPRRRRSQSLEHHHHHH Linker Segment Mutations
  • a linker segment may be optionally present between e.g. a modified core portion and a modified tail portion, for example between the amino acid residue 149 and another modified tail portion domain.
  • the linker segment is about 3 amino acids to about 15 amino acids in length (or any specific amino acid length disposed with the range) and can link e.g. a modified tail portion including a poly-lysine domain and/or a polyarginine domain to e.g. amino acid 149 of the HBV core protein, for example, to provide flexibility to the C-terminal tail.
  • poly-lysine and/or a poly-arginine domain can be followed by a poly histidine tag and/or followed by an Xhol restriction site.
  • a poly histidine tag can include at least six histidine residues added to the C- terminal tail.
  • linker segments may be represented by
  • Exemplary viral core proteins that include a linker segment ( with corresponding nucleic acids) are provided below:
  • Linker 1 K9 (SEQ ID NO: 59) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILCWGELMTLATWVGNNL CDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVSAG SAGKKKKKKKKKLEHHHHHH Linker 2 K9 has the following nucleic acid sequence: (SEQ ID NO: 60)
  • AAAAA AATTTT CCGGTT CCAAGG CCTTGG CCTTGG TGG TTT CAT ATC AGC TGC CTG ACC TTT GGC CGC GAA ACC
  • Linker 8 K9 (SEQ ID NO: 72) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG CCG AGC GAT TTC TTT CCG AGC GTG CGT GAT CTG CTG GAT ACC GCG AGC GCG CTG TAT CGC GAA GCG CTG GAA AGC CCG GAA CAT TGT AGC CCG CAC CAT ACC GCC CTG CGT CAG GCG ATT CTG TGC TGG GGT GAA CTG ATG ACC CTG GCG ACC TGG GTT GGC AAC AAC CTG TGC GAT CCG GCG AGC CGC GAT CTG GTT GTG AAT ACC AAC ATG GGC CTG AAA ATT CGT CAG CTG CTG TGG TTT CAT ATC AGC TGC CTG ACC TTT GGC CGC GAA ACC GTG CTG GAA TAT CTG GTG AGC TTT
  • Linker 9 K9 (SEQ ID NO: 74) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG CCG AGC GAT TTC TTT CCG AGC GTG CGT GAT CTG CTG GAT ACC GCG AGC GCG CTG TAT CGC GAA GCG CTG GAA AGC CCG GAA CAT TGT AGC CCG CAC CAT ACC GCC CTG CGT CAG GCG ATT CTG TGC TGG GGT GAA CTG ATG ACC CTG GCG ACC TGG GTT GGC AAC AAC CTG TGC GAT CCG GCG AGC CGC GAT CTG GTT GTG AAT ACC AAC ATG GGC CTG AAA ATT CGT CAG CTG CTG TGG TTT CAT ATC AGC TGC CTG ACC TTT GGC CGC GAA ACC GTG CTG GAA TAT CTG GTG AGC TGG
  • An exemplary non-His tagged K9 has the following nucleic acid sequence: (SEQ ID NO: (SEQ ID NO: 1)
  • Non-His tagged K9 viral core protein has the following amino acid sequence:
  • the modification of the tail portion may allow a nucleic acid to bind to the tail portion with a binding affinity that may allow release of the nucleic acid when the chimeric therapeutic (e.g. viral core protein bound to a nucleic acid) is administered e.g. in vivo.
  • a modified tail portion that includes lysine may bind a nucleic acid using substantially Coulombic forces only, such that the nucleic acid may, in some embodiments, be released when exposed to a ionic solution e.g. a salt solution.
  • a ionic solution e.g. a salt solution.
  • the disclosed chimeric therapeutics e.g. that do not include a modified tail portion with a substantial number of arginines such as those arranged as in the wild type tail portion, may be substantially free of endogenous nucleic acids.
  • a structural core portion of a viral core protein may be modified to for example, (a) strengthen and promote assembly of the viral core protein, e.g. HBV C-protein monomers, into a capsid; (b) enhance and promote the coating of one or more capsids with a layer comprising a lipid or lipid/cholesterol; (c) facilitate the attachment of other moieties, e.g., chemical modifiers and/or targeting agents; and/or (d) facilitate the disassembly of the entire capsid in the bloodstream following administration.
  • the viral core protein e.g. HBV C-protein monomers
  • the wild type HBV C-protein is typically 183 amino acids.
  • the first 149 amino acids typically form a globular fold or structural core.
  • a structural core portion includes the first 138 amino acids of e.g. a wild type HBV protein.
  • a structural core portion of a viral core protein based on amino acids 1-149 of SEQ ID NO: 1 or SEQ ID NO: 2, that may include one or more modifications.
  • a contemplated modified structural portion of a viral core protein may include amino acids 1-138 of SEQ ID NO: 1 or SEQ ID NO: 2, and that such a structural portion may include any one or more of the modifications indicated below.
  • HBV C-protein variant SEQ ID NO: X
  • SEQ ID NO: 78 An exemplary modified structural core protein can be, in some embodiments, represented by SEQ ID NO: 78, where X, independently for each occurrence, represents an amino acid.
  • a contemplated viral core protein may include a structural portion represented by e.g. SEQ ID NO: 78 and may additionally include a modified or unmodified tail portion, e.g. a modified C-terminal tail portion such as those described above.
  • a HBV capsid may be formed from protein dimers.
  • intermolecular interactions between dimers may stabilize the assembly and may be formed by disulfide bonds, salt bridges, and hydrophobic interactions between proteins.
  • a structural core portion may include mutation of interacting amino acid side chains to either stabilize or destabilize the interactions and therefore, the capsid or particle assembly.
  • destabilizing mutations may be introduced at Phel8, Tyrl32, and/or Ilel39.
  • a disulfide bond may be introduced at Serl21 and/or Serl41, which may, for example, stabilize inter-dimer associations between viral core proteins.
  • the native cysteine residues at positions 48, 61, and/or 107 may also be mutated, (for example to an alanine), without substantially affecting the ability of the core protein to form a capsid or particle.
  • Modifications of the structural core portion of a viral core protein can include the introduction of e.g., a pair of cysteines into a spike area of a formed dimer or the interface between dimers.
  • a first cysteine e.g. amino acid 23
  • a second cysteine amino acid 132 in this case
  • the second position may also participate in a disulfide bond, allowing the dimer to participate in four disulfide bridges and a total of 180 stabilizing covalent interactions. At least four different types of disulfide bonds may be created:
  • such mutations may affect the long-term stability of a capsid or particle formed from viral core proteins that include such viral structural portions.
  • Such stabilizing and destabilizing mutations can be introduced e.g. singly and/or in combination.
  • exemplary modified viral core proteins, that include a modified structural core portion include the following:
  • HBV C-protein variant of SEQ ID NO: 2 comprising mutation 1: phenylalanine 23 to cysteine; tyrosine 132 to cysteine.
  • SEQ ID NO: 79 MDIDPYKEFGATVELLSFLPSDCFPSVRDLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMTLATWVGNNLEDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWIRTPPACRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE SQC
  • HBV C-protein SEQ ID NO: 1 comprising mutation 1: phenylalanine 23 to cysteine; tyrosine 132 to cysteine. (SEQ ID NO: 80)
  • HBV C-protein variant SEQ ID NO: 2 comprising mutation 2: aspartic acid 29 to cysteine; arginine 127 to cysteine. (SEQ ID NO: 81)
  • HBV C-protein SEQ ID NO: 1 comprising mutation 2: aspartic acid 29 to cysteine; arginine 127 to cysteine.
  • SEQ ID NO: 82 MDIDPYKEFGASVELLSFLPSDFFPSIRCLLDTASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGVWICTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE SQC
  • HBV C-protein variant SEQ ID NO: 2 comprising mutation 3: threonine 33 to cysteine; valine 124 to cysteine.
  • HBV C-protein SEQ ID NO: 1 comprising mutation 3: threonine 33 to cysteine; valine 124 to cysteine.
  • SEQ ID NO: 84 MDIDPYKEFGASVELLSFLPSDFFPSIRDLLDCASALYREALESPEHCSPHHTALRQAIL CWGELMNLATWVGSNLEDPASRELVVSYVNVNMGLKIRQLLWFHI SCLTFGRETVLEYLV SFGCWIRTPPAYRPPNAPILSTLPETTVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRE SQC
  • HBV C-protein variant SEQ ID NO: 2 comprising mutation 4: leucine 37 to cysteine; valine 120 to cysteine. (SEQ ID NO: 85)
  • HBV C-protein SEQ ID NO: 1 comprising mutation 4: leucine 37 to cysteine; valine 120 to cysteine. (SEQ ID NO: 86)
  • modified viral core proteins that include a modified structural core portion, include the following viral core proteins together with corresponding nucleic acid sequences:
  • F18H K9 (SEQ ID NO: 87) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC CAT CTG
  • I139A S121C S141C K9 (SEQ ID NO: 105) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG
  • Y132V S121C S141C K9 (SEQ ID NO: 107) ATG GAT ATC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG
  • C61A K9 (SEQ ID NO: 112) MDIDPYKEFGATVELLSFLPSDFFPSVRDLLDTASALYREALESPEHCSPHHTALRQAILAWGELMTLATWVGNNL CDPASRDLVVNYVNTNMGLKIRQLLWFHI SCLTFGRETVLEYLVSFGVWIRTPPAYRPPNAPILSTLPETTVVDKL AAAKKKKKKKKKLEHHHHHH
  • C107A K9 (SEQ ID NO: 113) AATTGG GGAATT AATTCC GAT CCG TAT AAA GAA TTT GGC GCC ACC GTG GAA CTG CTG AGC TTT CTG
  • Alterations or mutations may be made on e.g., a. viral structural core that may, for example, facilitate disassembly of a capsid or particle formed disclosed viral core proteins after, for example, administering in vivo.
  • mutations are contemplated that may introduce blood protease recognition sequences, e.g., protease recognition sites at hinge and loop regions. Such sequences can be inserted, for example, into the spike region of the HBV C-protein (e.g. replacing amino acids 79 and 80 with these 12 amino acid insertion loops.
  • a viral core protein may include up to a further about 40, or about 46 residues and may still, in some embodiments, be capable of forming a particle or capsid.
  • Exemplary blood protease recognition sequences include for example, thrombin (SEQ ID NO: 125) and factor Xa (SEQ ID NO: 126.)
  • GPGAPGLVPRGS (SEQ ID NO: 125)
  • GPASGPGiEGRA SEQ ID NO: 1236
  • contemplated HBV C-proteins from SEQ ID NO:2 (and associated nucleic acids) that comprise such a blood protease recognition sequence can be represented by: (SEQ ID NO: 127)
  • a structural core portion of the viral core protein may be modified to include a conjugation site that allows the attachment of a moiety, e.g. a chemical linker moiety such as a lipid linker moiety.
  • a conjugation site that allows the attachment of a moiety, e.g. a chemical linker moiety such as a lipid linker moiety.
  • a chemical linker moiety such as a lipid linker moiety.
  • either of the amino acids cysteine or lysine may be placed in the structural core in such a way so that when formed in a capsid or particle these modifications may protrude away from the capsid surface e.g. toward a plasma membrane.
  • such modifications may permit the addition of one or more lipid linker moieties which can serve to promote or facilitate a lipid layer.
  • such a modification may permit the addition ⁇ e.g. the attachment of ) one or more targeting agents, as described below.
  • a structural core portion of a viral core protein may be used for the introduction of one or more cysteines and/or lysines, e.g. site 77, glutamic acid to cysteine; 78, aspartic acid to cysteine; and/or site 80, alanine to cysteine on a HBV C protein.
  • cysteine modifications for example, may be further functionalized.
  • Cysteine mutations can also be introduced at other locations in the C-protein.
  • Exemplary modified viral core proteins and associated nucleic acids include:
  • A80C (SEQ ID NO: 137)
  • HBV C-protein variant 1 (SEQ ID NO:1).
  • E77C generated within HBV C- protein variant 1 has the following amino acid sequence: (SEQ ID NO: 138)
  • D78C generated within HBV C-protein variant 1 has the following amino acid sequence: (SEQ ID NO: 139)
  • A80C generated within HBV C-protein variant 1 has the following amino acid sequence: (SEQ ID NO: 140)
  • a chemical linker may bind another moiety to a particle formed from viral core proteins that include a modified structure core portion, e.g. that include one or more cysteine residues.
