WO2023283550A1 - Signal activatable nucleic acid complexes - Google Patents

Signal activatable nucleic acid complexes Download PDF

Info

Publication number
WO2023283550A1
WO2023283550A1 PCT/US2022/073430 US2022073430W WO2023283550A1 WO 2023283550 A1 WO2023283550 A1 WO 2023283550A1 US 2022073430 W US2022073430 W US 2022073430W WO 2023283550 A1 WO2023283550 A1 WO 2023283550A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
strand
acid strand
acid complex
complex
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2022/073430
Other languages
English (en)
French (fr)
Inventor
Si-ping HAN
Robert Duff
Lisa Scherer
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Switch Therapeutics Inc
Original Assignee
Switch Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Switch Therapeutics Inc filed Critical Switch Therapeutics Inc
Priority to CN202280060504.5A priority Critical patent/CN117916376A/zh
Priority to CA3224950A priority patent/CA3224950A1/en
Priority to AU2022308843A priority patent/AU2022308843A1/en
Priority to KR1020247004152A priority patent/KR20240095155A/ko
Priority to IL309943A priority patent/IL309943A/en
Priority to EP22838546.4A priority patent/EP4367245A4/en
Priority to JP2024500651A priority patent/JP2024525647A/ja
Priority to US18/575,487 priority patent/US20250257353A1/en
Publication of WO2023283550A1 publication Critical patent/WO2023283550A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0085—Brain, e.g. brain implants; Spinal cord
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/113—Antisense targeting other non-coding nucleic acids, e.g. antagomirs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/321—2'-O-R Modification
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/322—2'-R Modification
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/32—Chemical structure of the sugar
    • C12N2310/323—Chemical structure of the sugar modified ring structure
    • C12N2310/3231—Chemical structure of the sugar modified ring structure having an additional ring, e.g. LNA, ENA
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol

Definitions

  • the present disclosure relates generally to the field of nucleic acid based technology, for example, signal activatable small interfering RNA complexes.
  • RNAi therapy can use nuclei acid logic switches to sense RNA transcripts (such as mRNAs and miRNAs), thereby restricting RNA interfering (RNAi) therapy to specific populations of disease-related cells.
  • RNAi RNA interfering
  • nucleic acid complex comprising: a first nucleic acid strand comprising 20-60 linked nucleosides; a second nucleic acid strand binding to a first region of the first nucleic acid strand to form a first nucleic acid duplex; and a third nucleic acid strand binding to a second region of the first nucleic acid strand to form a second nucleic acid duplex, wherein the third nucleic acid strand comprises a overhang, wherein the overhang is not complementary to the first nucleic acid strand and is capable of binding to an input nucleic acid strand to cause the displacement of the third nucleic acid strand from the first nucleic acid strand.
  • the first region of the first nucleic acid strand is 3’ of the second region of the first nucleic acid strand, the third nucleic acid strand does not bind to any region of the first nucleic acid strand that is 3’ of the first region of the first nucleic acid strand.
  • the first region of the first nucleic acid strand comprises a sequence complementary to a target RNA, where the sequence can be, for example, 10-35 nucleosides in length. In some embodiments, the sequence complementary to the target RNA is 10-21 nucleotides in length.
  • the second nucleic acid strand binds to 17-22 linked nucleotides in the first region of the first nucleic acid strand to form the first nucleic acid duplex.
  • the third nucleic acid strand binds to 10-30 linked nucleotides in the second region of the first nucleic acid strand to form the second nucleic acid duplex.
  • the third nucleic acid strand binds to about 14 linked nucleotides in the second region of the first nucleic acid strand to form the second nucleic acid duplex.
  • the first nucleic acid duplex, the nucleic acid complex, or both do not comprise a Dicer cleavage site.
  • the first region of the first nucleic acid strand is linked to the second region of the first nucleic acid strand via a linker.
  • the linker can, for example, comprise a C3 3-carbon linker, a nucleotide, a modified nucleotide, or a exonuclease cleavage- resistant moiety, or a combination thereof.
  • the modified nucleotide is a T -O-methyl nucleotide or a 2’-F nucleotide.
  • the T -O-methyl nucleotide is 2'-0-methyladenosine, 2'-0-methylguanosine, 2'-0-methyluridine, or 2'-0-methylcytidine.
  • the 2’-F nucleotide is 2’-F adenosine, 2’-F guanosine, 2’-F uridine, or 2’-F cytidine.
  • the 5’ terminus of the second nucleic acid strand comprises a blocking moiety.
  • the blocking moiety can, for example, comprise, or is, a fluorophore, an inverted-dT, a tri-ethylene-glycol, a fatty acid, a Cy3, or a combination thereof.
  • the fluorophore can be attached to the 5’ terminus of the second nucleic strand via a phosphorothioate linkage.
  • the first nucleic acid strand comprises a 3’ overhang in the first nucleic acid duplex.
  • the 3’ overhang of the first nucleic acid is one, two, or three nucleotides in length.
  • the 3’ overhang of the first nucleic acid comprises one or more phosphorothioate internucleoside linkages.
  • all of the internucleoside linkages in the 3 ’ overhang of the first nucleic acid are phosphorothioate internucleoside linkages.
  • the intemucleoside linkage(s) between the last two, three or four nucleosides at the 3’ terminus of the first nucleic acid strand is phosphorothioate intemucleoside linkage(s).
  • the first region of the first nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the intemucleoside linkage(s) between the last two or three nucleosides at the 5’ terminus, 3’ terminus, or both.
  • the first region of the first nucleic acid strand does not comprise phosphorothioate internucleoside linkages except for the intemucleoside linkage(s) between the last three nucleosides at the 5’ terminus and the last three nucleosides at 3’ terminus.
  • the second region of the first nucleic acid strand does not comprise phosphorothioate intemucleoside linkages.
  • the second nucleic strand is fully complementary to the first region of the first nucleic acid strand, thereby forming no overhang at the 5’ and 3’ termini of the second nucleic acid strand in the first nucleic acid duplex.
  • the second nucleic acid strand does not have an overhang at 3’ terminus, or 5’ terminus, or both in the first nucleic acid duplex.
  • the second nucleic acid strand comprises one or more phosphorothioate intemucleoside linkages.
  • the second nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the intemucleoside linkage(s) between the last two to three nucleosides at the 5’ terminus and the last two to three nucleosides at 3’ terminus.
  • the intemucleoside linkage(s) between the last two, three or four nucleosides at the 5’ terminus of the second nucleic acid strand, the 3’ terminus of the second nucleic acid strand, or both, are phosphorothioate intemucleoside linkages.
  • the 5’ terminus of the third nucleic acid strand comprises at least one phosphorothioate intemucleoside linkage.
  • the last two, three or four nucleosides at the 5’ terminus of the third nucleic acid strand are phosphorothioate intemucleoside linkages.
  • less than 5%, less than 10%, less than 25%, less than 50% of the intemucleoside linkages in the first nucleic acid strand are phosphorothioate intemucleoside linkages.
  • the first nucleic acid strand comprises no more than two phosphorothioate intemucleoside linkages, or does not comprise phosphorothioate intemucleoside linkages.
  • at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or all of the nucleosides of the first region of the first nucleic acid strand, the second region of the first nucleic strand, or both, are chemically modified.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or all of the nucleosides of one or more of the first nucleic acid strand, the second nucleic strand and the third nucleic strand can be chemically modified.
  • At least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or all of the nucleosides of the nucleic acid complex are chemically modified.
  • the chemical modifications are to resist nuclease degradation, to increase melting temperature (Tm), or both, of the nucleic acid complex.
  • at least 90%, at least 95%, or all of the nucleotides of the nucleic acid complex are non-DNA and non-RNA nucleotides.
  • at most 5%, at most 10%, or at most 15% of the nucleosides of the second nucleic strand are LNA.
  • the bases of the nucleic acid complex have a T -4’ bridging modifications. In some embodiments, about 10%-50% of the bases of the nucleic acid complex are locked nucleic acid (LNA) or analogues thereof. In some embodiments, about 10%-50% of the bases of the nucleic acid complex comprises T -O-methyl modification, 2’-F modification, or both.
  • the input nucleic acid strand is a RNA.
  • the target RNA is a cellular RNA transcript. In some embodiments, the target RNA is an mRNA, an miRNA, a non coding RNA, a viral RNA transcript, or a combination thereof.
  • the overhang of the second nucleic acid strand is capable of binding to the input nucleic acid strand to form a toehold, thereby causing the displacement of the second nucleic acid strand from the first nucleic acid strand.
  • the overhang of the third nucleic acid strand is 5 to 20 nucleosides in length in some embodiments, the overhang of the third nucleic acid strand is 8 to 16 nucleosides in length, preferably 12 nucleosides in length. In some embodiments, the overhang of the third nucleic acid strand is 12 nucleotides in length.
  • all intemucleoside linkages of the overhang of the third nucleic acid strand are phosphorothioate intemucleoside linkages.
  • the 5’ terminus, the 3’ terminus, or both of the third nucleic acid strand comprises a terminal moiety.
  • the terminal moiety comprises a ligand, a fluorophore, a exonuclease, a fatty acid, a Cy3, an inverted dT attached to a tri-ethylene glycol, or a combination thereof.
  • a method of modulating a target RNA includes: contacting a cell comprising a target RNA with any one or more of the nucleic acid complexes disclosed herein, wherein an input strand binds to the overhang of the third nucleic acid strand to cause displacement of the third nucleic acid strand from the first nucleic acid strand to release the sequence complementary to the target RNA into the cell, thereby modulating the target RNA.
  • contacting the cell with the nucleic acid complex is performed in vitro , in vivo , ex vivo , or a combination thereof.
  • contacting the cell with the nucleic acid complex occurs in the body of a subject.
  • the cell can be, for example, a disease cell, and optionally the cell is a cancer cell. In some embodiments, the cell is a neuron.
  • Also provided includes a method of treating a disease or a condition, wherein the method includes administering one or more of the nucleic acid complexes disclosed herein to a subject in need thereof, wherein the input strand binds to the overhang of the third nucleic acid strand to cause displacement of the third nucleic acid strand from the first nucleic acid strand to release the sequence complementary to a target RNA, thereby reducing the activity of the target RNA or protein expression from the target RNA in the subject to treat the disease or condition.
  • the disease or condition is a central nervous system (CNS) disease or disorder or cancer.
  • the target RNA is a mRNA or a miRNA.
  • the nucleic acid complex is administered to a subject via a lipid-mediated delivery system, optionally via liposomes, nanoparticles, or micelles.
  • the nucleic acid complex is administered to a subject via nanoparticles, inorganic nanoparticles, nucleic acid lipid particles, polymeric nanoparticles, lipid nanoparticles (LNPs), chitosan and inulin nanoparticles, cyclodextrins nanoparticles, carbon nanotubes, liposomes, micellar structures, capsids, polymers, polymer matrices, hydrogels, dendrimers, nucleic acid nanostructure, exosomes, GalNAc-conjugated melittin-like peptides, or combinations thereof.
  • the nucleic acid complex is administered to a subject in need thereof via a subcutaneous injection. In some embodiments, the nucleic acid complex is administered to a subject in need thereof via an intravenous injection. In some embodiments, the nucleic acid complex is administered to a subject in need thereof at a concentration about 0.1-10 nM, optionally about 0.1-1 nM.
  • FIG. 1 illustrates a schematic representation of a non-limiting exemplary nucleic acid complex construct disclosed herein.
  • FIG. 2 illustrates a schematic representation of non-limiting exemplary nucleic acid complex constructs based on two designs.
  • FIG. 3 is a schematic diagram showing the formation of an active RNAi duplex following the displacement of a sensor nucleic acid strand from a core nucleic acid strand and the degradation of the core nucleic acid strand overhangs.
  • FIG. 4 shows sequence diagrams of three non-limiting exemplary nucleic acid complex constructs.
  • Calc V3P3 passenger SEQ ID NO: 7; Alt anp sensl: SEQ ID NO: 8; Alt anp-calc core 1: SEQ ID NO: 9; Alt anp sens2: SEQ ID NO: 10; Alt mus-calc core2: SEQ ID NO: 11; Alt mus-calc core 3: SEQ ID NO: 12.
  • FIG. 5A illustrates a schematic representation of a non-limiting exemplary nucleic acid complex construct T1 CASi comprising a sensor strand, passenger strand and core strand. “+/- palmitic acid” indicates that palmitic acid can be optional at the terminus.
  • FIGS. 5B-5D illustrate the chemical formulas of an exemplary sensor strand (FIG. 5B: SEQ ID NO: 6), passenger strand (FIG. 5C: SEQ ID NO: 1) and core strand (FIG. 5D: SEQ ID NO: 2), respectively.
  • FIG. 5E shows the results from polyacrylamide gel electrophoresis (PAGE) analysis of T1 CASi constructs, individual strands and duplexes.
  • FIG. 5F shows an exemplary formulation of making a T1 CASi construct.
  • FIG. 6 shows a graphic representation of various brain regions evaluated with the exemplary CASi constructs disclosed herein.
  • FIG. 7 depicts graphs showing the target mRNA levels in various brain regions of the mice treated with the T1 CASi construct without a 3’ terminal palmitic acid (top panel) and with a 3’ terminal palmitic acid (bottom panel) 14 days after CASi injection.
  • FIG. 8 depicts a diagram showing the target mRNA levels in various brain regions of the mice treated with a CASi construct having a standard 8 nucleotide toehold (with or without palmitic acid) (“8 nt toe + PA” or “8 nt toe”), with a CASi construct having a 12 nucleotide toehold (12 nt toe), or with a CASi construct having a 16 nucleotide toehold (16 nt toe).
  • FIG. 9 depicts graphs showing the target mRNA levels in various brain regions of the mice treated with CASi constructs in comparison to saline treated mice 14 days, 30 days and 90 days after administration.
  • FIG. 10 depicts graphs showing the target mRNA levels in the spinal cord of the mice treated with the CASi constructs in comparison to saline treated mice 30 days and 90 days after the administration.
  • FIG. 11 depicts a graph showing the target mRNA levels in central and peripheral nervous systems 30 days after CASi administration.
  • FIG. 12 depict graphs showing the GFAP mRNA (top panel) and IBA-1 mRNA (bottom panel) levels in various brain regions of CASi treated animals (t) in comparison to saline treated animals (c).
  • RNA interference is an intrinsic cellular mechanism conserved in most eukaryotes, that helps to regulate the expression of genes critical to cell fate determination, differentiation, survival and defense from viral infection.
  • RNAi RNA interference
  • Emerging developments in the field of dynamic nuclei acid nanotechnology and biomolecular computing also offer a conceptual approach to design programmable RNAi agents.
  • challenges still remain in developing targeted RNAi therapy that can use nuclei acid logic switches to sense RNA transcripts (such as mRNAs and miRNAs) in order to restrict RNA silencing to specific populations of disease-related cells and spare normal tissues from toxic side effects.
  • Significant challenges include poorly suppressed background drug activity, weak activated state drug potency, input and output sequence overlap, high design complexity, short lifetimes ( ⁇ 24 hours) and high required device concentrations (> 10 nM).
  • conditionally activatable small interfering RNA (siRNA) complexes include conditionally activatable small interfering RNA (siRNA) complexes, components, compositions, and related methods and systems.
  • the conditionally activatable siRNA complex can switch from an inactivated state to an activated state when triggered by a complementary binding of an input nucleic acid strand (e.g. a disease biomarker gene specific to disease-related cells) to the siRNA complex, thereby activating the RNA interference activity of the siRNA complex to target a specific target RNA (e.g. a RNA to be silenced).
  • an input nucleic acid strand e.g. a disease biomarker gene specific to disease-related cells
  • the nucleic acid complexes herein described can mediate conditionally activated RNA interference activity to silence target RNA in specific populations of disease-related cells with improved potency at a low concentration as well as improved specificity that can reduce off-target effects.
  • the nucleic acid complex can comprise a first nucleic acid strand (e.g., core nucleic acid strand) comprising 20-60 linked nucleosides, a second nucleic acid strand (e.g., passenger nucleic acid strand) binding to a first region of the core nucleic acid strand to form a first nucleic acid duplex (e.g.
  • RNAi duplex RNAi duplex
  • a third nucleic acid strand e.g., sensor nucleic acid strand
  • the sensor nucleic acid strand can comprise an overhang that is not complementary to the core nucleic acid strand and is capable of binding to an input nucleic acid strand to cause the displacement of the third nucleic acid strand from the first nucleic acid strand.
  • the first region of the core nucleic acid strand is 3 of the second region of the core nucleic acid strand.
  • the sensor nucleic acid strand does not bind to any region of the core nucleic acid strand that is 3 of the first region of the core nucleic acid strand.
  • the first region of the core nucleic acid strand comprises a sequence complementary to a target RNA.
  • the sequence complementary to a target RNA can be 10-35 nucleosides in length.
  • Disclosed herein also includes a method of modulating a target RNA.
  • the method comprises contacting a cell comprising a target RNA with the nucleic acid complex herein described.
  • the input nucleic acid strand can bind to the overhang of the sensor nucleic acid strand to cause displacement of the sensor nucleic acid strand from the core nucleic acid strand to release the sequence complementary to the target RNA into the cell, thereby modulating the target RNA.
  • Disclosed herein also includes a method of treating a disease or a condition.
  • the method comprises administering the nucleic acid complex herein described to a subject in need thereof.
  • the input strand can bind to the overhang of the sensor nucleic acid strand to cause displacement of the sensor nucleic acid strand from the core nucleic acid strand to release the sequence complementary to a target RNA, thereby reducing the activity of the target RNA or protein expression from the target RNA in the subject to treat the disease or condition.
  • nucleoside refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar.
  • exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine.
  • nucleotide refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety.
  • exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates.
  • polynucleotide and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodi ester linkage between 5' and 3' carbon atoms.
  • RNA refers to a polymer of ribonucleotides.
  • DNA refers to a polymer of deoxyribonucleotides.
  • DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post- transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded or multi-stranded (e.g., double-stranded or triple-stranded).
  • mRNA or “messenger RNA” is single-stranded RNA molecule that is complementary to one of the DNA strands of a gene. “miRNA” or “microRNA” is a small single-stranded non-coding RNA molecule that functions in RNA silencing and post-transcriptional regulation of gene expression.
  • RNA analog refers to an polynucleotide having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA.
  • the nucleotide can retain the same or similar nature or function as the corresponding unaltered or unmodified RNA such as forming base pairs.
  • a single-stranded polynucleotide has a 5’ terminus or 5' end and a 3’ terminus or 3' end
  • the terms “5' end” “5’ terminus” and “3' end” “3’ terminus” of a single- stranded polynucleotide indicate the terminal residues of the single-stranded polynucleotide and are distinguished based on the nature of the free group on each extremity.
  • the 5 '-terminus of a single- stranded polynucleotide designates the terminal residue of the single-stranded polynucleotide that has the fifth carbon in the sugar-ring of the deoxyribose or ribose at its terminus (5' terminus).
  • the 3 '-terminus of a single-stranded polynucleotide designates the residue terminating at the hydroxyl group of the third carbon in the sugar-ring of the nucleotide or nucleoside at its terminus (3' terminus).
  • the 5' terminus and 3' terminus in various cases can be modified chemically or biologically e.g. by the addition of functional groups or other compounds as will be understood by the skilled person.
  • complementary binding and “bind complementarily” mean that two single strands are base paired to each other to form nucleic acid duplex or double-stranded nucleic acid.
  • base pair indicates formation of hydrogen bonds between base pairs on opposite complementary polynucleotide strands or sequences following the Watson-Crick base pairing rule.
  • adenine (A) forms a base pair with thymine (T)
  • guanine (G) forms a base pair with cytosine (C).
  • adenine (A) forms a base pair with uracil (U) and guanine (G) forms a base pair with cytosine (C).
  • U uracil
  • G guanine
  • C cytosine
  • the two strands that bind complementarily can have a mismatches can be, about, be at most, or be at most bout 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.
  • RNA interference refers to a selective intracellular degradation of RNA.
  • RNAi can occur in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA which direct the degradative mechanism to other similar RNA sequences. RNAi can also be initiated recombinantly, for example, to silence the expression of target genes.
  • small interfering RNA and “siRNA” refer to an RNA or RNA analog capable of reducing or inhibiting expression of a gene or a target gene when the siRNA is activated in the same cell as the target gene.
  • the siRNA used herein can comprise naturally occurring nucleic acid bases and/or chemically modified nucleic acid bases (RNA analogs).
  • nucleic acid complex that can be conditionally activated upon a complementary binding to an input nucleic acid strand (e.g., a mRNA of a disease biomarker gene specific to a target cell, including a disease-related cell) through a sequence in a sensor nucleic acid strand of the nucleic acid complex.
  • the activated nucleic acid complex can release the potent RNAi duplex formed by a core nucleic acid strand and a passenger nucleic acid strand, which can specifically inhibit or silence a target RNA.
  • the target RNA can have a sequence independent from the input nucleic acid strand.
  • the nucleic acid complexes described herein comprise a core nucleic acid strand (e.g. a first nucleic acid strand), a passenger nucleic acid strand (e.g. a second nucleic acid strand), and a sensor nucleic acid strand (e.g. a third nucleic acid strand) as shown in a non-limiting embodiment of FIG. 1.
  • These three strands base-pair with one another to form a RNAi duplex (e.g. a first nucleic acid duplex) and a sensor duplex (e.g. a second nucleic acid duplex).
  • the core nucleic acid strand, the passenger nucleic acid strand, and the sensor nucleic acid strand are RNA analogs comprising modified nucleotides.
  • nucleic acid duplex refers to two single-stranded polynucleotides bound to each other through complementarily binding.
  • the nucleic acid duplex can form a helical structure, such as a double-stranded RNA molecule, which is maintained largely by non-covalent bonding of base pairs between the two single-stranded polynucleotides and by base stacking interactions.
  • the core nucleic acid strand can comprise a first region and a second region and the first region is at the 3’ direction of the second region. In other words, the first region is at the 3’ end of the core nucleic acid strand and the second region is at the 5’ end of the core nucleic acid strand.
  • the first region of the core nucleic acid strand can be linked to the second region of the core nucleic acid strand via a connector, which can also be referred to as a 5’ connector.
  • the 5’ connector can be a normal phosphodiester internucleoside linkage connecting two adjacent nucleotides.
  • the core nucleic acid strand only comprises one connector (e.g. 5’ connector) and does not comprise a 3’ connector.
  • the first region of the core nucleic acids strand is complementarily bound to the passenger nucleic acid strand to form a RNAi duplex (e.g. a first nucleic acid duplex). Not the entire sequence of the core nucleic acid strand is complementarily bound to the passenger nucleic acid strand.
  • the second region of the core nucleic acid strand is not complementarily bound to the passenger nucleic acid strand.
  • the first region of the core nucleic acid strand is fully complementary to the passenger nucleic acid strand, thereby forming a RNAi duplex having a blunt end with no overhang at the 5’ and 3’ termini of the first region of the core nucleic acid strand.
  • the core nucleic acid strand of the RNAi duplex has a short overhang at the 3’ terminus (e.g. one, two, or three nucleosides), but the 3’ overhang does not extend back into the middle of the sensor duplex to bind with the sensor nucleic acid strand (see, for example, FIGS. 1-2).
  • the core nucleic acid strand does not have any region at the 3 ’ of the first region of the core nucleic acid strand.
  • the first region of the core nucleic acid strand can comprise a sequence complementary to a target nucleic acid (e.g. a RNA to be silenced).
  • the core nucleic acid strand of the nucleic acid complex therefore acts as a guide strand (antisense strand) and is used to base pair with a target RNA.
  • the passenger nucleic acid strand can therefore comprise a sequence homologous to the same target nucleic acid.
  • the released RNAi duplex can complementarily bind a target nucleic acid through the binding between the target nucleic acid and the first region of the core nucleic acid strand.
  • the sequence complementary to a target RNA in the core nucleic acid strand can be about 10-35 nucleosides in length.
  • the core nucleic acid strand comprises 20-60 linked nucleosides.
  • the sensor nucleic acid strand is complementarily bound to the second region of the core nucleic acid strand to form a sensor duplex (e.g. a second nucleic acid duplex).
  • the sensor nucleic acid strand does not bind to the first region of the core nucleic acid strand nor any region of the core nucleic acid strand that is 3’ of the first region of the core nucleic acid strand.
  • the sensor nucleic acid strand also does not bind to the passenger nucleic acid strand.
  • the sensor nucleic acid strand can comprise an overhang.
  • overhang refers to a stretch of unpaired nucleotides that protrudes at one of the ends of a double-stranded polynucleotide (e.g. a duplex).
  • An overhang can be on either strand of the polynucleotide and can be included at either the 3’ terminus of the strand (3’ overhang) or at the 5’ terminus of the strand (5’ overhang).
  • the overhang can be at the 3’ terminus of the sensor nucleic acid strand.
  • the overhang of the sensor nucleic acid strand does not bind to any region of the core nucleic acid strand.
  • the overhang of the sensor nucleic acid strand can be about 5-20 nucleosides in length. In some embodiments, the overhang of the sensor nucleic acid strand is about 8-16 nucleosides in length, for example 8, 9, 10, 11, 12, 13, 14, 15, 16, or a number or a range between any two of these values, nucleotides in length. In some embodiments, the overhang of the sensor nucleic acid strand is 12 nucleosides in length.
  • the sensor nucleic acid strand can comprise a sequence capable of binding to an input nucleic acid strand (e.g. a disease biomarker gene specific to disease-related cells). Upon activation, the binding of the sensor nucleic acid strand to the input nucleic acid strand can cause displacement and subsequent release of the sensor nucleic acid strand from the core nucleic acid strand, thereby releasing the potent RNAi duplex and switching on the RNA interfering activity of the RNAi duplex.
  • an input nucleic acid strand e.g. a disease biomarker gene specific to disease-related cells.
  • the nucleic acid complex herein described In the absence of an input nucleic acid strand or a detectable amount of the input nucleic acid strand, the nucleic acid complex herein described remains in an inactivated state (switched off) and the displacement of the sensor nucleic acid strand from the core nucleic acid strand does not take place. Therefore, the input nucleic acid strand can act as a trigger to activate (switch on) the RNA interfering activity of the nucleic acid complex (e.g. RNAi duplex).