  • exemplary chemical linkers include moieties such as those formed by contacting a cysteine residue with a maleimide containing compound such as phosphoethanolamine-maleimide (PE-maleimide or PE- mal).
  • Phospholipids for example, may be directly linked through a chemical linker to a modified structural core portion e.g. to link a lipid molecule and/or a targeting agent.
  • cysteine residues may be engineered into the structural core portion region to provide a covalent linker to a modified Hepatitis B Virus S-protein.
  • a S-protein may guide the coating of the lipid layer or lipid/cholesterol layer.
  • Contemplated S-proteins for attaching to a disclosed capsid or particle may be modified to have cysteines as well to complement the disulfide bridge formation between C-protein monomers.
  • a S-protein can be replaced by a peptide such as a transmembrane engineered peptide.
  • An exemplary transmembrane engineered peptide may have e.g., a flexible region that ends with a cysteine so as to form disulfide bridges with the cage, with the opposite end comprising primarily of hydrophobic residues.
  • a non-limiting example of such a HBV S- protein transmembrane engineered peptide has the amino acid sequence:
  • nucleic acid and amino acid sequences of the specific modified viral core proteins e.g. about 75% to about 99% identical, about 80% to about 95% identical, about 85% to about 90% identical, or about 95% to about 99% identical, or any specific percent identity disposed within these ranges, to disclosed viral core proteins capable of forming a capsid and capable of binding a nucleic acid are within the scope of the present invention.
  • Targeting Agents e.g. about 75% to about 99% identical, about 80% to about 95% identical, about 85% to about 90% identical, or about 95% to about 99% identical, or any specific percent identity disposed within these ranges
  • Various targeting agents can be incorporated into e.g. a coating layer of the disclosed multiplexes, e.g. incorporated or bound to a lipid layer or lipid/cholesterol layer coat to direct the multiplex to a tissue or cell target.
  • a targeting agent may be bound directly, e.g. chemically linked, directly or through a chemical linker moiety, to a disclosed particle.
  • An exemplary targeting agent may be an antibody.
  • exposed sulfhydryl groups on the heavy chain of an antibody can be used to link the antibody to e.g. a free sulfate group on a coating comprising one or more lipids.
  • a lipid can be attached to antibodies through different chemical means, such as reacting an activated lipid such as PE-maleimide to activated free amines of an antibody with agents such as Traut' s Reagent.
  • a reduced antibody heavy chain-light chain complex above can also be attached directly to the naked particle.
  • the modified viral core protein may incorporate cysteine residues with reactive sulfhydryl groups as described above which then can be linked with the partially disassociated antibody chains.
  • Antibodies suitable for use as targeting agents include antibodies directed to cell surface antigens which cause the antibody-multiplex complex to be internalized, either directly or indirectly.
  • Specific non-limiting examples of suitable antibodies include antibodies to CD19, CD20, CD22, CD33 and CD74.
  • CD33 and CD22 are over-expressed on lymphomas and binding to these antigens caused endocytosis and thereby internalization of the antibody- nanoparticle complex.
  • Methods for incorporating monoclonal antibodies to CD22 into the lipid coating can be found in U.S. Patent Publication No. 20070269370, which is incorporated herein by reference.
  • a coating of a multiplex, or the particle itself may be modified, to enhance e.g. the ability of the particles to enter target cells and/or to at least partially evade the immune system in vivo.
  • a large polymer e.g. PEG
  • PEG cholesterol-tagged or lipid-tagged polyethylene glycol
  • PTD protein transduction domains
  • Non-limiting examples of suitable PTDs are the Human Immunodeficiency Virus (HIV) transactivator of transcription (Tat) peptide and/or poly-arginine (poly-Arg).
  • the particles and/or coatings may be modified by attaching a PEG.
  • one or more cholesterol-tagged PEGs may be anchored into a lipid coating or particle, and/or one or more cholesterol tagged PTD may be anchored into a coating or particle.
  • a PTD may be engineered to bind to e.g. 77, 78, 79 on a HBV structure core portion of a viral core protein.
  • a particle and/or coating may be modified, e.g. covalently bonded through a chemical linker, to a carbohydrate and/or a sugar, e.g. a branched sugar, moiety.
  • Antibody mimetics and/or peptide mimetics that include complementarity determining region (CDR) subunits may also be, in some embodiments, associated with or bound to (e.g. via linker) to a coating or particle disclosed herein.
  • CDR complementarity determining region
  • an targeting agent that binds FcRN, s-protein or other moiety can be bound or associated with either a coating, e.g. a lipid coating, or may be bound directly to a modified viral core.
  • a coating e.g. a lipid coating
  • Such targeting agents include those in US Patent Application 20070254831. Nucleic Acids
  • the therapeutic chimerics, multiplexes, particles and compositions disclosed here include at least one nucleic acid substantially homologous to a particular target bound to, or associated with, a viral core protein.
  • an nucleic acid when bound to a viral core protein, is "substantially non-immunogenic" i.e., does not elicit, induce, or invoke a substantial immune response, for example, a humoral and/or a cellular immune response in a mammalian subject, such as a human subject.
  • a nucleic acid molecule e.g. an inhibitory nucleic acid that is not bound to the viral core protein, e.g.
  • nucleic acids when substantially released in vivo from a therapeutic disclosed herein, may be substantially non-immunogenic, or may have immunogenic properties.
  • Exemplary nucleic acids that may form part of the e.g. , disclosed therapeutics, multiplexes, particles and/or compositions disclosed herein include inhibitory nucleic acids.
  • Other exemplary nucleic acids contemplated for use include double stranded RNA, antisense nucleic acid, hairpin RNA, and microRNA.
  • Inhibitory nucleic acid include an inhibitory double-stranded RNA, i.e., a "interfering RNA" or “iRNA" of about 10 to about 60, about 15 to about 50, about 15 to about 40, about 15 to about 30, or about 15 to about 20 nucleotides in length.
  • an inhibitory double stranded RNA is about 25 to about 45, about 27 to about 40, about 30 to about 40, about 33 to about 40, or about 36 to about 40 nucleotides in length.
  • an inhibitory double stranded RNA is about 15 to about 30, about 15 to about 25, about 19 to about 25, about 19 to about 23, or about 19 to about 21 nucleotides in length.
  • an inhibitory double stranded RNA is about 20 to about 24 or about 21 to about 22 or to about 23 nucleotides in length.
  • An inhibitory double stranded RNA may be transcribed from a transcriptional cassette in a DNA plasmid. Such inhibitory double stranded RNA reduces, inhibits or silences expression of a target gene by mediating the degradation of mRNAs, which are complementary to the sequence of an inhibitory RNA, by the process of RNA interference.
  • RNA interference is a process by which double-stranded RNA (dsRNA) is used to silence gene expression. While not wanting to be bound by theory, RNAi begins with the cleavage of longer dsRNAs into smaller inhibitory dsRNAs by an RNaselll-like enzyme, dicer. Inhibitory dsRNAs that are usually about 19 to 28 nucleotides, or 20 to 25 nucleotides, or 21 to 22 nucleotides in length and often contain 2-nucleotide 3' overhangs, and 5' phosphate and 3' hydroxyl termini.
  • RISC RNA-induced silencing complex
  • RISC uses this RNA strand to identify mRNA molecules that are at least partially complementary to the incorporated RNA strand, and then cleaves these target mRNAs or inhibits their translation. Therefore, the RNA strand that is incorporated into RISC is known as the guide strand or the antisense strand.
  • the other RNA strand known as the passenger strand or the sense strand, is eliminated from the RNA and is at least partially homologous to the target mRNA.
  • dsRNA design e.g., decreased dsRNA duplex stability at the 5' end of the antisense strand
  • RISC reduced dsRNA duplex stability at the 5' end of the antisense strand
  • RISC-mediated cleavage of mRNAs having a sequence at least partially complementary to the guide strand leads to a decrease in the steady state level of that mRNA and of the corresponding protein encoded by this mRNA.
  • RISC can also decrease expression of the corresponding protein via translational repression without cleavage of the target mRNA.
  • Other RNA molecules and RNA-like molecules can also interact with RISC and silence gene expression. Examples of other RNA molecules that can interact with RISC include hairpin RNAs, single- stranded RNAs, microRNAs, and dicer- substrate 27-mer duplexes.
  • An inhibitory double stranded RNA can be formed by two complementary strands or by a single, self-complementary strand.
  • the relationship between a target mRNA and the sense strand of an inhibitory RNA is that of identity.
  • the sense strand of an inhibitory RNA is also called a passenger strand, if present.
  • the relationship between a target mRNA (a sense strand) and the antisense strand of an inhibitory RNA is that of complementarity.
  • the antisense strand of an inhibitory RNA is also called a guide strand.
  • Exemplary inhibitory double stranded RNA duplex may comprise 3' overhangs of about 1 to about 4 nucleotides, for example, of about 2 to about 3 nucleotides, and 5' phosphate termini.
  • an inhibitory double stranded RNA duplex may have no overhangs on one or both ends (blunt ends).
  • Some exemplary inhibitory double stranded RNAs may lack a terminal phosphate.
  • inhibitory double stranded RNA molecules include, without limitation, a double- stranded polynucleotide molecule assembled from two separate oligonucleotides, wherein one strand is the sense strand and the other is the complementary antisense strand; a double-stranded polynucleotide molecule assembled from a single oligonucleotide, where the sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker; a double- stranded polynucleotide molecule with a hairpin secondary structure having self- complementary sense and antisense regions; and a circular single-stranded polynucleotide molecule with two or more loop structures and a stem having self-complementary sense and antisense regions, where the circular polynucleotide may be processed in vivo or in vitro to generate an active inhibitory double-stranded RNA molecule.
  • an inhibitory double-stranded RNA to be delivered by the present invention must have a sufficient identity to a target nucleic acid in order to mediate target- specific RNA interference.
  • an inhibitory double-stranded RNA has an identity of at least about 85%, 90%, 95%, or 100% to the desired target nucleic acid.
  • the identity of a double- stranded RNA molecule to the target sequence may also be defined to include a 3' overhang, particularly an overhang having a length from 1-3 nucleotides, with a sequence identity of at least about 50%, about 70% , or about 85% or more to the target sequence.
  • the nucleotides from the 3' overhang and up to 2 nucleotides from the 5' and/or 3' terminus of the double strand may be modified without significant loss of activity.
  • Inhibitory nucleic acids may include one or more mismatch motifs or mismatch regions, which refer to a portion of an nucleic acid sequence that does not have 100% complementary to its target sequence.
  • a nucleic acid may have at least one, two, three, four, five, six, or more mismatch regions.
  • the mismatch regions may be contiguous or may be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides.
  • the mismatch motifs or regions may comprise a single nucleotide or may comprise two, three, four, five, or more nucleotides.
  • Inhibitory double stranded RNA contemplated herein may be sufficiently identical or sufficiently complementary, e.g. substantially homologous to a target nucleic acid, e.g., a target mRNA, such that the inhibitory double stranded RNA silences production of protein encoded by the target mRNA.
  • a contemplated inhibitory double stranded RNA may be identical or exactly complementary (excluding the RRMS containing subunit(s)) to a target RNA, e.g., the target RNA and the inhibitory double stranded RNA anneal, e.g., to form a hybrid made of Watson-Crick base pairs in the region of exact identity or complementarity.
  • a sufficiently identical or sufficiently complementary target RNA may include an internal region (e.g., of at least 10 nucleotides) that is exactly identical or complementary to a target.
  • an inhibitory double stranded RNA may specifically discriminate a single-nucleotide difference, for example, mediating RNA interference if exact identity or complementary is found in the region of the single-nucleotide difference (e.g., within 7 nucleotides of the single nucleotide difference).
  • Suitable inhibitory double stranded RNA sequences that target a gene of interest may be identified using any means known in the art. Typically, methods such as gene walking or the methods described in Elbashir et al., Nature 411 :494-498 (2001) and Elbashir et al., EMBO J 20: 6877-6888 (2001) are combined with rational design rules set forth in Reynolds et al., Nature Biotech. 22:326-330 (2004), each of which are incorporated herein by reference.