  • the length of the RNAi duplex of the nucleic acid complex herein described can vary in different embodiments.
  • the length of the RNAi duplex can be 10-30 nucleotides.
  • the length of the RNAi duplex can be, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • the length of the RNAi duplex can be 17-22 nucleotides.
  • the length of the sensor duplex of the nucleic acid complex herein described can vary in different embodiments.
  • the length of the sensor duplex can be 10-30 nucleotides.
  • the length of the sensor duplex can be, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.
  • the length of the sensor duplex is about 14 nucleotides.
  • the sensor duplex has a relatively short length with respect to the RNAi duplex.
  • linker molecule between a sensor duplex and a RNAi duplex of a nucleic acid complex except for the normal phosphodiester linkage connecting two adjacent nucleosides each located at a terminus of one of the two duplexes (see, for example, the CASi design shown in FIGS. 1-2).
  • FIGS. 1-2 An exemplary non-limiting embodiment of the nucleic acid complex herein described is illustrated in FIGS. 1-2.
  • the RNAi duplex formed by the passenger nucleic acid strand and the first region of the core nucleic acid strand has a length about 17-22 nucleotides (e.g. 21 nucleotides) and the sensor duplex formed by the second region of the core nucleic acid strand and the sensor nucleic acid strand has a length about 10-30 nucleotides (e.g. 14 nucleotides).
  • the nucleotides of the nucleic acid complex can be extensively modified. For example, all of the nucleotides in the nucleic acid complex can be modified.
  • the passenger nucleic acid strand can comprise two consecutive phosphorothioate internucleoside linkages at the 3’ terminus and three consecutive phosphorothioate internucleoside linkages at the 5’ terminus.
  • the 5’ terminus of the passenger nucleic acid strand can be attached to a blocking moiety, such as a fluorophore, via a phosphorothioate intemucleoside linkage to block interactions with RNAi pathway enzymes (e.g. Dicer, RISC).
  • the 5’ terminus of the passenger nucleic acid strand can also comprise one or two LNA or analogues thereof.
  • the core nucleic acid comprises a first region complementary bound to the passenger nucleic acid strand to form the RNAi duplex and a second region complementary bound to the sensor nucleic acid strand to form the sensor duplex.
  • the first region can be longer than the second region, therefore rendering a shorter sensor duplex.
  • the first region can be connected to the second region via a normal phosphodiester intemucleoside linkage between two adjacent nucleotides with no additional connector or linker.
  • the first region of the core nucleic acid strand can comprise two consecutive phosphorothioate intemucleoside linkages at the 3’ terminus and two consecutive phosphorothioate intemucleoside linkages at the 5’ terminus.
  • the second region of the core nucleic acid strand can have no phosphorothioate intemucleoside linkages.
  • the core nucleic acid strand can comprise a 3’ overhang that is about two nucleotides in length. The overhang does not extend back into the middle of the sensor duplex to bind with the sensor nucleic acid strand.
  • the sensor nucleic acid strand comprises a portion (e.g. 14 nucleotides) complementary bound to the second region of the core nucleic acid strand and a 3’ overhang (e.g. 12 nucleotides). All of the intemucleoside linkages in the 3’ overhang of the sensor nucleic acid strand can be phosphorothioate intemucleoside linkages.
  • the 3’ terminus of the sensor nucleic acid strand can be attached to a terminal moiety such as a delivery ligand, a dye (e.g. fluorophore) or exonuclease.
  • the 5’ terminus of the sensor nucleic acid strand can be attached to a terminal moiety such as a fatty acid, Cy3, or an inverted dT, a tri-ethylene glycol, or an inverted dT attached to a tri-ethylene glycol.
  • the sensor nucleic acid strand can comprise a higher percentage of LNA, analogues thereof, or other 2’-4’ bridged bases than the passenger nucleic acid strand and the core nucleic acid strand.
  • the core nucleic acid strand may comprise no LNA, analogues thereof, or other 2’-4’ bridged bases.
  • nucleic acid complexes herein described can be synthesized using standard methods for oligonucleotide synthesis well-known in the art including, for example, Oligonucleotide Synthesis by Herdewijin, Piet (2005) and Modified oligonucleotides: Synthesis and Strategy for Users, by Verma and Eckstein, Annul Rev. Biochem. (1998): 67:99-134, the contents of which are incorporated herein by reference in their entirety.
  • the synthesized nucleic acid complexes can be allowed to form its secondary structure under a desirable physiological condition as will be apparent to a skilled artisan.
  • the formed secondary structure can be tested using standard methods known in the art such as chemical mapping, NMR, or computational simulations.
  • the nucleic acid complex construct can be further modified, according to the test result, by introducing or removing chemical modifications or mismatches, as necessary, until the desired structure is obtained.
  • Suitable software suites can be used to aid in the design and analysis of nucleic acid structures.
  • Nupack can be used to check the formation of the duplexes and to rank the thermodynamic stability of the duplexes.
  • Oligonucleotide design tools can be used to optimize the placement of LNA modifications.
  • nucleic acid complexes that can be conditionally activated (e.g., via a signal for the presence of a mRNA of a gene specific for a target cell) to switch from an assembled, inactivated state to an activated state to act on (e.g. degrade or inhibit) a specific target nucleic acid in response to the detection of an input nucleic acid (e.g. nucleic acid specific to disease-related cells) having a sequence complementary to a sequence in the sensor nucleic acid strand of a nucleic acid complex.
  • an input nucleic acid e.g. nucleic acid specific to disease-related cells
  • the sensor nucleic acid strand of the nucleic acid complex inhibits enzymatic processing of the RNAi duplex, thereby keeping RNAi activity switched off.
  • the input nucleic acid strand can activate the nucleic acid complex by inducing separation of the sensor nucleic acid strand from the core nucleic acid strand via toehold mediated strand displacement. Displacement can start from a toehold formed at the 3’ or 5’ terminus of the sensor nucleic acid strand (e.g. a toehold formed at the 3’ terminus of the sensor nucleic acid strand) through a complementary binding between the input nucleic acid strand and an overhang of the sensor nucleic acid strand.
  • RISC RNA-induced silencing complex
  • FIG. 3 is a schematic diagram showing the formation of an active RNAi duplex following the displacement of a sensor nucleic acid strand from a core nucleic acid strand and the degradation of the core nucleic acid strand overhangs.
  • the passenger nucleic acid strand is then discarded, while the core nucleic acid strand (the first region of the core nucleic acid strand) is incorporated into RICS.
  • the core nucleic acid strand of the nucleic acid complex disclosed herein acts as a guide strand (antisense strand) and is used to base pair with a target RNA.
  • the passenger nucleic acid strand acts as a protecting strand prior to the loading of the core nucleic acid strand into RICS.
  • RICS uses the incorporated core nucleic acid strand as a template for recognizing a target RNA that has complementary sequence to the core nucleic acid strand, particularly the first region of the core nucleic acid strand.
  • the catalytic component of RICS Argonaute, is activated which can degrade the bound target RNA.
  • the target RNA can be degraded or the translation of the target RNA can be inhibited.
  • the nucleic acid complexes herein described do not have a dicer cleavage site, and therefore the RNAi interference mediated by the nucleic acid complexes can bypass Dicer-mediated cleavage.
  • Dicer is an endoribonuclease in the RNAse III family that can initiate the RNAi pathway by cleaving double-stranded RNA (dsRNA) molecule into short fragments of dsRNAs about 20-25 nucleotides in length.
  • the nucleic acid complexes herein described differentiate from the conditionally activated small interfering RNAs (Cond-siRNAs) disclosed in the related international application published as W0/2020/033938in that the nucleic acid complexes herein described can bypass the Dicer processing.
  • Cond-siRNAs conditionally activated small interfering RNAs
  • the nucleic acid complexes disclosed herein have structural features that discourage the Dicer binding.
  • the RNAi duplex does not create a Dicer substrate.
  • the RNAi duplex formed by the passenger nucleic acid strand and the first region of the core nucleic acid strand do not have a 3’ and/or 5’ overhang, but instead forming a blunt end that can render the passenger nucleic acid strand unfavorable for Dicer binding.
  • the passenger nucleic acid strand has about 17-22 nucleotides in length, making it short enough to bypass Dicer cleavage.
  • the passenger nucleic acid strand does not have G/C rich bases to the 3’ and/or 5’ end of the passenger nucleic acid strand. In some embodiments, the passenger nucleic acid strand is attached to a terminal moiety and/or a blocking moiety to avoid Dicer binding.
  • the nucleic acid complex herein described can inhibit a target nucleic acid in target cells, therefore resulting in a reduction or loss of expression of the target nucleic acid in the target cells.
  • the target cells are cells associated or related to a disease or disorder.
  • the term “associated to” “related to” as used herein refers to a relation between the cells and the disease or condition such that the occurrence of a disease or condition is accompanied by the occurrence of the target cells, which includes but is not limited to a cause- effect relation and sign/symptoms-disease relation.
  • the target cells used herein typically have a detectable expression of an input nucleic acid.
  • the expression of a target nucleic acid in target cells is inhibited by about, at least, or at least about, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
  • inhibition of gene expression refers to the absence or observable decrease in the level of protein and/or mRNA product from a target gene in target cells. The degree of inhibition can be evaluated by examination of the expression level of the target gene as demonstrated in the examples.
  • gene expression can be assayed by use of a reporter or drug resistance gene whose protein product is easily assayed.
  • reporter genes include, but no limiting to, acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof.
  • AHAS acetohydroxyacid synthase
  • AP alkaline phosphatase
  • LacZ beta galactosidase
  • GUS beta glucoronidase
  • CAT chloramphenicol acetyltransferase
  • GFP green fluorescent protein
  • HRP horseradish peroxidase
  • Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentarmicin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Quantitation of the amount of gene expression allows one to determine a degree of inhibition as compared to cells not treated with the nucleic acid complexes or treated with a negative or positive control.
  • RNA solution hybridization nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, enzyme linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), other immunoassays, and fluorescence activated cell analysis (FACS).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescence activated cell analysis
  • the nucleic acid complexes disclosed herein exhibit improved switching performance and reduced off-target effects.
  • the nucleic acid complexes disclosed herein can have a reduced unwanted RNAi activity when the nucleic acid complexes are in an inactivated state (switched off) and an enhanced RNAi activity when the nucleic acid complexes are activated upon detection of an input nucleic acid strand.
  • the expression of a target nucleic acid in non-target cells is inhibited about, at most, or at most about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a number or a range between any of these values.
  • Non-target cells can comprise cells of the subject other than target cells.
  • the nucleic acid complexes herein described have an enhanced potency, thus capable of evoking an RNAi activity at low concentrations.
  • Nonspecific, off-target effects and toxicity e.g. undesired proinflammatory responses
  • the concentration of the nucleic acid complexes disclosed herein can vary in different embodiments.
  • the nucleic acid complexes disclosed herein can be provided at a concentration of, about, at most, or at most about, 0.001 nM, 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0 nM, 5.5 nM, 6.0 nM, 6.5 nM, 7.0 nM, 7.5 nM
  • the nucleic acid complexes disclosed herein can be provided at a concentration between about 0.1-10 nM, preferably between about 0.1-1 nM. In some embodiments, the nucleic acid complex herein disclosed has a transfection concentration at 0.1 nM or lower.
  • the nucleic acid complex herein described can allow lasting and consistently potent inhibition effects at low concentrations.
  • the nucleic acid complex can remain active for an extended period of time such as 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 5 days, 6 days, 7 days, two weeks, or a number or a range between any of these values, or more.
  • the nucleic acid complex can remain active for at least 12 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, or at least 96 hours.
  • the nucleic acid complex can remain active for up to 30 days, up to 60 days, or up to 90 days.
  • the nucleic acid strands (the core nucleic acid strand, the passenger nucleic acid strand, and/or the sensor nucleic acid strand) comprised in the nucleic acid complexes herein described can be a non-standard, modified nucleic acid strand comprising non-standard, modified nucleotides (nucleotide analog) or non-standard, modified nucleosides (nucleoside analog).
  • nucleotide analog or “modified nucleotide” refers to a non-standard nucleotide comprising one or more modifications (e.g. chemical modifications), including non- naturally occurring ribonucleotides or deoxyribonucleotides.
  • nucleoside analog refers to a non-standard nucleoside comprising one or more modification (e.g. chemical modification), including non-naturally occurring nucleosides other than cytidine, uridine, adenosine, guanosine, and thymidine.
  • the modified nucleoside can be a modified nucleotide without a phosphate group.
  • the chemical modifications can include replacement of one or more atoms or moieties with a different atom or a different moiety or functional group (e.g. methyl group or hydroxyl group).
  • thermodynamic stability can be determined based on measurement of melting temperature T m. A higher T m can be associated with a more thermodynamically stable chemical entity.
  • the modification can render one or more of the nucleic acid strands in the nucleic acid complex to resist exonuclease degradation/cleavage.
  • exonuclease indicates a type of enzyme that works by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3' or the 5' end occurs. A 3' and 5' exonuclease can degrade RNA and DNA in cells, and can degrade RNA and DNA in the interstitial space between cells and in plasma, with a high efficiency and a fast kinetic rate.
  • exonuclease which cleaves phosphodiester bonds in the middle (endo) of a polynucleotide chain.