  • a sequence within about 50 to about 100 nucleotides 3' of the AUG start codon of a transcript from the target gene of interest is scanned for dinucleotide sequences (e.g., AA, CC, GG, or UU) (see, e.g., Elbashir, et al, EMBO J 20: 6877-6888 (2001)).
  • the nucleotides immediately 3' to the dinucleotide sequences are identified as potential inhibitory double stranded RNA target sequences.
  • the 19, 21, 23, 25, 27, 29, 31, 33, 35, 38, 40, 42, or more nucleotides immediately 3' to the dinucleotide sequences are identified as potential inhibitory double stranded RNA target sites.
  • the dinucleotide sequence may be, for example, an AA sequence and the 19 to about 40 nucleotides immediately 3' to the AA dinucleotide are identified as a potential inhibitory double stranded RNA target site.
  • inhibitory double stranded RNA target sites are spaced at different positions along the length of the target gene.
  • potential inhibitory double stranded RNA target sites may be analyzed to identify sites that do not contain regions of homology to other coding sequences.
  • a suitable inhibitory double stranded RNA target site of about 21 base pairs may not have more than 16-17 contiguous base pairs of homology to other coding sequences. If inhibitory double stranded RNA sequences are to be expressed from an RNA Pol III promoter, inhibitory double stranded RNA target sequences lacking more than 4 contiguous A's or T's may be selected.
  • inhibitory double stranded RNA sequences may be analyzed by a rational design algorithm to identify sequences that have one or more of the following features: (1) G/C content of about 25% to about 60% G/C; (2) at least 2 or 3 A/Us at positions 15- 19 of the sense strand; (3) no internal repeats; (4) an A at position 19 of the sense strand; (5) an A at position 3 of the sense strand; (6) a U at position 10 of the sense strand; (7) an A at position 14 of the sense strand; (8) no G/C at position 19 of the sense strand; and (9) no G at position 13 of the sense strand.
  • Inhibitory double stranded RNA design tools that incorporate algorithms that assign suitable values of each of these features and are useful for selection of inhibitory double stranded RNA can be found at, e.g., http://boz094.ust.hk/RNAi/siRNA. Techniques for selecting target sequences for inhibitory RNAs are provided by Tuschl, T. et al., "The siRNA User Guide,” revised May 6, 2004, available on the Rockefeller University web site; by Technical Bulletin #506, "siRNA Design Guidelines," Ambion Inc. at Ambion's web site; and by other web-based design tools at, for example, the Invitrogen, Dharmacon, Integrated DNA Technologies, Genscript, or Proligo web sites.
  • initial search parameters can include G/C contents between 35% and 55% and siRNA lengths between 19 and 27 nucleotides.
  • the target sequence may be located in the coding region or in the 5' or 3' untranslated regions of the mRNA.
  • sequences with one or more of the foregoing characteristics may be selected for further analysis and testing as potential inhibitory double stranded RNA sequences.
  • Inhibitory RNA sequences complementary to target nucleic acid sites may also be designed.
  • inhibitory double stranded RNA target sequences with one or more of the following criteria can often be eliminated as inhibitory double stranded RNA: (1) sequences comprising a stretch of 4 or more of the same base in a row; (2) sequences comprising homopolymers of Gs (i.e., to reduce possible non-specific effects due to structural characteristics of these polymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC, AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in a row; and (5) sequence comprising direct repeats of 4 or more bases within the candidates resulting in internal fold-back structures.
  • sequences with one or more of the foregoing characteristics may still be selected for further analysis and testing as potential inhibitory double stranded RNA sequences.
  • the importance of various criteria can vary greatly. For instance, a C base at position 10 of the sense strand may make a minor contribution to duplex functionality. In contrast, the absence of a C at position 3 of the sense strand is may be very important.
  • GC content, as well as a high number of AU in positions 15-19 of the sense strand may be important for easement of the unwinding of an inhibitory double stranded RNA duplex.
  • Duplex unwinding has been shown to be crucial for inhibitory double stranded RNA functionality in vivo.
  • the internal structure is measured in terms of the melting temperature (Tm) of the single strand of inhibitory double stranded RNA, which is the temperature at which 50% of the molecules will become denatured.
  • Tm melting temperature
  • All of the aforementioned criteria regarding sequence position specifics are with respect to the 5' end of the sense strand. Reference is made to the sense strand, because most databases contain information that describes the information of the mRNA.
  • An inhibitory nucleic acid molecule contemplated herein may be a variety of lengths.
  • the aforementioned criteria may assume an inhibitory nucleic acid molecule of at least 19 nucleotides in length so that it is important to keep the aforementioned criteria applicable to the correct bases. It is understood that a person skilled in the art will know how to apply the aforementioned criteria to inhibitory nucleic acid molecules of varying lengths.
  • RNA sequences are disclosed in Naito et al., Nucleic Acids Res 33: W589-591, 2005, Henschel et al, Nucleic Acids Res 32: Wl 13- 120, 2004, Naito et al., Nucleic Acids Res 32: W124-129, 2004 (for mammalian- specific interfering RNAs) and Naito et ah, Nucleic Acids Res 34: W448-450, 2006 (for viral- specific interfering RNAs), each of which is incorporated herein by reference.
  • a person skilled in the art may use one or more algorithms to select an inhibitory RNA sequence. Further, a person skilled in the art will appreciate the use of multiple parameters and algorithms in selecting and optimizing an inhibitory RNA sequence.
  • Inhibitory double stranded RNA selected according to the aforementioned criteria or one of the aforementioned algorithms are also, for example, useful in the simultaneous screening and functional analysis of multiple genes and gene families using high throughput strategies, as well as in direct gene suppression or silencing.
  • Useful applications for inhibitory nucleic acid molecules include, but are not limited to, target validation, gene functional analysis, research and drug discovery, gene therapy and therapeutics. Methods for using inhibitory nucleic acid molecules including inhibitory double-stranded RNA molecules in these applications are well known to persons of skill in the art.
  • Inhibitory double stranded RNA molecules contemplated herein may be applicable across a broad range of species, including but not limited to all mammalian species, such as humans, dogs, horses, cats, cows, mice, hamsters, chimpanzees and gorillas, as well as other species and organisms such as bacteria, viruses, insects, plants and C. elegans.
  • nucleic acids applicable for use for silencing a broad range of genes including but not limited to the roughly 45,000 genes of a human genome.
  • contemplated herein are nucleic acids that target to genes are associated with diseases such as the gene targets discussed herein. Analysis of Inhibitory Nucleic Acid Molecules
  • Potential inhibitory double stranded RNA target sequences may be further analyzed based on inhibitory double stranded RNA duplex asymmetry as described in, e.g., Khvorova et al., Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).
  • Potential inhibitory double stranded RNA target sequences may be further analyzed based on secondary structure at the mRNA target site as described in, e.g., Luo et al., Biophys. Res. Commun., 318:303-310 (2004).
  • mRNA secondary structure may be modeled using the Mfold algorithm (available at http://www.bioinfo.rpi.edu/applications/mfold/rna/forml.cgi) to select inhibitory double stranded RNA sequences which favor accessibility at the mRNA target site where less secondary structure in the form of base-pairing and stem-loops is present.
  • Mfold algorithm available at http://www.bioinfo.rpi.edu/applications/mfold/rna/forml.cgi
  • the sequence may be analyzed for the presence of any immunostimulatory properties, e.g., using an in vitro cytokine assay or an in vivo animal model.
  • Motifs in the sense and/or antisense strand of the inhibitory double stranded RNA sequence such as GU-rich motifs (e.g., 5'-GU-3', 5'- UGU-3', 5'-GUGU-3', 5'-UGUGU-3', etc.) may also provide an indication of whether the sequence may be immunostimulatory. If an inhibitory double stranded RNA molecule is found to be immunostimulatory, it may, in certain embodiments, be modified to decrease its immunostimulatory properties.
  • the detectable immune response may comprise production of a cytokine or growth factor such as, e.g., TNF- ⁇ , IFN-CC, IFN- ⁇ , IFN- ⁇ , IL-6, IL- 12, or a combination thereof.
  • a cytokine or growth factor such as, e.g., TNF- ⁇ , IFN-CC, IFN- ⁇ , IFN- ⁇ , IL-6, IL- 12, or a combination thereof.
  • an inhibitory double stranded RNA identified as being immunostimulatory can be modified to decrease its immunostimulatory properties by replacing at least one (but less than about 30%) of the nucleotides on the sense and/or antisense strand with modified nucleotides such as 2'0Me nucleotides (e.g., 2'OMe-guanosine, 2'0Me- uridine, 2'OMe-cytosine, and/or 2'OMe-adenosine), as described in further detail herein.
  • Suitable in vitro assays for detecting an immune response include, but are not limited to, the double monoclonal antibody sandwich immunoassay technique of David et al. (U.S. Pat. No. 4,376,110); monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkham and Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone, Edinburgh (1970)); the "Western blot" method of Gordon et al. (U.S. Pat. No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al, J. Biol. Chem.
  • Inhibitory nucleic acid molecules may be provided in several forms including, e.g., as one or more isolated RNA duplexes, e.g. siRNA, longer double- stranded RNA (dsRNA) or RNA transcribed from a transcriptional cassette in a DNA plasmid. Inhibitory nucleic acid molecules, such as inhibitory double stranded RNAs may also be chemically synthesized. The inhibitory double stranded RNA sequences may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al, Genes Dev. 15:188 (2001) or Nykanen et al, Cell 107:309 (2001), or may lack overhangs (i.e., to have blunt ends).
  • RNA duplexes e.g. siRNA, longer double- stranded RNA (dsRNA) or RNA transcribed from a transcriptional cassette in a DNA plasmid.
  • Inhibitory nucleic acid molecules
  • Exemplary RNA population may be used to provide long precursor RNAs, or long precursor RNAs that have substantial or complete identity to a selected target sequence may be used to make the inhibitory dsRNA.
  • the RNAs can be isolated from cells or tissue, synthesized, and/or cloned according to methods well known to those of skill in the art.
  • the RNA may be a mixed population (obtained from cells or tissue, transcribed from cDNA, subtracted, selected etc.), or may e.g. represent a single target sequence.
  • RNA may be naturally occurring, (e.g., isolated from tissue or cell samples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCR products or a cloned cDNA), or chemically synthesized.
  • the complement is transcribed in vitro and hybridized to form a dsRNA.
  • the RNA complements are also provided (e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g., by transcribing cDNAs corresponding to the RNA population, or by using RNA polymerases.
  • the precursor RNAs are then hybridized to form double stranded RNAs for digestion.
  • Inhibitory dsRNA can, in some embodiments, be transcribed as sequences that automatically fold into duplexes with hairpin loops from DNA templates in plasmids having RNA polymerase III transcriptional units, for example, based on the naturally occurring transcription units for small nuclear RNA U6 or human RNase P RNA Hl (see, Brummelkamp, et al, Science 296:550 (2002); Donze, et al, Nucleic Acids Res. 30:e46 (2002); Paddison, et al, Genes Dev.
  • a transcriptional unit or cassette will contain an RNA transcript promoter sequence, such as an Hl-RNA or a U6 promoter, operably linked to a template for transcription of a desired inhibitory dsRNA sequence and a termination sequence, comprised of 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal)
  • RNA transcript promoter sequence such as an Hl-RNA or a U6 promoter
  • a termination sequence comprised of 2-3 uridine residues and a polythymidine (T5) sequence (polyadenylation signal)
  • the selected promoter may e.g. provide for constitutive or inducible transcription.
  • Compositions and methods for DNA-directed transcription of RNA interference molecules is described in detail in U.S. Pat. No. 6,573,099.
  • the transcriptional unit is incorporated into a plasmid or DNA vector from which the interfering RNA is transcribed. Plasmids suitable for in vivo delivery of genetic material for therapeutic purposes are described in detail in U.S. Pat. Nos. 5,962,428 and 5,910,488, and such plasmids may provide for transient or stable delivery of a target cell.
  • plasmids originally designed to express desired gene sequences may be modified to contain a transcriptional unit cassette for transcription of inhibitory dsRNA.
  • Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, e.g., Gubler and Hoffman, Gene 25:263-269 (1983); Sambrook et al, supra; Ausubel et al, supra), as are PCR methods (see U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al, eds, 1990)).