  • 3' and 5' exonuclease and exonucleolytic complexes can degrade RNA and DNA in cells, and can degrade RNA and DNA in the interstitial space between cells and in plasma.
  • exoribonuclease refers to exonuclease ribonucleases, which are enzymes that degrade RNA by removing terminal nucleotides from either the 5' end or the 3' end of the RNA molecule. Enzymes that remove nucleotides from the 5' end are called 5 '-3' exoribonucleases, and enzymes that remove nucleotides from the 3' end are called 3 '-5' exoribonucleases.
  • the modification can comprise phosphonate modification, ribose modification (in the sugar portion), and/or base modification.
  • Preferred modified nucleotides used herein include sugar- and/or backbone-modified ribonucleotides.
  • the modified nucleotide can comprise modifications to the sugar portion of the nucleotides.
  • the 2’ OH-group of a nucleotide can be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, ME, NHR, NR2, COOR, or OR, wherein R is substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc.
  • the T OH-group of a nucleotide or nucleoside is replaced by T O-methyl group and the modified nucleotide or nucleoside is a T -O-methyl nucleotide or T -O-methyl nucleoside (2’-OMe).
  • the T -O-methyl nucleotide or T -O-methyl nucleoside can be 2'-0- methyladenosine, 2'-0-methylguanosine, 2'-0-methyluridine, or 2'-0-methylcytidine.
  • the T OH-group of a nucleotide is replaced by fluorine (F)
  • the modified nucleotide or nucleoside is a 2’-F nucleotide or 2’-F nucleoside (2’-deoxy-2’-fluoro or 2’-F).
  • the 2’-F nucleotide or 2’-F nucleoside can be 2'-F-adenosine, 2'-F-guanosine, 2'-F-uridine, or 2'-F- cytidine.
  • the modifications can also include other modifications such as nucleoside analog phosphoramidites.
  • glycol nucleic acids can be used.
  • the modified nucleotide can comprise a modification in the phosphate group of the nucleotide, e.g. by substituting one or more of the oxygens of the phosphate group with sulfur or a methyl group. In some embodiments, one or more of the nonbridging oxygens of the phosphate group of a nucleotide is replaced by a sulfur.
  • the nucleic acid strands herein described comprise one or more non-standard intemucleoside linkage that is not a phosphodiester linkage. In some embodiments, the nucleic acid strands herein described comprise one or more phosphorothioate intemucleoside linkages.
  • the term “phosphorothioate linkage” (PS) as used herein, indicates a bond between nucleotides in which one of the nonbridging oxygens is replaced by a sulfur. In some embodiments, both nonbridging oxygens may be replaced by a sulfur (PS2). In some embodiments, one of the nonbridging oxygens may be replaced by a methyl group.
  • phosphodiester linkage indicates the normal sugar phosphate backbone linkage in DNA and RNA wherein a phosphate bridges the two sugars.
  • the introduction of one or more phosphorothioate linkage in the core nucleic acid strand, the passenger nucleic acid strand, and/or the sensor nucleic acid strand can endow the modified nucleotides with increased resistance to nucleases (e.g. endonucleases and/or exonucleases).
  • the modified nucleotide can comprise modifications to or substitution of the base portion of a nucleotide.
  • uridine and cytidine residues can be substituted with pseudouridine, 2-thiouridine, N6-methyladenosine, 5-methycytidine or other base analogs of uridine and cytidine residues.
  • Adenosine can comprise modifications to Hoogsteen (e.g., 7-triazolo-8-aza-7-deazaadenosines) and/or Watson-Crick face of adenosine (e.g. N2-alkyl-2-aminopurines).
  • adenosine analogs also include Hoogsteen or Watson-Crick face-localized N-ethylpiperidine triazole-modified adenosine analogs, N- ethylpiperidine 7-EAA triazole (e.g., 7-EAA, 7-ethynyl-8-aza-7-deazaadenosine) and other adenosine analogs identifiable to a person skilled in the art.
  • Cytosine may be substituted with any suitable cytosine analogs identifiable to a person skilled in the art.
  • cytosine can be substituted with 6’-phenylpyrrolocytosine (PhpC) which has shown comparable base pairing fidelity, thermal stability and high fluorescence.
  • one or more nucleotides in the nucleic acid complex disclosed herein can be substituted with a universal base.
  • the term “universal base” refers to nucleotide analogs that form base pairs with each of the natural nucleotides with little discrimination between them.
  • Examples of universal bases include, but are not limited to, C- phenyl, C-naphthyl and other aromatic derivatives, inosine, azole carboxamides, and nitroazole derivatives such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as known in the art (see e.g., Loakes, 2001, Nucleic Acids Research, 29, 2437-2447).
  • base modification disclosed herein can reduce innate immune recognition while making the nucleic acid complex more resistant to nucleases.
  • Examples of base modifications that can be used in the nucleic acid complex disclosed herein are also described, for example, in Hu et al. (Signal Transduction and targeted Therapy 5: 101 (2020)), the content of which is incorporated by reference in its entirety.
  • the nucleic acid strands (the core nucleic acid strand, the passenger nucleic acid strand, and/or the sensor nucleic acid strand) comprised in the nucleic acid complexes herein described can comprise one or more locked nucleic acids or analogs thereof.
  • Exemplary locked nucleic acid analogs include, for example, their corresponding locked analog phosphoramidites and other derivatives apparent to a skilled artisan.
  • LNA locked nucleic acids
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' and 4' carbons (a 2’-0, 4’-C methylene bridge).
  • the bridge “locks” the ribose in the 3'-endo structural conformation and restricts the flexibility of the ribofuranose ring, thereby locking the structure into a rigid bicyclic formation.
  • LNA nucleotides can be mixed with DNA or RNA bases in the oligonucleotide whenever desired.
  • the incorporation of LNA into the nucleic acid complexes disclosed herein can increase the thermal stability (e.g.
  • LNA oligonucleotides display hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. Additional information about LNA can be found, for example, at www.sigmaaldrich.com/technical- documents/articles/biology/locked-nucleic-acids-faq.html.
  • glycol nucleic acids can be used.
  • the nucleic acid strands (the core nucleic acid strand, the passenger nucleic acid strand, and/or the sensor nucleic acid strand) comprised in the nucleic acid complexes herein described can comprise other chemically modified nucleotide or nucleoside with 2’-4’ bridging modifications (e.g. 2’-4’ bridged bases).
  • a 2’-4’ bridging modification refers to the introduction of a bridge connecting the 2' and 4' carbons of a nucleotide.
  • the bridge can be a 2’-0, 4’-C methylene bridge (e.g. in LNA).
  • the bridge can also be a 2’-0, 4’-C ethylene bridge (e.g. in ethylen-bridged nucleic acids (ENA)) or any other chemical linkage identifiable to a person skilled in the art.
  • the introduction of LNA, analogues thereof, or other chemically modified nucleotides with T -4’ bridging modifications in the nucleic acid complex herein described can enhance hybridization stability as well as mismatch discrimination.
  • a nucleic acid complex comprising a sensor nucleic acid strand with LNA, analogues thereof, or other chemically modified nucleotides with T -4’ bridging modifications can have an enhanced sensitivity to distinguish between matched and mismatched input nucleic acid strand (e.g. in the complementary binding between an input nucleic acid strand and a sensor nucleic acid strand).
  • one or more of the nucleic acid strands of the nucleic acid complex can comprise a chemical moiety linked to the 3’ and/or 5’ terminus of the strand.
  • the terminal moiety can include one or more suitable terminal linkers or modifications.
  • the terminal moiety can include a linker to link the oligonucleotide with another molecule or a particular surface (biotins, amino-modifiers, alkynes, thiol modifiers, azide, N- Hydroxysuccinimide, and cholesterol), a dye (e.g. fluorophore, Cy3, or a dark quencher), a fluorine modified ribose, a space (e.g.
  • the terminal moiety can serve as a fluorescent label used for imaging and detection.
  • the terminal moiety can also comprise one or more exonuclease cleavage-resistant group or moiety to protect a single-stranded nucleic acid from nuclease degradation (e.g. exonuclease cleavage-resistant moiety).
  • the terminal moiety can also serve as a blocking moiety to block interactions with RNAi pathway enzymes such as Dicer and RISC.
  • the terminal moiety can also comprise a ligand suitable for nucleic acid delivery and for use in targeting the nucleic acid complex to specific cell types described elsewhere in the present disclosure.
  • nucleotides and/or nucleosides can also be introduced to one or more strands of the nucleic acid complex herein described, such as modifications described in Hu et al. (Signal Transduction and targeted Therapy 5: 101 (2020)), the content of which is incorporated by reference in its entirety.
  • the percentage of the modified nucleosides of the nucleic acid complex can vary in different embodiments. In some embodiments, the percentage of the modified nucleosides of the nucleic acid complex herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%. For example, percentage of the modified nucleosides of the nucleic acid complex herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
  • nucleotides of the nucleic acid complex are modified (e.g. are non-DNA and non-RNA). In some embodiments, all of the nucleotides of the nucleic acid complex are modified (e.g. are non-DNA and non-RNA).
  • the percentage of the modified nucleosides in one or more strands of the nucleic acid complex can vary in different embodiments.
  • the percentage of the modified nucleosides in a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%.
  • the percentage of the modified nucleosides in a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,
  • nucleosides of a core nucleic acid strand are chemically modified.
  • the percentage of the modified nucleosides in the first region of a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%.
  • the percentage of the modified nucleosides in the first region of a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
  • nucleosides of the first region of a core nucleic acid strand are chemically modified.
  • the percentage of the modified nucleosides in the second region of a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%.
  • the percentage of the modified nucleosides in the second region of a core nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
  • nucleosides of the second region of a core nucleic acid strand are chemically modified.
  • the percentage of the modified nucleosides in a passenger nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%.
  • the percentage of the modified nucleosides in a passenger nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,
  • nucleosides of a passenger nucleic acid strand are chemically modified.
  • the percentage of the modified nucleosides in a sensor nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 60%, 70%, 80%, 85%, 90%, or 95%.
  • the percentage of the modified nucleosides in a sensor nuclei acid strand herein described can be, be about, be at least, or be at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
  • nucleosides of a sensor nucleic acid strand are chemically modified.
  • the modified nucleosides in one or more of the core nucleic acid strand, the passenger nucleic acid strand, and the sensor nucleic acid strand can comprise T -O-methyl nucleoside and/or 2’-F nucleoside.
  • the percentage of T -O-methyl nucleoside and/or 2’-F nucleoside in the nucleic acid complex herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%-50%.
  • the percentage of 2’-0-methyl nucleoside and/or 2’-F nucleoside in the nucleic acid complex herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • the percentage of 2’-0-methyl nucleoside and/or 2’-F nucleoside in a core nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%-50%.
  • the percentage of 2’-0-methyl nucleoside and/or 2’-F nucleoside in a core nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%,
  • the percentage of T -O-methyl nucleoside and/or 2’-F nucleoside in a passenger nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%-50%.
  • the percentage of 2’-0- methyl nucleoside and/or 2’-F nucleoside in a passenger nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
  • the percentage of 2’-0-methyl nucleoside and/or 2’-F nucleoside in a sensor nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%-50%.
  • the percentage of 2’-0- methyl nucleoside and/or 2’-F nucleoside in a sensor nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
  • the percentage of phosphate modification to the nucleotides in the nucleic acid complex described herein can vary in different embodiments.
  • the phosphate modification comprises or is a phosphorothioate internucleoside linkage.
  • the percentage of phosphorothioate internucleoside linkages in a core nucleic acid strand is less than 5%, less than 10%, less than 25%, less than 50%, or a number or a range between any two of these values.
  • percentage of phosphorothioate intemucleoside linkages in a core nucleic acid strand is about, less than, or less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%,
  • the core nucleic acid strand comprises no more than two phosphorothioate intemucleoside linkages. In some embodiments, the core nucleic acid strand does not comprise a phosphorothioate intemucleoside linkage modification.
  • the percentage of phosphodiester intemucleoside linkages in a core nucleic acid strand can be about, at least, or at least about 50%, 80% or 95%, or a number or a range between any two of these values.
  • percentage of phosphodiester intemucleoside linkages in a core nucleic acid strand is about, at least, or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,
  • all the intemucleoside linkages in the core nucleic acid strand are phosphodiester intemucleoside linkage.
  • the 3’ terminus of the first region of the core nucleic acid strand comprises at least one phosphorothioate intemucleoside linkage (e.g. one, two or three phosphorothioate intemucleoside linkage).
  • the phosphorothioate intemucleoside linkage can be between the last two, three, or four nucleosides at the 3 ’ terminus of the first region of the core nucleic acid strand.
  • the 5’ terminus of the first region of the core nucleic acid strand comprises at least one phosphorothioate intemucleoside linkage (e.g.
  • each of the 5’ terminus of the first region of the core nucleic acid strand and the 3’ terminus of the first region of the core nucleic acid strand independently comprises one or more phosphorothioate intemucleoside linkages (e.g. one, two or three phosphorothioate intemucleoside linkage).
  • the first region of the core nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the phosphorothioate intemucleoside linkage(s) between the last two or three nucleosides at the 5’ terminus, 3’ terminus, or both, of the first region.
  • the first region of the core nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the phosphorothioate intemucleoside linkage(s) between the last three nucleosides at the 5’ terminus and the last three nucleosides 3’ terminus of the first region.