  • contemplated inhibitory nucleic acid molecules are chemically synthesized.
  • the single stranded molecules that comprise a modified inhibitory nucleic acid molecule may be synthesized using any of a variety of techniques known in the art, such as those described in Usman et al, J. Am. Chem. Soc. 109:7845 (1987); Scaringe et al., Nuc. Acids Res. 18:5433 (1990); Wincott et al, Nuc. Acids Res. 23:2677-2684 (1995); and Wincott et al, Methods MoI. Bio. 74:59 (1997).
  • oligonucleotides makes use of common nucleic acid protecting and coupling groups, such as dimethoxytrityl at the 5'-end and phosphoramidites at the 3'-end.
  • small scale syntheses may be conducted on an Applied Biosystems synthesizer using a 0.2 ⁇ mol scale protocol with a 2.5 min. coupling step for 2'-O-methylated nucleotides.
  • syntheses at the 0.2 ⁇ mol scale may be performed on a 96- well plate synthesizer from Protogene (Palo Alto, Calif.).
  • a larger or smaller scale of synthesis is also within the scope of the present invention.
  • Suitable reagents for oligonucleotide synthesis, methods for RNA deprotection, and methods for RNA purification are known to those of skill in the art.
  • an inhibitory dsRNA may be synthesized via a tandem synthesis technique, wherein both strands are synthesized as a single continuous oligonucleotide fragment or strand separated by a cleavable linker that is subsequently cleaved to provide separate fragments or strands that hybridize to form the inhibitory dsRNA duplex.
  • a linker can be a polynucleotide linker or a non-nucleotide linker.
  • a tandem synthesis of modified inhibitory dsRNA may be readily adapted to both multiwell/multiplate synthesis platforms as well as large scale synthesis platforms employing batch reactors, synthesis columns, and the like.
  • the modified inhibitory dsRNA can be assembled from two distinct oligonucleotides, wherein one oligonucleotide comprises the sense strand and the other comprises the antisense strand of the inhibitory dsRNA.
  • each strand can be synthesized separately and joined together by hybridization or ligation following synthesis and/or deprotection.
  • the modified inhibitory dsRNA can be synthesized as a single continuous oligonucleotide fragment, wherein the self-complementary sense and antisense regions hybridize to form an inhibitory dsRNA duplex having hairpin secondary structure.
  • Inhibitory dsRNAs described herein may comprise at least one modified nucleotide in the sense and/or antisense strand.
  • Exemplary contemplated modifications include the introduction of phosphorothioate linkages and 2'-substitutions on the ribose unit, e.g., 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-0-MOE), 2'-O-aminopropyl (2'-0-AP), 2'-O-dimethylaminoethyl (2'-0-DMAOE), 2'-O-dimethylaminopropyl (2'-0-DMAP), 2'-O- dimethylaminoethyloxyethyl (2'-0-DMAEOE), 2'-O-N-methylacetamido (2'-0-NMA) substitutions, 5-C-methyl, 2'-methoxyethyl, 4'-thio
  • Modified nucleotides having a Northern conformation such as those described in, e.g., Saenger, Principles of Nucleic Acid Structure, Springer- Verlag Ed. (1984), are also suitable for use in an inhibitory dsRNA of the present invention.
  • modified nucleotides include, without limitation, locked nucleic acid (LNA) nucleotides (e.g., 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'-deoxy- 2'-fluoro nucleotides, 2'-deoxy-2'-chloro nucleotides, and 2'-azido nucleotides.
  • LNA locked nucleic acid
  • MOE 2'-methoxyethoxy
  • MOE 2'-methyl-thio-ethyl nucleotides
  • 2'-deoxy- 2'-fluoro nucleotides 2'-deoxy-2'-chloro nucleotides
  • 2'-azido nucleotides include one or more G-clamp nucleotides.
  • a G-clamp nucleotide refers to a modified cytosine analog wherein the modifications confer the ability to hydrogen bond both Watson-Crick and Hoogsteen faces of a complementary guanine nucleotide within a duplex (see, e.g., Lin et al., J. Am. Chem. Soc. 120:8531-8532 (1998)).
  • nucleotides having a nucleotide base analog such as, for example, C-phenyl, C- naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res. 29:2437-2447 (2001)) can be incorporated into the inhibitory dsRNA.
  • a nucleotide base analog such as, for example, C-phenyl, C- naphthyl, other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res. 29:2437-2447 (2001))
  • a cholesterol moiety e.g., on the 3'-end of the sense strand
  • a T- modification e.g., a 2'-O-methyl or 2'-deoxy-2'-fluoro-modification
  • a phosphorothioate e.g., on the 3'-most one or two nucleotides of the sense and antisense strands
  • a cholesterol moiety e.g., on the 3'-end of the sense strand
  • a T- modification e.g., a 2'-O-methyl or 2'-deoxy-2'-fluoro-modification
  • a phosphorothioate e.g., on the 3'-most one or two nucleotides of the sense and antisense strands
  • 2'- substitutions may be made to the 5' nucleotide of a 5'-UA-3' dinucleotide, a 5'-UG-3' dinucleotide, a 5'-CA-3' dinucleotide, a 5'-UU-3' dinucleotide, or a 5'- CC-3' dinucleotide on the sense strand and, optionally, also on the antisense strand of the inhibitory dsRNA, or to all pyrimidine-base comprising nucleotides.
  • the 5'-most pyrimidines in substantially occurrences of the sequence motifs 5'-UA-3', 5'-CA-3', 5'-UU-3', and 5'-UG-3' may be 2'-modified nucleotides, or for example, substantially all pyrimidines in the sense strand are 2'-modified nucleotides, and 5'-most pyrimidines in substantially all occurrences of the sequence motifs 5'-UA-3' and 5'-CA-3', e.g.
  • all pyrimidines in the sense strand are 2'-modified nucleotides
  • the 5'-most pyrimidines in all occurrences of the sequence motifs 5'-UA-3', 5'-CA-3', 5'-UU-3', and 5'-UG-3' are 2'-modified nucleotides in the antisense strand.
  • Inhibitory dsRNA may include one or more chemical modifications such as terminal cap moieties, phosphate backbone modifications, and the like.
  • terminal cap moieties include, without limitation, inverted deoxy abasic residues, glyceryl modifications, 4',5'-methylene nucleotides, l-( ⁇ -D-erythrofuranosyl) nucleotides, 4'-thio nucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L- nucleotides, OC- nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seco nucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties,
  • Non-limiting examples of phosphate backbone modifications include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et ah, Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)).
  • the sense and/or antisense strand may include, for example, a 3'-terminal overhang having about 1 to about 4 (e.g.,. 1, 2, 3, or 4) 2'-deoxy ribonucleotides and/or any combination of modified and unmodified nucleotides. Additional examples of modified nucleotides and types of chemical modifications that can be introduced into the modified inhibitory dsRNA are described, e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos. 20040192626 and 20050282188.
  • Modified inhibitory dsRNA described herein may include one or more non- nucleotides in one or both strands of the inhibitory dsRNA.
  • non- nucleotide refers to any group or compound that can be incorporated into a nucleic acid chain in the place of one or more nucleotide units, including sugar and/or phosphate substitutions, and allows the remaining bases to exhibit their activity.
  • the group or compound is abasic in that it does not contain a commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine and therefore lacks a base at the 1 '-position.
  • Chemical modification of the inhibitory dsRNA may include attaching a conjugate to the chemically-modified inhibitory dsRNA.
  • the conjugate may be attached at the 5' and/or 3'-end of the sense and/or antisense strand of the chemically-modified inhibitory dsRNA via a covalent attachment such as, e.g., a biodegradable linker.
  • the conjugate may also be attached to the chemically-modified inhibitory dsRNA, e.g., through a carbamate group or other linking group (see, e.g., U.S. Patent Publication Nos. 20050074771, 20050043219, and 20050158727).
  • the conjugate is a molecule that facilitates the delivery of the chemically- modified inhibitory dsRNA into a cell.
  • conjugate molecules suitable for attachment to a chemically-modified inhibitory dsRNA include, without limitation, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetyl galactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S.
  • Other examples include the lipophilic moiety, vitamin, polymer, peptide, protein, nucleic acid, small molecule, oligosaccharide, carbohydrate cluster, intercalator, minor groove binder, cleaving agent, and cross-linking agent conjugate molecules described in U.S. Patent Publication Nos. 20050119470 and 20050107325.
  • Yet other examples include the 2'-O-alkyl amine, 2'-O- alkoxyalkyl amine, polyamine, C5-cationic modified pyrimidine, cationic peptide, guanidinium group, amidininium group, cationic amino acid conjugate molecules described in U.S. Patent Publication No. 20050153337. Additional examples include the hydrophobic group, membrane active compound, cell penetrating compound, cell targeting signal, interaction modifier, and steric stabilizer conjugate molecules described in U.S. Patent Publication No. 20040167090. Further examples include the conjugate molecules described in U.S. Patent Publication No. 20050239739.
  • the type of conjugate used and the extent of conjugation to the chemically- modified inhibitory dsRNA molecule may be evaluated for improved pharmacokinetic profiles, bioavailability, and/or stability of the inhibitory dsRNA.
  • one skilled in the art can screen chemically-modified inhibitory dsRNA having various conjugates attached thereto to identify ones having improved properties using any of a variety of well-known in vitro cell culture or in vivo animal models.
  • inhibitory dsRNAs may enhance stabilization towards degradation in biological environments and may improve pharmacological properties, e.g. pharmacodynamic properties.
  • Other suitable modifications to a sugar, base, or backbone of an inhibitory dsRNA are described in PCT Publication No. WO 2004/064737.
  • an inhibitory dsRNA may include a non-naturally occurring base, such as the bases described in PCT Publication No. WO 2004/094345 and/or may include a non-naturally occurring sugar, such as a non-carbohydrate cyclic carrier molecule.
  • Exemplary features of non-naturally occurring sugars for use in inhibitory dsRNAs are described in PCT Publication No. WO 2004/094595.
  • An inhibitory dsRNA may include, in some embodiments, an internucleotide linkage (e.g., the chiral phosphorothioate linkage) useful for increasing nuclease resistance.
  • an inhibitory dsRNA may include, for example, a ribose mimic for increased nuclease resistance.
  • An inhibitory dsRNA may include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis, and/or may be complexed with an amphipathic moiety.
  • the sense and antisense sequences of an inhibitory dsRNA may be palindromic, and/or may have non-canonical pairings, such as between the sense and antisense sequences of the iRNA duplex. Examples of these modifications are described in PCT Publication No. WO 2004/080406 and U.S. Patent Publication No. 2005/0107325. Enhanced Nuclease Resistance
  • An inhibitory dsRNA e.g., an inhibitory dsRNA that targets a gene of interest
  • increased resistance may include identifying cleavage sites and modifying such sites to inhibit cleavage.
  • the dinucleotides 5'-UA-3', 5'-UG-3', 5'-CA-3', 5'-UU-3', or 5'-CC-3' can serve as cleavage sites, as described in PCT Publication No. WO 2005/115481.
  • an inhibitory dsRNA e.g., the sense and/or antisense strands of the inhibitory dsRNA
  • the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.
  • n may be any integer, e.g. 0 to 10.
  • T modifications may be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate).
  • all the pyrimidines of an inhibitory dsRNA may carry a T- modification which may have enhanced resistance to endonucleases.
  • enhanced nuclease resistance may also be achieved by modifying the 5' nucleotide, resulting, for example, in at least one 5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine is a 2'-modified nucleotide; at least one 5'-uridine-guanine-3' (5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide; at least one 5'-cytidine-adenine-3' (5'-CA-3') dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide; at least one 5'-uridine-uridine- 3' (5'-UU-3') dinucle
  • the inhibitory dsRNA may include, in some embodiments, at least 2, at least 3, at least 4 or at least 5 of such dinucleotides.
  • 5'-most pyrimidines in substantially all occurrences of the sequence motifs 5'-UA-3', 5'-CA-3', 5'-UU-3', and 5'-UG-3' may be T- modified.
  • an inhibitory dsRNA may be further modified by including a 3' cationic group, or by inverting the nucleoside at the 3'- terminus with a 3'-3' linkage.
  • the 3'-terminus may be blocked with an aminoalkyl group, e.g., a 3' C5-aminoalkyl dT, or other 3' conjugates such as naproxen or ibuprofen, small alkyl chains, aryl groups, or heterocyclic conjugates or modified sugars (D- ribose, deoxyribose, glucose etc.).
  • a 5' conjugate may be included, such as naproxen or ibuprofen, may inhibit exonucleolytic cleavage by sterically blocking the exonuclease from binding to the 5'-end of oligonucleotide.
  • an inhibitory dsRNA may have increased resistance to nucleases when a duplexed inhibitory dsRNA includes a single- stranded nucleotide overhang on at least one end.
  • the nucleotide overhang includes 1 to 4, e.g. about 2 to 3, unpaired nucleotides.
  • an unpaired nucleotide of the single- stranded overhang that is directly adjacent to the terminal nucleotide pair contains a purine base, and the terminal nucleotide pair is a G-C pair, or at least two of the last four complementary nucleotide pairs are G-C pairs.
  • a nucleotide overhang may have 1 or 2 unpaired nucleotides, and in an exemplary instance a nucleotide overhang is 5'-GC-3', for example on the 3'-end of the antisense strand.
  • the inhibitory dsRNA includes the motif 5'- CGC-3' on the 3'-end of the antisense strand, such that a 2-nt overhang 5'-GC-3' is formed.
  • an inhibitory dsRNA may include monomers which have been modified so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRMs, or Nuclease Resistance promoting Monomers or modifications.
  • inhibitory dsRNA will modulate other properties of the inhibitory dsRNA as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC, or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.
  • a protein e.g., a transport protein, e.g., serum albumin, or a member of the RISC
  • the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.
  • Modifications that may be useful for producing inhibitory dsRNA that invoke nuclease resistance may include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone: [0185] (1) chiral (Sp) thioates, e.g., that include nucleotide dimers with a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., Sp or Rp, at the position X, where this is the position normally occupied by the oxygen.
  • the atom at X may be for example, selected from S, Se, Nr2, or Br3.
  • the linkage may be an enriched or chirally pure Sp linkage.
  • NRMs including monomers at the terminal position derivatized at a cationic group.
  • a 5'-end of an antisense sequence has a terminal —OH or phosphate group so that a NRM is not used at the 5'-end of an antisense sequence.
  • the group should be attached at a position on the base which minimizes interference with H bond formation and hybridization, e.g., away form the face which interacts with the complementary base on the other strand, e.g., at the 5' position of a pyrimidine or a 7-position of a purine.
  • nonphosphate linkages at the termini for example a NRM that includes non- phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond.
  • NRM non- phosphate linkages
  • a-nucleosides e.g. L nucleosides and dimeric nucleotides derived from L-nucleosides
  • 2'-5' phosphate, non-phosphate and modified phosphate linkages e.g., thiophosphates, phosphoramidates and boronophosphates
  • conjugate groups e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;
  • abasic linkages e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein; and
  • NRM's may include monomers, e.g. at the terminal position, e.g., the 5' position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, may be removed as a result of the action of a component in the subject's body, e.g., a carboxyesterase or an enzyme present in the subject's body.
  • one or more different NRM modifications may be introduced into an inhibitory dsRNA or into a sequence of an inhibitory dsRNA.
  • An NRM modification may be used more than once in a sequence or in an inhibitory dsRNA.
  • NRMs interfere with hybridization the total number incorporated should be such that acceptable levels of inhibitory dsRNA duplex formation are maintained.
  • NRM modifications may be introduced into the terminal cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject, which may reduce off- target silencing.
  • Nuclease resistant modifications may include those placed only at the terminus and others which may be placed at any position.
  • a NRM may be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the inhibitory dsRNA is maintained. In some instances it is desirable to put a NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene, as it may minimize off-target silencing.
  • any nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an antisense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfere with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference). Such modifications may be introduced into the terminal regions, e.g., at the terminal position or within 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.
  • Inhibitory dsRNA may, in some embodiments, include a 5' phosphorylate or include a phosphoryl analog at the 5' prime terminus. Possible 5'-phosphate modifications of the antisense strand include those which are compatible with RISC mediated gene silencing.
  • Suitable modifications include: 5'-monophosphate ((HO) 2 (O)P-O-S'); 5'-diphosphate ((HO) 2 (O)P-O-P(HO)(O)-O-5'); 5'-triphosphate ((HO) 2 (O)P-O-(HO)(O)P-O-P(HO)(O)- 0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O- (HO)(O)P- O— P(HO)(O)- 0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- monothiophosphate (phosphorothioate; (HO) 2 (S)P- 0-5'
  • a sense strand can be modified in order to inactivate the sense strand and prevent formation of an active RISC, thereby potentially reducing off-target effects, for example, by a modification which prevents 5'-phosphorylation of the sense strand, e.g., by modification with a 5'-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation may also be used, e.g., simply substituting the 5'-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5'-phosphate turning it into a phosphodiester linkage.
  • a modification which prevents 5'-phosphorylation of the sense strand e.g., by modification with a 5'-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP
  • inhibitory dsRNA More detailed and specific modifications for an inhibitory dsRNA, such as phosphate group and/or sugar group modifications, replacement of the phosphate group and/or ribophosphate backbone, terminal modifications or base modifications, as well as preferred inhibitory dsRNA formula, can be found in U.S. Patent Publication No. 2007/0275914. Evaluation of candidate inhibitory nucleic acid molecule
  • a candidate inhibitory dsRNA may be evaluated for its ability to downregulate target gene expression.
  • a candidate inhibitory dsRNA may be contacted with a cell that expresses the target gene either endogenously or because it has been transfected with a construct from which the gene can be expressed.
  • the level of target gene expression prior to and following contact with the candidate inhibitory dsRNA can be compared, e.g. on an mRNA or protein level. If it is determined that the amount of RNA or protein expressed from the target gene is lower following contact with the inhibitory dsRNA, then it may be concluded that the inhibitory dsRNA downregulates target gene expression.
  • the level of target RNA or protein in the cell may be determined by any method desired. For example, the level of target RNA may be determined by Northern blot analysis, reverse transcription coupled with polymerase chain reaction (RT-PCR), or RNAse protection assay. The level of protein can be determined, for example, by Western blot analysis.
  • a functional assay may also be used in some embodiments, to evaluate a modified candidate inhibitory dsRNA.
  • a functional assay may be applied to determine if the modification alters the ability of the molecule to silence gene expression.
  • a cell e.g., a mammalian cell, such as a mouse or human cell
  • a plasmid expressing a fluorescent protein, e.g., GFP
  • a candidate inhibitory dsRNA homologous to the transcript encoding the fluorescent protein see, e.g., WO 00/44914.
  • a modified inhibitory dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate inhibitory dsRNA, e.g., controls with no inhibitory dsRNA added and/or controls with a non-modified inhibitory dsRNA added.
  • Efficacy of the candidate inhibitory dsRNA on gene expression may be assessed by comparing cell fluorescence in the presence of the modified and unmodified inhibitory dsRNA molecules. Stability Testing, Modification, and Retesting of inhibitory nucleic acid molecules
  • a candidate inhibitory dsRNA may be evaluated with respect to stability, e.g., its susceptibility to cleavage by an endonuclease or exonuclease, such as when the inhibitory dsRNA is introduced into the body of a subject.
  • stability e.g., its susceptibility to cleavage by an endonuclease or exonuclease, such as when the inhibitory dsRNA is introduced into the body of a subject.
  • methods can be employed to e.g., identify sites that are susceptible to modification, particularly cleavage, e.g., cleavage by a component found in the body of a subject.
  • a further inhibitory dsRNA may be designed and/or synthesized wherein the potential cleavage site is made resistant to cleavage, e.g. by introduction of a 2'-modification on the site of cleavage, e.g. a 2'-O-mathyl group.
  • This further inhibitory dsRNA may be retested for stability, and this process may be iterated until an inhibitory dsRNA is found exhibiting the desired stability.
  • a candidate inhibitory dsRNA e.g., a modified inhibitory dsRNA
  • a selected property may be scanned for a selected property by e.g. exposing the inhibitory dsRNA or modified inhibitory dsRNA and a control molecule to the appropriate conditions and evaluating for the presence of the selected property.
  • resistance to a degradent may be evaluated as follows.
  • a candidate modified inhibitory dsRNA e.g., a control molecule, usually the unmodified form
  • may be exposed to degradative conditions e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease.
  • a biological sample e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells.
  • the candidate and control could then be evaluated for resistance to degradation by any of a number of approaches.
  • the candidate and control may be labeled, e.g. prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5.
  • Control and modified inhibitory dsRNA may be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent.
  • a physical parameter, e.g., size, of the modified and control molecules are then determined, and may be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally.
  • Northern blot analysis may be used to assay the length of an unlabeled modified molecule.
  • an inhibitory dsRNA identified as being capable of inhibiting target gene expression may be tested for functionality in vivo in an animal model (e.g., in a mammal, such as in mouse or rat).
  • the inhibitory dsRNA may be administered to an animal, and the inhibitory dsRNA evaluated with respect to its biodistribution, stability, and its ability to inhibit target gene expression.
  • an inhibitory dsRNA may be administered directly to the target tissue, such as by injection, or an inhibitory dsRNA may be administered to the animal model in the same manner that it would be administered to a human.
  • An inhibitory dsRNA can also be evaluated for its intracellular distribution.
  • Such evaluation may include determining whether the inhibitory dsRNA was taken up into the cell and/or may include determining the stability (e.g., the half-life) of the inhibitory dsRNA.
  • an evaluation of an inhibitory dsRNA in vivo can be facilitated by use of an inhibitory dsRNA conjugated to a traceable marker (e.g., a fluorescent marker such as fluorescein; a radioactive label, such as 35 S, 32 P, 33 P, or 3 H; gold particles; or antigen particles for immunohistochemistry), or by using realtime PCR to quantitatively amplify the dsRNA directly.
  • a traceable marker e.g., a fluorescent marker such as fluorescein; a radioactive label, such as 35 S, 32 P, 33 P, or 3 H
  • gold particles such as 35 S, 32 P, 33 P, or 3 H
  • antigen particles for immunohistochemistry or by using realtime PCR to quantitatively amplify the dsRNA directly.
  • an inhibitory dsRNA useful for monitoring biodistribution may lack gene silencing activity in vivo.
  • the inhibitory dsRNA may target a gene not present in the animal (e.g., an inhibitory dsRNA injected into mouse may target luciferase), or an inhibitory dsRNA may have a non-sense sequence, which does not target any gene, e.g., any endogenous gene). Localization/biodistribution of the inhibitory dsRNA may be monitored, e.g. by a traceable label attached to the inhibitory dsRNA, such as a traceable agent described above.
  • Inhibitory dsRNA may be evaluated with respect to its ability to modulate, e.g. down regulate the gene expression of a particular target.
  • Levels of target gene expression in vivo may be measured, for example, by in situ hybridization, or by the isolation of RNA from tissue prior to and following exposure to the inhibitory dsRNA. Where the animal needs to be sacrificed in order to harvest the tissue, an untreated control animal may serve for comparison.
  • Target mRNA can be detected by any desired method, including but not limited to RT-PCR, Northern blot, branched-DNA assay, or RNAase protection assay. Alternatively, or additionally, target gene expression can be monitored by performing Western blot analysis on tissue extracts treated with the inhibitory dsRNA.
  • a candidate inhibitory dsRNA homologous to an endogenous mouse gene e.g., a maternally expressed gene, such as c-mos
  • an immature mouse oocyte can be injected into an immature mouse oocyte to assess the ability of the inhibitory dsRNA to inhibit gene expression in vivo (see, e.g., WO 01/36646).
  • a phenotype of the oocyte e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the inhibitory dsRNA is inhibiting expression.
  • cleavage of c-mos mRNA by the inhibitory dsRNA may cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et ⁇ l. Nature 370: 65-68, 1994; Hashimoto et ⁇ l. Nature, 370:68-71, 1994).
  • the effect of a modified inhibitory dsRNA on target RNA levels may be verified by, for example, a Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control.
  • Such controls may include e.g. cells in which no inhibitory dsRNA is added and/or cells in which a non-modified inhibitory dsRNA is added.
  • Disclosed chimeric therapeutics, multiplexes, particles and/or compositions include a nucleic acid, for example a RNA, as described above.