  • the percentage of phosphorothioate intemucleoside linkages in the second region of a core nucleic acid strand is less than 5%, less than 10%, or a number or a range between any two of these values. In some embodiments, the second region of a core nucleic acid strand does not comprise phosphorothioate internucleoside linkages.
  • the passenger nucleic acid strand comprises one or more phosphorothioate intemucleoside linkage.
  • the percentage of phosphorothioate intemucleoside linkages in a passenger nucleic acid strand can be less than 5%, less than 10%, less than 25%, less than 50%, or a number or a range between any two of these values.
  • percentage of phosphorothioate intemucleoside linkages in a passenger nucleic acid strand is about, less than, or less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or a number or a range between any two of these values.
  • the 5’ terminus of the passenger nucleic acid strand comprises one or more phosphorothioate intemucleoside linkage (e.g. one, two, or three phosphorothioate intemucleoside linkage).
  • the 3’ terminus of the passenger nucleic acid strand comprises at least one phosphorothioate intemucleoside linkage (e.g. one, two, or three phosphorothioate intemucleoside linkage).
  • each of the 5’ terminus of the passenger nucleic acid strand and the 3’ terminus of the passenger nucleic acid strand independently comprises one or more phosphorothioate intemucleoside linkages (e.g. one, two or three phosphorothioate intemucleoside linkage).
  • the passenger nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the phosphorothioate intemucleoside linkage(s) between the last two, three, or four nucleosides at the 5’ terminus, 3’ terminus, or both, of the passenger nucleic acid strand.
  • the passenger nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the phosphorothioate intemucleoside linkage(s) between the last two to three nucleosides at the 5’ terminus and the last two to three nucleosides at 3’ terminus of the passenger nucleic acid strand.
  • the sensor nucleic acid strand can comprise one or more phosphorothioate intemucleoside linkage.
  • the percentage of phosphorothioate intemucleoside linkages in a sensor nucleic acid strand can be less than 5%, less than 10%, less than 25%, less than 50%, less than 60%, less than 70% or a number or a range between any two of these values.
  • percentage of phosphorothioate intemucleoside linkages in a sensor nucleic acid strand is about, less than, or less than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,
  • the 5’ terminus of the sensor nucleic acid strand comprises at least one phosphorothioate internucleoside linkage (e.g. one, two or three phosphorothioate intemucleoside linkage).
  • the 3’ terminus of the sensor nucleic acid strand comprises at least one phosphorothioate intemucleoside linkage (e.g. one to twenty phosphorothioate intemucleoside linkage.
  • each of the 5’ terminus of the sensor nucleic acid strand and the 3’ terminus of the sensor nucleic acid strand independently comprises one or more phosphorothioate intemucleoside linkages (e.g.
  • the sensor nucleic acid strand does not comprise phosphorothioate intemucleoside linkages except for the phosphorothioate intemucleoside linkage(s) at the 5’ terminus, 3’ terminus, or both, of the passenger nucleic acid strand.
  • the phosphorothioate intemucleoside linkages at the 3’ terminus of the passenger nucleic acid strand are in the singled-stranded overhang of the passenger nucleic acid strand.
  • the percentage of the LNA or analogues thereof of the nucleic acid complex can vary in different embodiments. In some embodiments, the percentage of the LNA or analogues thereof of the nucleic acid complex herein described can be about 10%-50%. For example, the percentage of the LNA or analogues thereof of the nucleic acid complex herein described can be about, at most, at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
  • the percentage of the LNA or analogues thereof in one or more strands of the nucleic acid complex can vary in different embodiments.
  • the percentage of the LNA or analogues thereof in a core nucleic acid strand herein described can be, be about, be at most, or be at most about 5%, 10%, or 15%.
  • the percentage of the LNA or analogues thereof of a core nucleic acid strand herein described can be, be about, be at most, or be at most about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a number or a range between any two of these values.
  • the percentage of the LNA or analogues thereof in a passenger nucleic acid strand herein described can be, be about, be at most, or be at most about 5%, 10%, or 15%.
  • the percentage of the LNA or analogues thereof of a passenger nucleic acid strand herein described can be, be about, be at most, or be at most about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, or a number or a range between any two of these values.
  • a percentage of the LNA or analogues thereof in a passenger nucleic acid strand herein described greater than 5%, greater than 10%, or greater than 15% can decrease the RNAi activity of the nucleic acid complex.
  • the percentage of the LNA or analogues thereof in a sensor nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%-50%.
  • the percentage of the LNA or analogues thereof of a sensor nucleic acid strand herein described can be, be about, be at least, be at least about, be at most, or be at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or a number or a range between any two of these values.
  • the percentage of T -4’ bridging modification of the nucleic acid complex can vary in different embodiments. In some embodiments, the percentage of the 2’-4’ bridging modification of the nucleic acid complex herein described can be about 10%-50%. For example, the percentage of the T -4’ bridging modification of the nucleic acid complex herein described can be about, at most, at most about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% or a number or a range between any two of these values.
  • the core nucleic acid strand of the nucleic acid complex described herein can comprise a first region and a second region.
  • the first region is at the 3’ direction of the second region.
  • the length of the core nucleic acid strand can vary.
  • the core nucleic acid strand comprises 20-60 linked nucleosides.
  • the core nucleic acid strand can comprise 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, linked nucleosides.
  • the length of the first region of the core nucleic acid strand can vary. In some embodiments, the first region of the core nucleic acid strand comprises 10-30 linked nucleosides.
  • the first region of the core nucleic acid strand can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, linked nucleosides.
  • the first region of the core nucleic acid strand comprises 17-22 linked nucleosides.
  • the length of the second region of the core nucleic acid strand can vary in different embodiments.
  • the length of the second region of the core nucleic acid strand comprises 10-30 linked nucleosides.
  • the second region of the core nucleic acid strand can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, linked nucleosides.
  • the first region and the second region of the core nucleic acid strand can have a same length or a different length.
  • the second region of the core nucleic acid strand has a relatively short length with respect to the first region of the core nucleic acid strand.
  • the second region of the core nucleic acid strand has about 14 linked nucleosides.
  • the first region of the core nucleic acid strand comprises a sequence complementary to a target RNA.
  • the length of the sequence complementary to a target RNA can vary in different embodiments.
  • the sequence complementary to a target RNA is 10-35 nucleotides in length.
  • the sequence complementary to a target RNA is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35, nucleotides in length.
  • the sequence complementary to a target RNA is 10-21 nucleotides in length.
  • the first region of the core nucleic acid strand comprises a sequence complementary to a passenger nucleic acid strand.
  • the length of the sequence complementary to a passenger nucleic acid strand can vary in different embodiments. In some embodiments, the sequence complementary to a passenger nucleic acid strand is 17-22 nucleotides in length. For example, the sequence complementary to a passenger nucleic acid strand is 17, 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, the sequence of the core nucleic acid strand complementary to a passenger nucleic acid strand is about 21 nucleotides in length.
  • the first region of the core nucleic acid strand is linked to the second region of the core nucleic acid strand via a connector.
  • the first region of the core nucleic acid strand is linked the second region of the core nucleic acid strand via a 5’ connector.
  • the core nucleic acid strand only comprises one connector (e.g. 5’ connector) and does not comprise a 3’ connector.
  • the 5’ connector can comprise a three-carbon linker (C3 linker), a nucleotide, any modified nucleotide described herein, or any moiety that can resist exonuclease cleavage when the core nucleic acid strand is single-stranded (e.g. after displacement of the sensor nucleic acid strand from the core nucleic acid strand).
  • the 5’ connector can comprise a T - F nucleotide such as 2'-F-adenosine, 2'-F-guanosine, 2'-F-uridine, or 2'-F-cytidine.
  • the 5’ connector can comprise a 2’-0-methyl nucleotide such as 2'-0-methyladenosine, 2'-0- methylguanosine, 2'-0-methyluridine, or 2'-0-methylcytidine.
  • the 5’ connector can comprise a naturally occurring nucleotide such as cytidine, uridine, adenosine, or guanosine.
  • the 5’ connector of the core nucleic acid strand can comprise a phosphodiester linkage (phosphodiester 5’ and 3’ connection) cleavable by an exonuclease when in a single-stranded form.
  • the 5’ connector and/or the 3’ connector of the core nucleic acid strand can comprise any suitable moiety that can resist exonuclease cleavage when in single-stranded form.
  • the 5’ connector of the core nucleic acid strand comprises no linker molecule except for the normal phosphodiester linkage connecting two adjacent nucleosides (see, for example, the CASi design shown in FIGS. 1-2).
  • the 5’ connector can comprise or is, a C3 3-carbon linker, a nucleotide, a modified nucleotide (e.g. 2’-0-methyl nucleotide, 2’-F nucleotide), a nucleotide with a phosphodiester 5’ and 3’ connection cleavable by an exonuclease when in a single stranded form, or a combination thereof.
  • the 5’ connector can comprise or is a 2’- O-methyl nucleotide such as 2'-0-methyladenosine, 2'-0-methylguanosine, 2'-0-methyluridine, or 2'-0-methylcytidine.
  • the 5’ connector can comprise or is 2’-F nucleotide such as 2'-F-adenosine, 2'-F-guanosine, 2'-F-uridine, or 2'-F-cytidine.
  • the 5’ connector of the core nucleic acid strand does not comprise or is not a C3 3-carbon linker. In some embodiments, it is advantageous not to have a C3 3 -carbon linker as the 5’ connector.
  • a nucleic acid complex not having a C3 3-carbon linker as the 5’ connector may exhibit higher RNA interfering activity.
  • not having a C3 3-carbon linker as the 5’ connector can increase RNA interfering activity of the nucleic acid complex by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or a number or a range between any of these value, greater than nucleic acid complexes having a C3 3 -carbon linker as the 5’ connector.
  • the core nucleic acid strand do not comprise a 5’ connector. Instead, the first region of the core nucleic acid strand is linked to the second region via a standard phosphodiester linkage connecting two adjacent nucleosides.
  • not having a 5’ connector between the first region and the second region of the core nucleic acid strand can increase RNA interfering activity of the nucleic acid complex by at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or a number or a range between any of these value, greater than nucleic acid complexes having a linker (e.g. C3 3 -carbon linker, a nucleotide, a modified nucleotide, or other moieties) as the 5’ connector.
  • a linker e.g. C3 3 -carbon linker, a nucleotide, a modified nucleotide, or other moieties
  • the core nucleic acid strand has an overhang.
  • the overhang can be at the 3’ terminus of the core nucleic acid strand (3’ overhang).
  • the core nucleic acid strand can have a short overhang at the 3’ terminus (e.g. 1-3 nucleosides), but the 3’ overhang does not extend back into the middle of the sensor duplex to bind with the sensor nucleic acid strand (see, for example, FIGS. 1-2).
  • the length of the overhang can vary in different embodiments.
  • the 3’ overhang is about one to three nucleotides in length.
  • the 3’ overhang can be one, two or three nucleotides in length.
  • the overhang can comprise one or more modified nucleotides, such as T - O-methyl nucleotides.
  • the 3’ overhang can comprise two 2’-0-methyl nucleotides (see, for example, the CASi design shown in FIGS. 1-2).
  • the overhang can comprise modified internucleoside linkages, such as phosphorothioate intemucleoside linkages.
  • all of the nucleotides in the overhang are chemically modified.
  • all of intemucleoside linkages in the 3’ overhang of the core nucleic acid strand are phosphorothioate intemucleoside linkages.
  • the passenger nucleic acid strand of the nucleic acid complex described herein is complementary bound to the first region of the core nucleic acid strand to form a RNAi duplex (e.g. a first nucleic acid duplex).
  • the first region of the core nucleic acid strand comprises a sequence complementary to a target nucleic acid strand
  • the passenger nucleic strand of the nucleic acid complex can comprise a sequence homologous to the target nuclei acid strand.
  • sequence identity refers to sequence identity between at least two sequences.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the nucleotide bases or residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • the sequence identity between a passenger nucleic acid strand and a target nucleic acid or a portion there of can be, be about, be at least, or be at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, or a number or a range between any two of these values.
  • the passenger nucleic acid strand of a nucleic acid complex can have a sequence substantially identical, e.g. at least 80%, 90%, or 100%, to a target nucleic acid or a portion thereof.
  • the length of the passenger nucleic acid strand can vary in different embodiments.
  • the passenger nucleic acid strand comprises 10-35 linked nucleosides.
  • the core nucleic acid strand can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 linked nucleosides.
  • the passenger nucleic acid strand comprises 17-21 linked nucleosides.
  • the passenger nucleic acid strand has a 3’ overhang, a 5’ overhang, or both in the RNAi duplex. In some embodiments, the passenger nucleic acid strand has a 3’ overhang, and the 3’ overhang is one to five nucleosides in length.
  • the overhang of the passenger nucleic acid strand is capable of binding to the input nucleic acid strand to form a toehold, thereby initiating a toehold mediated strand displacement and causing the displacement of the passenger nucleic acid strand from the core nucleic acid strand.
  • the overhang of the passenger nucleic acid strand is 5 to 20 nucleosides in length.
  • the overhang of the passenger nucleic acid strand can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length.
  • the overhang of the passenger nucleic acid strand is 9 nucleosides in length.