  • a nucleic acid is targeted to specific gene target, such as apoB, for example, a provided nucleic acid is substantially homologous to a region of the apoB gene, for example, a mammalian ⁇ e.g. human or mouse) apoB gene.
  • chimeric therapeutics, multiplexes, particles and/or compositions include a nucleic acid targeted to one or more of: prothrombin, FIX, angiotensinogen, renin (see US20070270365), TFPI (see US7022672), CCR5, HCV (see US20070149470), SYK (see US 7173015), RANKL, IL-23 (see WO2004/094636), Complement C3 (see US20070178068), Factor H, IL-4Ralpha (see US 2005014333), RBP4, glucagon/glucagon receptor (see US2008/0113372), ghrelin (see US20080140056), GOAT, gastrin, PTPlB, leptin, PCSK-9, IGF-IR, cMet, DR4, DR5, VEGF-A, HGF, sclerostin, and/or myostatin.
  • prothrombin FIX
  • compositions, multiplexes, particles or therapeutics disclosed herein may localize in the liver or to the gut, e.g., the intestine, such as to the jejunum of the intestine after administration to a patient.
  • Such methods can further include administration (e.g., concurrently or consecutively) with conventional agents used to treat such diseases or disorders.
  • nucleic acids targeting particular targets may be identified using the methods set forth herein.
  • contemplated nucleic acids e.g. RNAs targeting specific gene targets include those recited in the following patents and patent applications, hereby incorporated by reference, and targeting the following genes: VEGF (see U.S. Patent
  • HIFl see U.S. Patent Publication No. 20080188430
  • SARS see U.S. Patent Publication No. 20070270360
  • HDAC see U.S. Patent Publication No. 20070185049
  • Nogo and Nogo Receptor see U.S. Patent Publication No. 20070185043
  • WHN see U.S. Patent Publication No. 20070179104
  • PCSK9 see U.S. Patent Publication No. 20070173473
  • CETP see U.S. Patent Publication No. 2007017346
  • XIAP see U.S. Patent Publication No.
  • CHK-I see U.S. Patent Publication No. 20060216747
  • HR see U.S. Patent Publication No. 20060160757
  • CDK2 see U.S. Patent Publication No. 20060142225
  • PGF see U.S. Patent Publication No. 20050267058
  • PTP-IB see U.S. Patent Publication No. 20060025361
  • TGF-beta and TGF-beta Receptor see U.S. Patent Publication No. 20050287128)
  • STAT3 see U.S. Patent Publication No. 20050196781
  • GAB2 see U.S.
  • Patent Publication No. 20050196767 ICAM (see U.S. Patent Publication No. 20050187174), BCL-2 (see U.S. Patent Publication No. 20050176025), ADAM33 (see U.S. Patent Publication No. 20050164968), EZH2 (see U.S. Patent Publication No. 20050159382), PCNA (see U.S. Patent Publication No. 20050158735), c-SRC (see U.S. Patent Publication No. 20040101850), Notch2 (see U.S. Patent Publication No. 20040101847), IL22 (see U.S. Patent Publication No. 20040097447), Adam9 (see U.S. Patent Publication No.
  • Contemplated nucleic acids include those targeting targets associated with viral infections, for example, those nucleic acids targeting the following viruses and recited in the following patent applications: Influenza Virus (see U.S. Patent Publication No. 20070197460), HCV (see U.S. Patent Publication No. 20080207542), RSV (see U.S. Patent Publication No. 20060287267), and HIV (see U.S. Patent Publication No. 20050191618), wherein each patent and patent application is incorporated by reference. Administration and Dosage
  • Disclosed chimeric therapeutics, multiplexes, compositions and/or particles may be administered to a patient by any conventional route. These include, but are not limited to, the systemic routes, e.g. subcutaneous, intradermal, intramuscular or intravenous route, and mucosal routes, e.g. oral, nasal, pulmonary or anogenital route.
  • an intratumoral route may be used in e.g. the treatment of solid tumors.
  • the choice of the route of administration will depend on the nature of the disease; for example, multiplexes and/or compositions may be administered via a pulmonary route in the case of cystic fibrosis (e.g.
  • disclosed particles may be administered a composition that comprises a biocompatible aqueous solution.
  • Contemplated solutions may include water and/or saline, and may optionally contain pharmaceutical excipients known to those skilled in the art, for example, buffers, stabilizing molecules, preservatives, sugars, amino acids, proteins, carbohydrates and vitamins, and the like.
  • the administration of disclosed multiplexes or compositions can be carried out at a single dose or at a dose repeated once or several times after a certain time interval.
  • the appropriate dosage varies according to various parameters, for example the therapeutically effective dosage is dictated by and directly dependent on the individual treated, the mode of administration, the unique characteristics of the nucleic acid and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Appropriate doses can be established by persons skilled in the art of pharmaceutical dosing such as physicians.
  • the multiplexes can be included in a container, pack, or dispenser together with instructions for administration.
  • the multiplexes and compositions disclosed herein may allow for sustained release of a nucleic acid, e.g. an inhibitory nucleic acid, to e.g. a specific body target site, e.g., the liver.
  • a nucleic acid e.g. an inhibitory nucleic acid
  • the disclosed compositions and/or multiplexes may allow for release of a nucleic acid over about 1 day to about 7 days or more, e.g. about 1 day to about 3 days or more, or about 1 day to about 3 weeks or more.
  • long-term storage stability of disclosed multiplexes may be increased and/or enhanced by for example, freezing and lyophilizing particles or multiplexes disclosed herein in the presence of one or more protective agents such as sucrose, mannitol, trehalose or the like.
  • the suspension may, for example, retain essentially all nucleic acid previously encapsulated and/or may retain substantially the same particle size. Rehydration may be accomplished by e.g., adding purified or sterile water or 0.9% sodium chloride injection or 5% dextrose solution followed by gentle swirling of the suspension.
  • 77C His-tagged core protein is cloned into the Ndel/Xhol restriction sites of vector pET21b (Novagen). This plasmid is transformed into E. coli BL21 (DE3) pLysS cells
  • DNA fragments containing the genes for K5, K7, K9, KlO, KI l, K13, K20, KA9, KG9 and K4-5 core protein mutants described previously are synthesized via PCR using the Cassette 1 template and the primer sequences described in Table 1.
  • Each PCR reaction is composed of 12.5 ⁇ l of 5X GC polymerase buffer (Finnzyme), 1.25 ⁇ l of a 1OmM dNTP mixture, 1.5 ⁇ l of 5 ⁇ M forward primer, 1.5 ⁇ l of 5 ⁇ M reverse primer, 0.6 ⁇ l of Stratagene mini-prepped template, 0.8 ⁇ l of2 unit/ ⁇ l Phusion Hot Start polymerase (Finnzyme), and 44.25 ⁇ l of water.
  • the PCR reaction consist of a one-time incubation at 98 0 C for 1 minute, followed by incubation at 98°C for 25 seconds, incubation at 70°C for 30 seconds, and incubation at 72 0 C for 1 minute and 10 seconds. These last three steps are repeated 24 times followed by a final incubation at 72°C for 7 minutes.
  • the Cassettel template consists of the following nucleic acid sequence inserted into the Ndel/Xhol restriction sites of vector pET22b:
  • the PCR products and a pET22b vector are both digested with restriction enzymes Ndel and Xhol at 37 0 C for 2 hours.
  • the digested products are run on an agarose gel.
  • the bands are excised and purified via gel extraction (Stratagene).
  • Ligation reactions are composed of 5 ⁇ l of digested and purified PCR product, 1 ⁇ l of digested and purified pET22b vector, 1 ⁇ l of T4 DNA ligase buffer (NEB), 1 ⁇ l of T4 DNA ligase (NEB), and 2 ⁇ l of water and are incubated at room temperature for 12 hours.
  • the ligation reactions are transformed into XLI Blue E. coli cells (Stratagene) and the resulting colonies are grown in IX LB broth.
  • the plasmids are purified via mini-prep (Stratagene).
  • the purified plasmids are sequenced (see below) and transformed into E. coli BL21 (DE3) pLysS cells (Stratagene) for protein expression. This strategy can be used for proteins containing from 0 to 30 lysine residues.
  • DNA fragments containing point mutations of the K9 construct are synthesized via PCR using the K9 template (or in the case of double or triple mutants, the appropriate single or double mutant K9 template) and the primer sequences described in Table 2.
  • Each PCR reaction consists of 5 ⁇ l of 1OX Pfu Turbo polymerase buffer (Stratagene), 1 ⁇ l of a 10 mM dNTP mixture, 1.5 ⁇ l of 5 ⁇ M forward primer, 1.5 ⁇ l of 5 ⁇ M reverse primer, 1 ⁇ l of Stratagene mini-prepped template, 1 ⁇ l of 2.5 unit/ ⁇ l Pfu Turbo polymerase (Stratagene), and 39 ⁇ l of water.
  • the PCR reaction consists of a one-time incubation at 98°C for 1 minute, followed by incubation at 98°C for 30 seconds, incubation at 64-72°C (depending on primer T m ) for 1 minute, and incubation at 72°C for 6 minutes. These last three steps are repeated 20 times.
  • the K9 template consists of the following nucleic acid sequence inserted into the Ndel/Xhol restriction sites of vector pET22b:
  • the PCR products are digested with the restriction enzyme Dpnl at 37 0 C for 1.5 hours to eliminate any un-mutated template.
  • the digested products are run on a 1% agarose gel.
  • the bands are excised and purified via gel extraction (Stratagene).
  • the PCR products are then transformed into E. coli BL21 (DE3) pLysS cells
  • Protocol 1 A pET-lla vector containing the full-length HBV C-protein gene is transformed into E. coli DE3 cells and grown at 37 0 C in LB media that is fortified with 2-4% glucose, trace elements and 200 ⁇ g/mL of carbenicillin. Protein expression is induced by the addition of 2mM IPTG (isopropyl-beta-D-thiogalactopyranoside). Cells are harvested by pelleting after three hours of induction. SDS-PAGE is used to assess expression of C-protein. [0232] Core protein is purified from E.
  • the mixture is stirred for one hour and then centrifuged for one hour at 2600OxG.
  • the pellet is resuspended in a solution of 100 mM Tris-HCI at pH 7.5, 100 mM NaCl, 50 mM sucrose and 2 mM DTT (Buffer A) and loaded onto a Sepharose CL-4B (Phannacia Biotech, Piscataway, NJ) column (5 cm diameter X 95 cm) equilibrated with Buffer A.
  • the column is eluted at 2mL/minute.
  • HBV viral capsids and aggregates are separated, as well as soluble proteins of lower molecular weight.
  • the fractions are pooled according to chromatographic profile and SDS-PAGE analysis.
  • the solution is concentrated by ultrafiltration using Diaflo YM 100 ultrafiltration membrane (Amicon, Beverly, MA) to about 10 mg/mL.
  • Concentrated C-protein is dialyzed against 50 mM Tris-HCI, pH 7.5 and 0.15 M sucrose.
  • the solution is then adjusted to pH 9.5 by adding ION NaOH and urea to reach a final concentration of 3.5 M.
  • the solution is then filtered using a Millex-HA 0.45 ⁇ m pore size filter unit (Millipore, Bedford, MA) and applied to a column (6.0 cm diameter X 60 cm) of Superdex 75 (Pharmacia Biotech, Piscataway, NJ) equilibrated with a solution consisting of 100 mM sodium bicarbonate, pH 9.5, and 2 mM DTT.
  • the column is eluted at 5 niL/minute.
  • the fractions containing dimeric protein as assessed by SDS-PAGE are pooled.
  • the pET vector containing the gene for K9 protein is kept in BL21 (DE3) PlysS cells for expression.
  • the starter culture can be inoculated from a colony on a IX Luria Broth (IXLB) agar plate or from a 10% glycerol stock, stored at -80°C.
  • the IXLB is autoclaved in a 2 L flask and cooled. 100 mg of ampicillin (Amp) is added to the IXLB.
  • a starter culture is inoculated and allowed to grow at 37°C for up to 24 hours with shaking at 200 rpm.