  • one or more intemucleoside linages of the overhang of the passenger nucleic acid strand are phosphorothioate intemucleoside linkage which can protect the overhang from degradation.
  • all intemucleoside linages of the overhang of the passenger nucleic acid strand can be phosphorothioate intemucleoside linkage.
  • the passenger nucleic acid strand is fully complementary o the first region of the core nucleic acid strand, thereby forming no overhang at the 5’ and 3’ termini of the passenger nucleic acid strand in the RNAi duplex. Therefore, in some embodiments, the passenger nucleic acid strand does not have a 3’ overhang, a 5’ overhang, or both in the RNAi duplex. In some embodiments, having a blunt end with no overhang can render the passenger nucleic acid strand unfavorable for Dicer binding, thereby bypassing the Dicer-mediated cleavage.
  • the passenger nucleic acid strand is attached to a terminal moiety and/or a blocking moiety.
  • Any suitable terminal moiety described herein that is capable of blocking the passenger nucleic acid strand from interacting with a RNAi pathway enzyme e.g. Dicer, RISC
  • the blocking moiety can include one or more suitable terminal linkers or modifications such as a blocker that can protect a single-stranded nucleic acid from nuclease degradation such as an exonuclease blocking moiety.
  • suitable blocking moieties include, but are not limited to, a dye (e.g.
  • a linker to link the oligonucleotide with another molecule or a particular surface biotins, amino-modifiers, alkynes, thiol modifiers, azide, N-Hydroxysuccinimide, and cholesterol
  • a space e.g. C3 spacer, Spacer 9, Spacer 18, dSpacer, tri-ethylene glycol spacer, hexa-ethylene glycol spacer
  • a fatty acid e.g. T -O-methyl, 2’-F, PS backbone connection, LNA, and/or T -4’ bridged base
  • modified nucleotides e.g. T -O-methyl, 2’-F, PS backbone connection, LNA, and/or T -4’ bridged base
  • the 5’ terminus of the passenger nucleic acid is attached to an inverted-dT, a tri-ethylene-glycol, or a fluorophore.
  • a fluorophore can be attached to the 5’ terminus of the passenger nucleic acid strand via a phosphorothioate linkage.
  • the sensor nucleic acid strand of the nucleic acid complex described herein comprises a region complementary bound to the second region of the core nucleic acid strand to form a sensor duplex (e.g. a second nucleic acid duplex).
  • the length of the region complementary bound to the second region of the core nucleic acid strand can vary in different embodiments.
  • the region complementary bound to the second region of the core nucleic acid strand comprises 10-35 linked nucleosides.
  • the region in the sensor nucleic acid strand complementary bound to the second region of the core nucleic acid strand can comprise 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 linked nucleosides.
  • the region in the sensor nucleic acid strand complementary bound to the second region of the core nucleic acid strand can comprise 10-30 linked nucleosides. In some embodiments, the region in the sensor nucleic acid strand complementary bound to the second region of the core nucleic acid strand comprise about 14 linked nucleosides.
  • the sensor nucleic acid strand doe not bind to the first region of the core nucleic acid strand nor any region 3’ of the first region of the core nucleic acid strand.
  • the sensor nucleic acid strand can comprise an overhang.
  • the overhang can be at the 3’ end or 5’ end, or both, of the sensor nucleic acid strand.
  • the overhang can be at the 3’ of the region complementary bound to the second region of the core nucleic acid strand.
  • the overhang is not complementary to the core nucleic acid strand and is capable of binding to an input nucleic acid strand, thereby initiating a toehold mediated strand displacement and causing the displacement of the sensor nucleic acid strand from the core nucleic acid strand.
  • the length of the overhang in the sensor nucleic acid strand can vary in different embodiments.
  • the length of the overhang can be 5-20 linked nucleotides.
  • the length of the overhang in the sensor nucleic acid strand can comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length.
  • the overhang of the sensor nucleic acid strand is 12 nucleotides in length.
  • the overhang of the sensor nucleic acid strand can comprise nucleotide modification introduced to improve the base-pairing affinity, nuclease resistance of the singled- stranded overhang, and thermodynamic stability to avoid spurious exonuclease induced activation of the strand.
  • exemplary modifications include, but not limited to, 2'-0-methyl modification, 2'-Fluoro modifications, phosphorothioate internucleoside linkages, inclusions of LNA, and the like that are identifiable by a skilled person.
  • at least 50% of the intemucleoside linkages in the overhang of the sensor nucleic acid strand are phosphorothioate intemucleoside linkages. For example, at least 50%, 51%, 52%, 53%, 54%,
  • intemucleoside linkages in the overhang of the sensor nucleic acid strand are phosphorothioate intemucleoside linkages.
  • all intemucleoside linkages in the overhang of the sensor nucleic acid strand are phosphorothioate intemucleoside linkages.
  • the 5’ terminus and/or the 3’ terminus of the sensor nucleic acid strand can comprise a terminal moiety.
  • Any suitable terminal moiety described herein can be used.
  • the terminal moiety can include a tri- or hexa-ethylene glycol spacer, a C3 spacer, an inverted dT, an amine linker, a ligand (e.g. a delivery ligand), a fluorophore, an exonuclease, a fatty acid, a Cy3, an inverted dT attached to a tri-ethylene glycol, or a combination thereof.
  • the 3’ terminus of the sensor nucleic acid strand can be attached to a delivery ligand, a dye (e.g. fluorophore), or exonuclease.
  • the 5’ terminus can be attached to a fatty acid, a dye (e.g. Cy3), an inverted dT, a tri-ethylene glycol, or an inverted dT attached to a tri-ethylene glycol.
  • the delivery ligand attached to the 3’ terminus can be any suitable ligand for use in targeting the nucleic acid complex to specific cell types described elsewhere in the present disclosure.
  • the delivery ligand is a palmitic acid.
  • the palmitic acid is attached to the 3’ terminus of the sensor nucleic acid strand.
  • a nucleic acid complex construct comprising a sensor nucleic acid strand with a palmitic acid attached to the 3’ terminus of the sensor nucleic acid can achieve a higher degree of inhibition of a target nucleic acid in comparison to corresponding constructs without a 3’ terminal palmitic acid.
  • the sequence of the sensor nucleic acid strand can be designed to sense an input nucleic acid strand or a portion thereof. For example, from the sequence of an input biomarker, a list of all possible sensor segments which are antisense to the input strand can be generated. The sensor sequences for uniqueness in the transcriptome of the target animal can be ranked using NCBI BLAST. For human cancer cell lines, sequences can be checked against human transcript and genomic collection using the BLASTn algorithm. In some embodiments, sensor segments that have more than 17 bases of sequence complementarity and complete overhang complementarity to known or predicted RNA transcripts may be eliminated. Examples of design features of the sensor nucleic acid strand that can be used in the nucleic acid complexes described herein are described, for example, in WO/2020/033938, the content of which is incorporated herein by reference.
  • the input nucleic acid strand described herein acts as a trigger to activate (switch on) the RNA interfering activity of the nucleic acid complex (e.g. RNAi duplex) upon binding to a sequence of the sensor nucleic acid in the nucleic acid complex.
  • the nucleic acid complex e.g. RNAi duplex
  • the input nucleic acid strand comprises a sequence complementary to a sequence in the sensor nucleic acid of the nucleic acid complex, such as, in the overhang region of the sensor nucleic acid.
  • the complementary binding between the input nucleic acid strand and the sensor nucleic acid strand e.g. an overhang
  • the input nucleic acid strand can be cellular RNA transcripts that are present at relatively high expression levels in a set of target cells (e.g. cancer cells) and at a relatively low level of expression in a set of non-target cells (e.g. normal cells).
  • target cells e.g. cancer cells
  • non-target cells e.g. normal cells
  • the nucleic acid complex herein described is activated (switched on) in target cells; whereas in the non-target cells, the nucleic acid complex remains inactivated (switched off).
  • the input nucleic acid strand can be expressed at a level of, about, at least, or at least about 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60- fold, 70-fold, 80-fold, 90-fold, or 100-fold higher than in the non-target cells.
  • the input nucleic acid strand can be expressed at a level of, about, at least, at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 transcripts.
  • the input nucleic acid strand is expressed at a level of less than 50, less than 40, less than 30, less than 20, or less than 10 transcripts.
  • the non-target cells have no detectable expression of the input nucleic acid strand.
  • the input nucleic acid strand can comprise an mRNA, an miRNA, or a non coding RNA such as a long non-coding RNA, an RNA fragment, or an RNA transcript of a virus.
  • the input nucleic acid strand is an RNA transcript that is expressed in a set of cells that are causing the progression of a disease and are therefore targeted for RNAi therapy.
  • the non-target cells are usually a set of cells where silencing of a target RNA can cause side effects that are not beneficial for therapy.
  • the nucleic acid complex can be designed such that the sensor nucleic acid strand comprises a sequence complementary to the input RNA sequence.
  • the input nucleic acid strand comprises a biomarker.
  • biomarker refers to a nucleic acid sequence (DNA or RNA) that is an indicator of a disease or disorder, a susceptibility to a disease or disorder, and/or of response to therapeutic or other intervention.
  • a biomarker can reflect an expression, function or regulation of a gene.
  • the input nucleic acid strand can comprise any disease biomarker known in the art.
  • the input nucleic acid strand is a mRNA, for example a cell type or cell state specific mRNA.
  • a cell type or cell-state specific mRNA include, but are not limited to, C3, GFAP, NPPA, CSF1R, SLC1A2, PLP1, and MBP mRNA.
  • the input nucleic acid is a microRNA (also known as miRNA), including but is not limited to, hsa-mir-23a-3p, hsa-mir-124-3p, and hsa-mir-29b-3p.
  • the input nucleic acid strand is a non-coding RNA, for example MALATl (metastasis associated lung adenocarcinoma transcript 1, also known as NEAT2 (noncoding nuclear-enriched abundant transcript 2).
  • the first region of the core nucleic acid strand can, for example, comprise a sequence complementary to a target RNA in order to direct target-specific RNA interference.
  • the target RNA is a cellular RNA transcript.
  • the target RNA can be an mRNA, an miRNA, a non-coding RNA, a viral RNA transcript, or a combination thereof.
  • a “target RNA” refers to a RNA whose expression is to be selectively inhibited or silenced through RNA interference.
  • a target RNA can be a target gene comprising any cellular gene or gene fragment whose expression or activity is associated with a disease, a disorder or a condition.
  • a target RNA can also be a foreign or exogenous RNA or RNA fragment whose expression or activity is associated with a disease, a disorder or a certain condition (e.g. a viral RNA transcript or a pro-viral gene).
  • the target RNA can comprise an oncogene, a cytokinin gene, an idiotype protein gene (Id protein gene), a prion gene, a gene that expresses a protein that induces angiogenesis, an adhesion molecule, a cell surface receptor, a gene of a protein involved in a metastasizing and/or invasive process, a gene of a proteinase, a gene of a protein that regulates apoptosis and the cell cycle, a gene that expresses the EGF receptor, a multi-drug resistance 1 gene (MDR1), a gene of a human papilloma virus, a hepatitis C virus, or a human immunodeficiency virus, a gene involved in cardiac hypertrophy, or a fragment thereof.
  • MDR1 multi-drug resistance 1 gene
  • the target RNA can comprise a gene encoding for a protein involved in apoptosis.
  • exemplary target RNA genes include, but are not limited to, bcl-2, p53, caspases, cytotoxic cytokines such as TNF-a or Fas ligand, and a number of other genes known in the art as capable of mediating apoptosis.
  • the target RNA can comprise a gene involved in cell growth.
  • Exemplary target RNA genes include, but not limited to, oncogenes (e.g., genes encoding for ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN, NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES), as well as genes encoding for tumor suppressor proteins (e g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53, and WTI).
  • oncogenes e.g., genes encoding for ABLI, BCLI, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA,
  • the target RNA can comprise a human major histocompatibility complex (MHC) gene or a fragment thereof.
  • MHC genes include MHC class I genes such as genes in the HLA-A, HLA-B or HLA-C subregions for class I cc chain genes, or b2- microglobulinand and MHC class II genes such as any of the genes of the DP, DQ and DR subregions of class II a chain and b chain genes (i.e. DPa, ⁇ Rb, DQa, ⁇ z)b, DRa, and DRb).
  • the target RNA can comprise a gene encoding for a pathogen-associated protein.
  • Pathogen associated protein include, but are not limited to, a viral protein involved in immunosuppression of the host, replication of the pathogen, transmission of the pathogen, or maintenance of the infection, or a host protein which facilitates entry of the pathogen into the host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of infection in the host, or assembly of the next generation of pathogen.
  • the pathogen can be a virus, such as a herpesvirus (e.g., herpes simplex, varicella- zoster virus, Epstein-Barr virus, cytomegalovirus (CMV)), hepatitis C, HIV, JC virus), a bacteria or a yeast.
  • a herpesvirus e.g., herpes simplex, varicella- zoster virus, Epstein-Barr virus, cytomegalovirus (CMV)), hepatitis C, HIV, JC virus
  • CMV cytomegalovirus
  • the target RNA can comprise a gene associated with a disease or a condition of the central nervous system (CNS).