  • Two 50 ml tubes (approximately 20 mL each) of cell paste are thawed. The following steps are applied to each tube. 40 mL of resuspension buffer (5 M urea, 50 mM NaHCO 3 (pH 9.5), 10 mM imidazole) is added into each tube. The cells are suspended by continuous pipetting and poured into a 400 mL beaker. More resuspension buffer is added until there is -100 mL total cell resuspension in the beaker.
  • resuspension buffer 5 M urea, 50 mM NaHCO 3 (pH 9.5), 10 mM imidazole
  • the beaker containing resuspended cells is placed in an ice bath and sonicated for 5 minutes using a Branson probe sonifier (pulse mode at approximately 40% duty cycling and power setting of 5).
  • the cell mixture is sonicated in several intervals and is allowed to rest on ice if it appears that the sample is heated to higher than room temperature.
  • the cell lysate is diluted 2 fold to 200 mL total, and 200 ⁇ L of 100 mg/mL DNase is added to the suspension. This suspension is stirred on ice for 10 minutes.
  • the sonication step is repeated for 5 more minutes while on ice.
  • the lysate is transferred to six 50 niL plastic centrifuge tubes, and centrifuged at 32000 g for 45 minutes. Supernatant is discarded.
  • a 50 rnL Ni + -NTA agarose (Qiagen) column is washed and equilibrated in the resuspension buffer. 12 L of cells is lysed for each run of the column. The centrifuged lysate from 12 L of cells is combined and diluted to 500 mL with resuspension buffer. The centrifuged cell lysate is loaded onto the column, and the protein solution is allowed to sink to the top of the nickel matrix. 50 mL of resuspension buffer is passed through the column.
  • An optional salt wash can be performed by washing the column with 250 mL of NaCl wash buffer (5 M urea, 50 mM NaHCO 3 (pH 9.5), 20 mM imidazole, 250 mM NaCl).
  • This salt wash reduces the A260/A280 ratio of the final purified protein by a value of 0.1 A.U..
  • the column is washed with 250 mL of wash buffer (5 M Urea, 50 mM NaHCO 3 (pH 9.5), 20 mM imidazole). Subsequently, 200 mL of elution buffer (5 M Urea, 2 mM NaHCO 3 (pH 9.5), 250 mM imidazole) is passed through the column.
  • Fractions are collected at every 5 mL, and of these, which 5 to 8 fractions should contain protein. [0238] The presence and/or concentration of protein is detected by measuring the absorbance of the fractions. SDS polyacrylamide gel electrophoresis (SDS PAGE) analysis is performed on the proteins to determine purity. Fractions containing protein are pooled, and transferred to dialysis tubing. Dialysis is performed in 4 L of storage buffer (5 M Urea, 2 mM NaHCO 3 (pH 9.5)) for at least 4 hours at 4°C. The protein can then be concentrated in an Amicon stirred cell concentrator (Millipore) to a final protein concentration of up to 75 mg/ml. A 12 L cell growth yields approximately 500 mg of pure protein. Pure dialyzed protein can be stored at -8O 0 C for 6-8 months.
  • SDS PAGE SDS polyacrylamide gel electrophoresis
  • PE phosphatidyl ethanolamine
  • TAA triethylamine
  • the solution is maintained under an argon or nitrogen atmosphere.
  • the reaction can also be done in dry chloroform.
  • 50 mg of SMPB (Pierce) is added to the PE solution and mixed well to dissolve.
  • the solution is maintained under an argon or nitrogen atmosphere while the reaction proceeds for 2 hours at room temperature. Methanol is removed from the reaction solution by rotary evaporation and the solids are redissolved in chloroform (5 mL).
  • the water-soluble reaction by-products is extracted from the chloroform with an equal volume of 1% NaCl. Extraction is performed twice.
  • the MPB-PE derivative is purified by chromatography on a column of silicic acid (Martin FJ et al., Immuno specific targeting of liposomes to cells: A novel and efficient method for covalent attachment of Fab' fragments via disulfide bonds. Biochemistry, 1981; 20:4229-38). Chloroform is removed from the MBP-PE by rotary evaporation, and the derivative is stored at -20°C under a nitrogen atmosphere until use.
  • the solution is purged under a nitrogen or argon atmosphere for 20 minutes.
  • the maleimide-containing linker is dissolved in the same buffer as above, and purged under a nitrogen or argon atmosphere for 20 minutes, to obtain a 10-fold molar excess.
  • the instant example describes a general method for forming a therapeutic multiplex particle containing inhibitory dsRNA .
  • the protein is allowed to thaw to 25 0 C.
  • the inhibitory dsRNA-containing solution is added to the protein solution at a molar ratio of 9.58:1 or 9.42:1 for non-modified or modified inhibitory dsRNA, respectively.
  • the solution is mixed for 1 hour at 25 0 C.
  • BME is added at a molar ratio of 3: 1 to the protein to protect cysteine functional groups.
  • This reaction is incubated for 1 hour at 25 0 C.
  • a 2:1 volume ratio of 10 mM NaCl solution is added to reaction mixture containing protein and inhibitory dsRNA.
  • the solution is kept in a water bath set to 25 0 C for 48 hours.
  • PE- MAL l,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[4-(p-maleimidophenyl)- butyramide] (Sodium Salt)
  • DMF dimethylformamide
  • lipid coating material cholesterol (Avanti Lipids, Alabaster, AL, USA), HSPC (L- ⁇ -Phosphatidylcholine, Hydrogenated (Soy), Avanti Lipids, Alabaster, AL, USA) and POPG (l-Palmitoyl-2-Oleoyl-sft-Glycero-3-[Phospho-rac-(l -glycerol)] (Sodium Salt), Avanti Lipids, Alabaster, AL, USA) in dry power forms are premixed in a 3:1:1 molar ratio, respectively, in a glass beaker. The mixture is predissolved and homogenized with 2.0 mL of chloroform.
  • the chloroform is allowed to evaporate (20 to 30 minutes on a hot plate set to 5O 0 C).
  • 0.5X PBS pH 9.5 phosphate buffered saline with 5 mM NaHCO 3
  • This solution is sonicated for at least 3.0 min at 62 0 C.
  • the lipid coating material is added immediately after sonication to the solution containing functionalized protein and inhibitory dsRNA at a mass ratio of 5: 1 protein:lipid coating material. The material is allowed to cool to 25 0 C.
  • the chimeric protein for forming the capsid is purified via FPLC (fast performance liquid chromatography) (Amersham Pharmacia).
  • FPLC fast performance liquid chromatography
  • the large FPLC column (Pharmacia XK- 16 16mm x 700mm) is ran at 1.0 niL/min using 0.5X PBS pH 9.5 buffer as the mobile phase, and Sepharose CL-4B (Amersham Pharmacia) matrix as the stationary phase.
  • Fractions containing particles are collected and combined (typically eluting at 70 mL based on the stationary phase and column configuration described above). Combined fractions are then concentrated on an Amicon Cell Concentrator using a Millipore polyethersulfone filter membrane with a molecular weight cutoff of 5000 (Millipore Cat No. PBCC02510).
  • the fractions are loaded into the concentrator and concentrated at 70 psi.
  • the protein concentrate is removed from the concentrator and filtered through a 0.2 ⁇ m PES (Poly
  • Protein concentration is determined using the Agilent 2100 Bioanalyzer system. Samples are diluted 1:1 with 0.5X PBS pH 9.5 and ran on a Protein 80 chip in triplicates as described by the manufacturer. Protein concentration after filtration is 4.5 to 5.2 mg/mL. [0251] The concentration of inhibitory dsRNA encapsulated within a capsid is determined by methods described in Example 6. The size of the capsid is determined via dynamic light scattering (Dynapro Titan, Wyatt Instruments, Goleta, CA).
  • the instant example describes two general methods for quantifying the inhibitory dsRNA contained within a capsid.
  • the first method describes SDS Extraction of inhibitory dsRNA from a capsid. A standard curve of inhibitory dsRNA is generated for concentrations of 175, 250, 350, 500, 700 nM; samples are prepared in triplicates. 100 ⁇ L of the therapeutic particle is mixed with 5 ⁇ L of 10% SDS solution. The solution is heated at 7O 0 C for 30 minutes and cooled to room temperature. 30 ⁇ L of a 75% glycerol stock is added to the particle/SDS solution.
  • the lysed particles and the inhibitory dsRNA standards are run on a 15% urea-PAGE gel (Biorad 161- 1135) at 230 V for 35 minutes in IX TBE buffer.
  • the gel is then stained with SYBR Green II (140 mL of water and 4 ⁇ L of stock SYBR Green II) on an orbiting platform for 30 minutes.
  • the stained gel is scanned on a Typhoon Trio using 488 nm excitation with a 526 nm SP filter and the PMT set at 350 V.
  • V 7.0 Image Quant TL
  • the second method describes extraction using phenol/chloroform.
  • a standard curve of inhibitory dsRNA is generated for concentrations of 175, 250, 350, 500, 700 nM; samples are prepared in triplicates.
  • 100 ⁇ L of therapeutic particles are mixed with 100 ⁇ L of phenol: chloroform (95:5) solution. These solutions are vortexed for 5 minutes and spun at 13,000 g for 60 seconds.
  • 50 ⁇ L of aqueous solution is mixed with 15 ⁇ L of a 75% glycerol stock.
  • the lysed particles and the inhibitory dsRNA standards are run on a 15% urea-PAGE gel (Biorad 161-1135) at 230 V for 35 minutes in IX TBE buffer.
  • the gel is stained with SYBR Green II (140 mL of water and 4 ⁇ L of stock SYBR Green II) on an orbiting platform for 30 minutes.
  • the stained gel is scanned on a Typhoon Trio using 488 nm excitation with a 526 nm SP filter and the PMT set at 350 V.
  • the Image Quant TL (V 7.0) software the densitometry of the bands is measured and the concentration of the inhibitory dsRNA is determined using a standard curve.
  • the instant example describes two general methods for analyzing the lipid contained within multiplexes.
  • the first example is a NMR- Analysis (dry extraction). 30 mL of multiplexes is dried under vacuum in a speedvac. The resulting material is scraped into a 10 mL glass beaker. 3 mL of water and 3 mL of chloroform are added to the solids. This solution is sonicated for 20 seconds and the mixture is incubated for 30 minutes at room temperature. The solution is centrifuged and the chloroform layer is isolated and filtered through a glass plug. The chloroform is removed under vacuum and the NMR spectrum is measured of the material.
  • the second example is a NMR- Analysis (wet extraction). 30 mL of multiplexes is mixed with 10 mL of chloroform. The solution is sonicated for 20 seconds and the mixture is incubated for 30 minutes at room temperature. The solution is centrifuged and the chloroform layer is isolated and filtered through a glass plug. The chloroform is removed under vacuum and the NMR spectrum is measured of the material.
  • the instant example describes a general method for quantifying the protein contained within a capsid.
  • Protein analysis is done according to Agilent Protein 80 Assay Protocol (Protein 80 kit 5067-1515; protocol revision 04/2007). Samples are diluted 1:1 in 0.5X phosphate buffered saline (PBS, Fisher Scientific BP3994, 5mM NaHCO 3 , pH 9.5) and analyzed. The Chips are run on an Agilent 2100 Bioanalyzer and protein concentration is obtained.
  • Example 9 Methods of providing targeting of particles are described.
  • antibodies at a concentration of 4 mg/mL in 1 X PBS buffer pH 7.4 are treated with 20 mole equivalents of Traut's reagent, 2-iminothiolane HCI, for 1 hour.
  • the antibodies are purified via column chromatography (8 x 200 rnm) G-50 (Amersham Pharmacia) in 0.25X PBS buffer pH 7.4.
  • One mole equivalent of purified coated multiplex is treated with 200 mole equivalents of PE-maleimide lipid (1,2-Dipalrnitoyl-sra-Glycero 3-Phosphoethanolamine-N-[4- (p-maleimidophenyl)-butyramide] (Sodium Salt)) (dissolved in DMF).
  • Example 10 K9 protein-RNA complex using electrophoresis of labeled inhibitory dsRNA and K9 protein.
  • Inhibitory dsRNA 3 '-labeled with Dy547 is purchased from Dharmacon (Lafayette,CO). Labeled and unlabeled inhibitory dsRNA solutions are diluted to 4 ⁇ M in 4 M Urea, 10 mM Hepes, pH 8.15. The protein is labeled using 3 ⁇ M of the K9 protein incubated with 30 ⁇ M maleimido-cy5 (Pierce) in 4 M Urea, 10 mM Hepes, pH 8.15 for 1 hour. Labeled and unlabeled protein solutions are diluted to 3 ⁇ M in 4M Urea.
  • RNA-Protein On a 1.5% agarose gel, a shifted band ("RNA-Protein") appears only when both RNA and K9 protein are present. Distinct fluorescent labeling shows that both RNA and protein migrate in this band, indicating that the K9 protein and inhibitory dsRNA form a complex in solution.
  • Example 11 Capsid Stability Assay.
  • capsids formed by core proteins with destabilizing and/or stabilizing mutations are assessed, and the relative stabilities of various capsids with that of the wild type capsid are compared.
  • Capsids are formed in the presence of fluorescently-labeled RNA molecules. The core protein is incubated with the RNA at a ratio of 10: 1 protein:RNA and then 70 mM NaCl is added at a 1:1 ratio to the protein/RNA mixture to initiate capsid formation. Capsid formation can be confirmed by light scattering and/or size-exclusion chromatography. The pre-formed capsids are incubated in 4M urea at a temperature of 55 0 C to force degradation of the capsids.
  • RNA in the intact capsid band can be quantified by densitometry and plotted over time (see Figure 6).
  • Benzonase protection assays are performed to determine if a K9 core protein protects the encapsulated inhibitory dsRNA molecules from the benzonase nuclease.
  • a sample is prepared with no benzonase as a negative control. The mixture is incubated for 1 hour at room temperature. The samples are run on a 1.0% TAE-agarose gel containing ethidium bromide. The gel is imaged and the intensity of the RNA bands is determined.
  • RNA band is degraded at about 20 units/nmol.
  • RNA associated with the K9 core protein does not degrade at any nuclease concentrations tested, indicating that the RNA is effectively protected, as quantitated in Figure 7.
  • This assay shows that RNA is significantly protected against nuclease activity by encapsulation with K9 core protein.
  • Example 13 Serum Protection Assay.
  • RNA sample is mixed with human serum. The total volume of sample and serum is between 2-4 mL. Freeze several aliquots of sample and serum immediately for time zero time points, and place the remaining samples at 37 0 C. Multiple 50 ⁇ L aliquots are removed from samples at regular intervals, labeled and froze at - 80 0 C. To process the samples, 10% SDS is added to achieve a final concentration of 0.7% SDS. The mixture is incubated at room temperature for 5 minutes. Samples are ran at 200 V for 30 minutes on a 1.0% TAE-agarose gel containing ethidium bromide. The lifetime of the RNAs is quantitated to determine the amount of protection.
  • Figure 8 demonstrates that the control samples are degraded by the first time point (1 day) while the particle-protected RNA survived without appreciable degradation for the duration of the experiment, 4 days. These results indicate that the particle protects the RNA cargo from serum degradation. Additional experiments indicate that RNA stability is achieved at 14 days without the degradation of the RNA payload. Free RNA, in the presence and absence of empty particle, is completely degraded by 1 day.
  • Example 14 [0276] The following assays determine the K d , for K7, K9 and KI l constructs with fluorescent inhibitory dsRNA. The purpose of this study is to determine the affinity of a fluorescent inhibitory dsRNA construct for the HBV core protein mutants. Below is the sequence of fluorescent inhibitory dsRNA that was used in these experiments.
  • f-RNA buffer A solution of 20 nM fluorescent duplex (Siglo cycB, RNA from Dharmacon) in 10 mM Tris is referred to as f-RNA buffer.
  • K9 protein stock is diluted to 6 ⁇ M in f-RNA buffer. The dilution is performed quickly on ice, so that particle assembly is less apt to form. Successive dilutions of K9 is made in f-RNA buffer. The RNA -protein dilutions are removed from ice and incubated at room temperature for 5 minutes.
  • the reactions are run on a gel under the following conditions: 15 ⁇ L of the samples in duplicates are loaded per lane on a 1.5% TAE-agarose gel. The gel is run at 200 V for 35 minutes and documented on a Molecular Dynamics Typhoon scanner.
  • Figure 9 depicts that the fluorescent inhibitory dsRNA binds to K9 with a K d of 115 nM. This is a tight affinity, which is characteristic of RNA-protein interactions. This tight binding affinity is well below the concentrations of RNA and protein used for assembly of particles. Therefore, these data suggests that during assembly the RNA binding sites of K9 protein are saturated with RNA.
  • RNA loading buffer (xylene cyanol in 55% glycerol 20 mM Tris, pH 7.7) is added to the samples, and the samples are loaded on a 1.0% TAE-agarose gel (13cmxl6cm) at 80 ⁇ L/lane. The gel is run at 180 V for 35 minutes and documented on a Molecular Dynamics Typhoon scanner.
  • Figure 10 depicts that the fluorescent inhibitory dsRNA bound to K7 with a K d of 370 nM and to Kl 1 with a IQ of 69 nM. This is a tight affinity which is characteristic of RNA- protein interactions. The increase affinity observed for Kl 1 relative to K9 and K7 is attributable to the larger number of cationic residues at the C-terminal end of this protein. As with the mutant K9, the affinity is high enough to fully saturate the RNA binding sites for both mutants during the process of particle assembly. Table 2 provides a summary of K7, K9, and KI l mutant binding conditions as well as the K d values. Table 2
  • Cryopreserved mouse primary hepatocytes (Cellz Direct) are thawed and grown in William's Media with 10% fetal bovine serum (according to the manufacturer's protocol) at 37 0C and 5% CO2 for 24 hours. Thawed cells in suspension are counted and plated at a density of 35,000 cells/well in 96-well collagen- 1 coated plates using a proprietary mixture of media supplements (CellzDirect- Thawing/Plating Supplement Pack). Cells are allowed to settle and attach to the collagen-1 substrate for 48 hours as media is replaced after 24 and 48 hours.
  • FVII inhibitory dsRNA sense 5'- GGAUCAUCUCAAGUCUUACT*T-3' (SEQ ID NO: 198 and antisense 5'- GUAAGACUUGAGAUGAUCCT*T -3' (SEQ ID NO: 199 bold letters denote 2'-F-modified nucleotides and asterisks represent phosphrothioate linkages
  • SiGenome 5 sense 5'- UGGUUUACAUGUCGACUAA- 3' (SEQ ID NO: 200) are loaded into the therapeutic multiplexes described previously.
  • RNA is then purified from individual samples using Qiagen RNEasy columns on a QiaCube (Qiagen) automated RNA purification system.
  • RNA purification Following RNA purification, total RNA is quantified using a Nanodrop spectrophotometer (ThermoFisher) and equal amounts of RNA will be reverse transcribed for every sample within each experiment into cDNA using the iScript reverse transcriptase kit (BioRad).
  • the cDNA is added to SybrGreen qPCR master mix (BioRad-as recommended by the manufacturer) and quantitative real-time qPCR (qPCR) is performed using the 96-well plate format on MyIQ real-time qPCR machines (BioRad). Included in all qPCR experiments are pre-designed and pre-validated primers for 3 housekeeping genes, glyceraldehydes-3-p- dehydrogenase (GAPDH), Cyclophillin A, and tyrosine 3-monooxygenase/tryptophan 5- monooxygenase (YWHAZ), as well as pre-designed and pre-validated primers specific for the mouse Factor VII mRNA sequence.
  • GPDH glyceraldehydes-3-p- dehydrogenase
  • Cyclophillin A Cyclophillin A
  • YWHAZ tyrosine 3-monooxygenase/tryptophan 5- monooxygenase
  • FIG. 11 demonstrates that FVII mRNA expression is reduced in primary mouse hepatocytes following 72 hours of incubation with therapeutic multiplex loaded with FVII inhibitory dsRNA, when compared to therapeutic multiplex loaded with SiGenome 5 inhibitory dsRNA (SiG5-Dharmacon/ThermoFisher), and PBS. .
  • Endotoxin levels are based on the Limulus Amebocyte Lysate Pyrogent Plus Single Test Kit (Lonza, US License No. 1701, Catalog No. N289-06).
  • the endotoxin vial is reconstituted with 1.0 ml of LAL reagent water (Lonza #W50-640) and vortexed for at least 15 minutes.
  • Endotoxin is diluted with the LAL reagent water to a concentration of lEU/ml.
  • 0.25 ml of an endotoxin standard that contains twice the labeled minimum sensitivity is used.
  • 0.23 ml of LAL reagent water is used.
  • sample serial dilutions are prepared in duplicates, beginning with a 1:4 dilution and end with a 1:64 dilution of sample:LAL reagent water. 0.25 ml of each sample is added to the test vial. Mix the samples by tilting and gently swirling the vial until the contents are in solution. Each vial of sample is incubated for 60 minutes (+/- 2 minutes) at 37 0 C (+/- 1 0 C). At the end of the incubation period, each vial is carefully removed and inverted 180 degrees. A positive reaction is characterized by the formation of a firm gel that remains intact momentarily when the tube is inverted, which should be observed in the positive sample control vial.
  • a negative test is characterized by the absence of solid clot after inversion.
  • the lysate may show an increase in turbidity or viscosity. This is considered a negative result.
  • the endotoxin concentration is calculated by the multiple of the lysate sensitivity and the geometric mean of the endpoint:

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  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Dispersion Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne des multiplexes chimères thérapeutiques comprenant au moins deux capsides constituées, par exemple, d'une protéine centrale virale modifiée et d'un acide nucléique lié à la protéine centrale virale modifiée. L'acide nucléique peut être sensiblement homologue à une cible du gène spécifique. Dans certains modes de réalisation, l'acide nucléique lié à la protéine centrale virale modifiée est essentiellement non-immunogène.
PCT/US2009/060040 2008-10-08 2009-10-08 Multiplexes chimères, compositions et procédés permettant de les utiliser Ceased WO2010042743A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10384308P 2008-10-08 2008-10-08
US61/103,843 2008-10-08

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WO2010042743A2 true WO2010042743A2 (fr) 2010-04-15
WO2010042743A3 WO2010042743A3 (fr) 2010-10-28

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014027211A1 (fr) * 2012-08-17 2014-02-20 Cancer Research Technology Limited Complexes biomoléculaires
JP2014527072A (ja) * 2011-09-09 2014-10-09 バイオメド リアルティー， エル．ピー． ウイルスタンパク質の集合を制御するための方法および組成物
WO2022012539A1 (fr) * 2020-07-17 2022-01-20 养生堂有限公司 Nouveau peptide de pénétration cellulaire et utilisation associée
JP2022519992A (ja) * 2018-12-28 2022-03-28 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 操作されたｂ型肝炎コアポリペプチド

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2352738A1 (fr) * 1998-12-04 2000-06-08 Biogen, Inc. Particules d'antigene capsidique du virus de l'hepatite b avec composants immunogenes multiples lies par des ligands peptides
US7320793B2 (en) * 2001-01-19 2008-01-22 Cytos Biotechnology Ag Molecular antigen array
US9045727B2 (en) * 2002-05-17 2015-06-02 Emory University Virus-like particles, methods of preparation, and immunogenic compositions
EP2392345A3 (fr) * 2002-07-18 2012-03-07 Cytos Biotechnology AG Conjugués vecteurs d'haptène comportant des particules de type viral et utilisations associées

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014527072A (ja) * 2011-09-09 2014-10-09 バイオメド リアルティー， エル．ピー． ウイルスタンパク質の集合を制御するための方法および組成物
EP2747774A4 (fr) * 2011-09-09 2015-02-11 Biomed Realty L P Procédés et compositions de régulation d'un assemblage de protéines virales
WO2014027211A1 (fr) * 2012-08-17 2014-02-20 Cancer Research Technology Limited Complexes biomoléculaires
JP2022519992A (ja) * 2018-12-28 2022-03-28 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 操作されたｂ型肝炎コアポリペプチド
JP7491932B2 (ja) 2018-12-28 2024-05-28 ザ ボード オブ トラスティーズ オブ ザ レランド スタンフォード ジュニア ユニバーシティー 操作されたｂ型肝炎コアポリペプチド
WO2022012539A1 (fr) * 2020-07-17 2022-01-20 养生堂有限公司 Nouveau peptide de pénétration cellulaire et utilisation associée

Also Published As

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