  • CNS disease or a condition include, but are not limited to, APP, MAPT, SOD1, BACE1, CASP3, TGM2, NFE2L3, TARDBP, ADRBl, CAMK2A, CBLN1, CDK5R1, GABRA1, MAPKIO, NOS1, NPTX2, NRGN, NTS, PDCD2, PDE4D, PENK, SYT1, TTR, FUS, LRDD, CYBA, ATF3, ATF6, CASP2, CASP1, CASP7, CASP8, CASP9, HRK, C1QBP, BNIP3, MAPK8, MAPK14, Racl, GSK3B, P2RX7, TRPM2, PARG, CD38, STEAP4, BMP2, GJA1, TYROBP, CTGF, ANXA2, RHOA, DUOX1, RTP801, RTP801, RTP80
  • compositions and methods of administration are provided.
  • compositions comprising the nucleic acid complex as herein described, in combination with one or more compatible and pharmaceutically acceptable carriers.
  • nucleic acid complex herein described can be suitably formulated and introduced into cell environment by any means that allows for a sufficient portion of the constructs to enter the cells to induce gene silencing, if it occurs.
  • the nucleic acid complex can be admixed, encapsulated, conjugated, or associated with other molecules, molecule structures, mixtures of compounds or agent, or other formulations for assistance in uptake, distribution, and/or absorption during delivery.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases “pharmaceutically acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth: (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com 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
  • pharmaceutically acceptable carrier comprise a pharmaceutical acceptable salt.
  • a “pharmaceutical acceptable salt” includes a salt of an acid form of one of the components of the compositions herein described. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids.
  • pharmaceutically acceptable salts to be used with the nucleic acid complex herein described include but are not limited to (1) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (2) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (3) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalene disulfonic acid, polygal
  • nucleic acid complex herein described such as antibody conjugates, micelles, natural polysaccharides, peptides, synthetic cationic polymers, microparticles, lipid-based nanovectors among others.
  • Delivery systems and the related excipients used for delivery of the nucleic acid complex herein described can vary in different embodiments. Delivery systems can be selected based on the mode of administration utilized, types of formulations, target sites, and types of diseases or disorders to be treated to facilitate tissue penetration, cellular uptake and to prevent extravasation and endosomal escape.
  • the nucleic acid complex can be formulated with one or more polymers to form a supramolecular complex containing the nucleic acid complex and a multi-dimensional polymer network.
  • the polymer can be linear or branched.
  • the supramolecular complex can take any suitable form, and preferably, is in the form of particles.
  • the nucleic acid complex can be delivered via a lipid-mediated delivery system.
  • the nucleic acid complex can be encapsulated or associated with liposomes.
  • the nucleic acid complex can be condensed with a polycationic condensing agent, suspended in a low-ionic strength aqueous medium and cationic liposomes formed of a cationic vesicle-forming lipid.
  • liposomes refers to lipid vesicles having an outer lipid shell, typically formed on one or more lipid bilayers, encapsulating an aqueous interior.
  • the liposomes are cationic liposomes composed of between about 20-80 mole percent of a cationic vesicle-forming lipid, with the remaining neutral vesicle-forming lipids and/or other components.
  • vesicle-forming lipid refers to any amphipathic lipid having hydrophobic and polar head group moieties and which by itself can form spontaneously into bilayer vesicles in water (e.g. phospholipids).
  • a preferred vesicle forming lipid is a diacyl-chain lipid, such as a phospholipid, whose acyl chains are typically between about 14-22 carbon atoms in length, and have varying degrees of unsaturation.
  • a cationic vesicle-forming lipid is a vesicle-forming lipid whose polar head group with a net positive charge, at the operational pH, e.g., pH 4-9.
  • examples include phospholipids (e.g. phosphatidylethanolamine), glycolipids (e.g. cerebrosides and gangliosides having a cationic polar head-group), cholesterol amine and related cationic sterols (e.g.
  • DOTAP 1,2- diolelyloxy-3-(trimethylanuno) propane
  • DMRIE N-[l-(2,3,-ditetradecyloxy)propyl]-N,N- dimethyl-N-hydroxyethylammonium bromide
  • DORIE N-[l-(2,3,-dioleyloxy)propyl]-N,N- dimethyl-N-hydroxy ethylammonium bromide
  • DORIE N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
  • DC-Choi dimethyldioctadecylammonium
  • a neutral vesicle-forming lipid is a vesicle-forming lipid having no net charge or including a small percentage of lipids having a negative charge in the polar head group.
  • vesicle-forming lipids include phospholipids, such as phosphatidylcholine (PC), phosphatidyl ethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), and cholesterol, cholesterol derivatives, and other uncharged sterols.
  • the delivery systems used herein include, but are not limited to, nanoparticles (NPs), inorganic nanoparticles (e.g. slica NPs, gold NPs, Qdots, superparamagnetic iron oxide NPs, paramagnetic lanthanide ions) and other nanomaterials, nucleic acid lipid particles, polymeric nanoparticles, lipidoid nanoparticles (LNPs), chitosan and inulin nanoparticles, cyclodextrins nanoparticles, carbon nanotubes, liposomes, micellar structures, capsids, polymers (e.g.
  • nucleic acid complex can be formulated in buffer solutions such as phosphate buffered saline solutions.
  • the nucleic acid complex herein described is delivered via lipidoid nanoparticles (LNPs).
  • LNPs can comprise ionizable LNPs, cationic LNPs, and/or neutral LNPs.
  • Ionizable LNPs are nearly uncharged during circulation but become protonated in a low pH environment, e.g., in the endosomes and lysosomes.
  • Cationic LNPs exhibit a constitutive positive charge in blood circulation and in endosomes or lysosomes.
  • Neutral LNPs are neutral, uncharged during circulation and in endosomes or lysosomes.
  • the nucleic acid complex herein described can be provided naked or conjugated to a ligand.
  • Naked siRNA refer to a system that contains no delivery system that is associated with the siRNA either covalently or noncovalently.
  • the naked siRNAs can be locally injected to a target site such as specific organs that are relatively closed off and contain few nucleases (e.g. eye).
  • the nucleic acid complex herein described can be attached to (e.g. fused or conjugated) a ligand to form ligand-siRNA conjugates that can transport siRNA to desired tissues and cells by specific recognition and interactions between the ligand and the surface receptor of the cells or tissues.
  • ligand-siRNA conjugates can transport siRNA to desired tissues and cells by specific recognition and interactions between the ligand and the surface receptor of the cells or tissues.
  • Common targeting ligands include carbohydrate, aptamers, antibodies or antibody fragments, peptides (e.g. cell -penetrating peptides, endosomolytic peptides), and small molecules (e.g. N-Acetylgalactosamine (GalNAc)), and others as will be apparent to a skilled artisan.
  • the nucleic acid complex is conjugated to an aptamer.
  • aptamers refers to oligonucleotide or peptide molecules that bind a specific target with high affinity and specificity.
  • nucleic acid aptamers can comprise, for example, nucleic acid species that have been engineered through repeated rounds of in vitro selection or equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms.
  • Peptide aptamers are peptides that are designed to specifically bind to and interfere with protein-protein interactions inside cells.
  • peptide aptamers can be derived, for example, according to a selection strategy that is derived from the yeast two-hybrid (Y2H) system. Aptamers are useful in biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the antibodies.
  • the nucleic acid complex is conjugated to a small molecule.
  • small molecule indicates an organic compound that is of synthetic or biological origin and that, although may include monomers and/or primary metabolites, is not a polymer.
  • small molecules can comprise molecules that are not protein or nucleic acids, which play a biological role that is endogenous (e.g. inhibition or activation of a target) or exogenous (e.g. cell signaling), which are used as a tool in molecular biology, or which are suitable as drugs in medicine.
  • Small molecules can also have no relationship to natural biological molecules. Typically, small molecules have a molar mass lower than 1 kg/mol.
  • Exemplary small molecules include secondary metabolites (such as actinomicyn-D), certain antiviral drugs (such as amantadine and rimantadine), teratogens and carcinogens (such as phorbol 12-myristate 13-acetate), natural products (such as penicillin, morphine and paclitaxel) and additional molecules identifiable by a skilled artisan.
  • the nucleic acid complex herein described is conjugated to GalNAc.
  • ligands suitable for use in targeting the nucleic acid complex to specific cell types include, but are not limited to, folate capable of binding to folate receptor of epithelial carcinomas and bone marrow stem cells, water soluble vitamins capable of binding to vitamin receptors of various cells, pyridoxal phosphate capable of binding to CD4 of CD4 + lymphocytes, apolipoproteins capable of binding to LDL of liver hepatocytes and vascular endothelial cells, insulin capable of binding to insulin receptor, transferrin capable of binding to transferrin receptor of endothelial cells, galactose capable of binding to asialoglycoprotein receptor of liver hepatocytes, sialyl-Lewis x capable of binding to E, P selectin of activated endothelial cells, Mac-1 capable of binding to L selectin of neutrophils and leukocytes, VEGF capable of binding to Flk-1,2 of tumor epithelial cells, basic FGF capable of binding to FGF receptor of tumor
  • integrin of vascular endothelial cells PECAM-1/CD31 capable of binding to a v b3 integrin of vascular endothelial cells and activated platelets, osteopontin capable of binding to a v bi integrin and a v b integrin of endothelial cells and smooth muscle cells in atherosclerotic plaques, RGD sequences capable of binding to a v b 3 integrin of tumor endothelial cells and vascular smooth muscle cells, or HIV GP 120/41 or GP120 capable of binding to CD4 of CD4 + lymphocytes, and others identifiable to a skilled artisan.
  • the delivery of the nucleic acid complex herein described is such that at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the target cells incorporate the nucleic acid complex. In some embodiments, about 0.1-10 nm nucleic acid complex is delivered to the target cells.
  • the pharmaceutical compositions of the present disclosure may be specially fonnulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension: (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the hydrogel composition.
  • the pharmaceutical compositions can comprise one or more pharmaceutically-acceptable carriers.
  • Formulations useful in the methods of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the RNAi constructs which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1% to about 99% of active ingredient, preferably from about 5% to about 70%, most preferably from about 10% to about 30%.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a respiration uncoupling agent as an active ingredient.
  • a nucleic acid complex composition may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface- active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.
  • compositions of the present disclosure may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • Tablets, and other solid dosage forms may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, oils (in particular, cottonseed, groundnut, com, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emuls
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more respiration uncoupling agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of hydrogel compositions include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a respiration uncoupling agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and
  • compositions herein described comprise a therapeutically-effective amount of the nucleic acid complexes.
  • therapeutically-effective amount means that amount of nucleic acid complex disclosed herein which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.
  • the therapeutically- effective amount also varies depending on the structure of the constructs, the route of administration utilized, the target sites, and the specific diseases or disorders to be treated as will be understood to a person skilled in the art. For example, if a given clinical treatment is considered effective when there is at least a 20% reduction in a measurable parameter associated with a disease or disorder, a therapeutically-effective amount of the constructs for the treatment of that disease or disorder is the amount necessary to achieve at least a 20% reduction in that measurable parameter.
  • the pharmaceutical composition herein described comprises the nucleic acid complex in a suitable dosage sufficient to inhibit expression of the target gene in a subject (e.g. animal or human) being treated.
  • a suitable dosage of the nucleic acid complex is in the range of 0.001 to 0.25 milligrams per kilogram body weight of the subject per day, or in the range of 0.01 to 20 micrograms per kilogram body weight per day, or in the range of 0.01 to 10 micrograms per kilogram body weight per day, or in the range of 0.10 to 5 micrograms per kilogram body weight per day, or in the range of 0.1 to 2.5 micrograms per kilogram body weight per day.
  • the pharmaceutical compositions comprising the nucleic acid complex can be administered once daily, twice daily, three times daily or as needed or prescribed by a physician.
  • the pharmaceutical composition herein described can also be provided in dosage units comprising two, three, four, five, six or more sub-doses administered at appropriate intervals throughout the day.
  • the dosage unit can also be compounded for a single dose (e.g. using sustained or controlled release formulation) which can be sustainably released over several days in a controlled manner.
  • a suitable dosage unit of the pharmaceutical composition herein described can be estimated from data obtained from cell culture assays and further determined from data obtained in animal studies.
  • toxicity and therapeutic efficacy of the pharmaceutical compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compositions that exhibit large therapeutic indices are preferred.
  • Suitable dosages of the compositions in combination with particular delivery systems can be selected in order to minimize toxicity, such as to minimize potential damage to untargeted cells and to reduce side effects.
  • nucleic acid complexes herein described and compositions thereof can be administrated to a subject using any suitable administration routes.
  • the nucleic acid complexes and compositions thereof can be administered to a target site locally or systematically.
  • local administration or “topic administration” as used herein indicates any route of administration by which a composition is brought in contact with the body of the individual, so that the resulting composition location in the body is topic (limited to a specific tissue, organ or other body part where the imaging is desired).
  • exemplary local administration routes include injection into a particular tissue by a needle, gavage into the gastrointestinal tract, and spreading a solution containing hydrogel composition on a skin surface.
  • systemic administration indicates any route of administration by which a nucleic acid complex composition is brought in contact with the body of the individual, so that the resulting composition location in the body is systemic (i.e. non limited to a specific tissue, organ or other body part where the imaging is desired).
  • Systemic administration includes enteral and parenteral administration.
  • Enteral administration is a systemic route of administration where the substance is given via the digestive tract, and includes but is not limited to oral administration, administration by gastric feeding tube, administration by duodenal feeding tube, gastrostomy, enteral nutrition, and rectal administration.
  • Parenteral administration is a systemic route of administration where the substance is given by route other than the digestive tract and includes but is not limited to intravenous administration, intra-arterial administration, intramuscular administration, subcutaneous administration, intradermal, administration, intraperitoneal administration, and intravesical infusion.
  • the methods of administration can comprise aerosol delivery, nasal delivery, vaginal delivery, rectal delivery, buccal delivery, ocular delivery, local delivery, topical delivery, intraci sternal delivery, intraperitoneal delivery, oral delivery, intramuscular injection, intravenous (IV) injection, subcutaneous (SC) injection, intranodal injection, intratumoral injection, intraperitoneal injection, and/or intradermal injection, or any combination thereof.
  • the administration can also be site-specific injection (e.g. in the eye or the cerebral spinal fluid).
  • the administration can be Ex vivo transduction, cell injection, subcutaneous injection, intravenous injection, intrathecal delivery, intracerebroventricular injection, intradermal injection, intravitreal delivery, intratumoral delivery, or topical application (e.g. topical eye drop).
  • lipid formulations can be administered to animals such as by intravenous, intramuscular, or intraperitoneal injection, or orally or by inhalation or other methods as known in the art.
  • the administration can be SC injection into the adipose tissue below the epidermis and dermis.
  • SC administration can be associated with ligand-conjugated nucleic acid complex herein described.
  • SC administration can render a slower release rate of the drugs into the systemic circulation and an entering into the lymphatic system, giving more time for recycling of cellular receptors that mediate uptake.
  • SC administration can be faster and easier to administer, reducing treatment burden.
  • the administration can be any administration route allowing the penetration of drugs through the blood brain barrier.
  • the route of administration can be direct brain injection, transmembrane diffusion, or intraventricular infusion of therapeutic substances directly into the cerebrospinal fluid.
  • the administration can be intrastriatal injection, intrathecal injection, intracerebral injection, intraparenchymal injection, intranasal delivery or intracerebroventricular injection.
  • the administration can be intracerebroventricular injection into the CNS to bypass the blood-brain barrier and other mechanisms that limit drug distribution to the brain, allowing a higher drug concentration to enter the central compartment.
  • IV administration can be associated with nanoparticle and lipid nanoparticle formulated nucleic acid complex herein described. In some embodiments, IV administration can avoid first-pass metabolism in the liver and affords quick access to target tissue through the systemic circulation.
  • Target sites can be in vitro , in vivo or ex vivo.
  • Exemplary target sites can include cells grown in an in vitro culture, including, primary mammalian, cells, immortalized cell lines, tumor cells, stem cells, and the like.
  • Additional exemplary target sites include cells, tissues and organs in an ex vivo culture and cells, tissues, organs, or organs systems in vivo in a subject, for example, lungs, brain, kidney, liver, heart, the central nervous system, the peripheral nervous system, the gastrointestinal system, the circulatory system, the immune system, the skeletal system, the sensory system, within a body of an individual and additional environments identifiable by a skilled person.
  • the target site is the central nervous system (e.g., brain and spinal cord), peripheral nervous system (e.g., nerves that branch off from the spinal cord) and connective tissues/organs involving in the function and pathways between the central and peripheral nervous systems (e.g., dorsal root and ventral root).
  • the target site can include the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction and muscles.
  • the target site is the central nervous system.
  • the target site can comprise a site of disease or disorder or can be proximate to a site of a disease or disorder.
  • the location of the one or more sites of a disease or disorder can be predetermined.
  • the location of the one or more sites of a disease or disorder can be determined during the method (e.g., by an imaging-based method such as ultrasound or MRI).
  • the target site can comprise a tissue, such as, for example, adrenal gland tissue, appendix tissue, bladder tissue, bone, bowel tissue, brain tissue, breast tissue, bronchi, coronal tissue, ear tissue, esophagus tissue, eye tissue, gall bladder tissue, genital tissue, heart tissue, hypothalamus tissue, kidney tissue, large intestine tissue, intestinal tissue, larynx tissue, liver tissue, lung tissue, lymph nodes, mouth tissue, nose tissue, pancreatic tissue, parathyroid gland tissue, pituitary gland tissue, prostate tissue, rectal tissue, salivary gland tissue, skeletal muscle tissue, skin tissue, small intestine tissue, spinal cord, spleen tissue, stomach tissue, thymus gland tissue, trachea tissue, thyroid tissue, ureter tissue, urethra tissue, soft and connective tissue, peritoneal tissue, blood vessel tissue and/or fat tissue.
  • a tissue such as, for example, adrenal gland tissue, appendix tissue, bladder tissue, bone, bowel tissue, brain tissue, breast tissue,
  • the tissue can be inflamed tissue.
  • the tissue can comprise (i) grade I, grade II, grade III or grade IV cancerous tissue; (ii) metastatic cancerous tissue; (iii) mixed grade cancerous tissue; (iv) a sub-grade cancerous tissue; (v) healthy or normal tissue; and/or (vi) cancerous or abnormal tissue.
  • the nucleic acid complex and a composition thereof accumulates in vasculature of cancerous tissue.
  • the target site can comprise a solid tumor.
  • the target site can comprise a tissue, such as, for example, grey matter, white matter, ganglion, nerves, endoneurium, perineurium, epineurium.
  • target sites where the nucleic acid complex or compositions thereof can be administered can vary in different embodiments depending on the mode of administration utilized and the types of diseases or disordered to be treated.
  • the target sites can be related to ocular tissues, respiratory system, muscle, liver, central nerve system, solid tumors, hematopoietic system, skin, eye, placenta, bone, or other target sites in an individual as will be apparent to a skilled artisan.
  • the term “individual” or “subject” or “patient” as used herein in the context of imaging includes an animal and in particular higher animals and in particular vertebrates such as mammals and more particularly human beings.
  • the ratio of the concentration of the nucleic acid complex at the subject’s target site to the concentration of the nucleic acid complex outside the target site can vary. In some embodiments, the ratio of the concentration of the nucleic acid complex at the subject’s target site to the concentration of the nucleic acid complex outside the target site (e.g.
  • blood circulation, serum, or plasma can be, or be about, be at least, be at least about, be at most, or be at most about, 1:1, 1.1:1, 1.2:1, 1.3: 1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1,
  • the target site can comprise target cells.
  • the target cells can be tumor cells (e.g., solid tumor cells).
  • the administration of the nucleic acid complex and/or compositions herein described to a target site of the subject results in the death of at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or a range between any two of these values, of the target cells.
  • the ratio of target cell death to non-target cell death after administration of the nucleic acid complex and/or compositions can be at least about 2:1.
  • the ratio of target cell death to non-target cell death after administration of the nucleic acid complex and/or compositions can be, or be about, or be at least, or be at least about, or be at most, or be at most about, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4: 1, 1.5:1, 1.6:1, 1.7:1, 1.8: 1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1,
  • the target cells can include nerve cells and glial cells, including pyramidal cells, purkinje cells, granule cells, spindle neurons, nedium spiny neurons, intemeurons, astrocyte, ependymal cells, microglia, oligodendrocyte, and oligodendrocyte progenitor cells.
  • the target cells can also include dorsal root ganglion, ventral root ganglion, and automonic ganglion.
  • the administration of the nucleic acid complex and/or compositions herein described to a target site of the subject results in at least about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or a number or a range between any two of these values, reduction in the target nucleic acid expression in the target cells.
  • the ratio of reduction in the target nucleic acid in the target cells to non-target cell after administration of the nucleic acid complex and/or compositions can be at least about 2:1.
  • the ratio can be, or be about, or be at least, or be at least about, or be at most, or be at most about, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7: 1, 1.8:1, 1.9:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,
  • Also provided herein is a method of modulating a target RNA using the nucleic acid complex or a composition thereof herein described.
  • the method can comprise contacting a cell comprising a target RNA with the nucleic acid complex herein describe.
  • an input strand can bind to the overhang of the sensor nucleic acid strand to cause displacement of the sensor nucleic acid strand from the core nucleic acid strand to release the sequence complementary to the target RNA into the cell, thereby modulating the target RNA.
  • the cells can be cells grown in an in vitro culture, including, primary mammalian, cells, immortalized cell lines, tumor cells, stem cells, and the like.
  • the cells can comprise cells, tissues and organs in an ex vivo culture and cells, tissues, organs, or organs systems in vivo in a subject, for example, lungs, brain, kidney, liver, heart, the central nervous system, the peripheral nervous system, the gastrointestinal system, the circulatory system, the immune system, the skeletal system, the sensory system, within a body of an individual and additional environments identifiable by a skilled person.
  • the cell can be a disease cell or a cell of disorder.
  • the cell can be a cancer cell.
  • Contacting the cell with the nucleic acid complex can occur can also occur in vitro , ex vivo, or in vivo (e.g., in the body of a subject).
  • Also provided herein is a method of treating a disease or a condition using the nucleic acid complex or a composition thereof herein described.
  • the method can comprise administering the nucleic acid complex described herein to a subject in need thereof.
  • the input nucleic acid strand can bind to the overhang of the sensor nucleic acid strand to cause displacement of the sensor nucleic acid strand from the core nucleic acid strand to release the sequence complementary to a target RNA, thereby reducing the activity of the target RNA or protein expression from the target RNA in the subject to treat the disease or condition.
  • condition indicates a physical status of the body of an individual (as a whole or as one or more of its parts), that does not conform to a standard physical status associated with a state of complete physical, mental and social well-being for the individual.
  • Conditions herein described include but are not limited disorders and diseases wherein the term “disorder” indicates a condition of the living individual that is associated to a functional abnormality of the body or of any of its parts, and the term “disease” indicates a condition of the living individual that impairs normal functioning of the body or of any of its parts and is typically manifested by distinguishing signs and symptoms.
  • treatment refers to an intervention made in response to a disease, disorder or physiological condition manifested by a patient.
  • the aim of treatment may include, but is not limited to, one or more of the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and the remission of the disease, disorder or condition.
  • the term “treat” and “treatment” includes, for example, therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.
  • treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already affected by a disease or disorder or undesired physiological condition as well as those in which the disease or disorder or undesired physiological condition is to be prevented. As used herein, the term “prevention” refers to any activity that reduces the burden of the individual later expressing those symptoms.
  • tertiary prevention can take place at primary, secondary and/or tertiary prevention levels, wherein: a) primary prevention avoids the development of symptoms/disorder/condition; b) secondary prevention activities are aimed at early stages of the condition/disorder/symptom treatment, thereby increasing opportunities for interventions to prevent progression of the condition/disorder/symptom and emergence of symptoms; and c) tertiary prevention reduces the negative impact of an already established condition/disorder/symptom by, for example, restoring function and/or reducing any condition/disorder/symptom or related complications.
  • the term “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.
  • the target RNA can comprise any gene described herein or known in the art whose expression or activity is associated with a disease or disorder (e.g., a neurological disease or cancer).
  • a disease or disorder e.g., a neurological disease or cancer.
  • the neurological disease is Huntington’s disease and the target RNA comprises a HTT gene.
  • the sensor nucleic acid strand of the nucleic acid complex can be designed to detect any biomarker described herein or known in the pertinent art which is related to a disease or disorder such as a disease or disorder of the central nervous system or cancer.
  • the biomarker can be a universal mRNA that is not cell type specific or selective (e.g., mir-23a-3p).
  • the biomarker can also be a cell type or cell-state specific or selective mRNA such as mRNAs specific for cells of the central nervous system (e.g., GFAP mRNA).
  • the nucleic acid complex is administered to the subject in need thereof at a concentration about 0.001-10 nM.
  • the nucleic acid complex can be provided at a concentration of, about, at most, or at most about, 0.001 nM, 0.002 mM, 0.004 mM, 0.006 mM, 0.008 mM, 0.01 nM, 0.02 nM, 0.03 nM, 0.04 nM, 0.05 nM, 0.06 nM, 0.07 nM, 0.08 nM, 0.09 nM, 0.1 nM, 0.2 nM, 0.3 nM, 0.4 nM, 0.5 nM, 0.6 nM, 0.7 nM, 0.8 nM, 0.9 nM, 1.0 nM, 1.5 nM, 2.0 nM, 2.5 nM, 3.0 nM, 3.5 nM, 4.0 nM, 4.5 nM, 5.0
  • the nucleic acid complex is administered to the subject in need thereof at a dosage about 1-100 mg/kg body weight of the subject, preferably 10-50 mg/kg body weight of the subject.
  • dosages may be based and calculated based upon the subject being treated, the severity and responsiveness of the condition to be treated, the manner of administration, and the judgement of the prescribing physician, as understood by those of skill in the art.
  • the subject can be administered with the nucleic acid complex one, two, three, four or more times for the treatment. In some embodiments, at most one, two, three or four administrations are needed to achieve a desired treatment outcome. In some embodiments, only one administration is needed.
  • Two administrations of the nucleic acid complex can be separated by a suitable time period.
  • the suitable time period between two administrations can be the same as or different from the suitable time period between another two administrations.
  • the time period between two administrations can be about, at least or at least about 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or longer. In some embodiments, the time period between any two administrations can be at least 6 months.
  • Various diseases and disorders can be treated with any one or more of the nucleic acid complex compositions provided herein.
  • Diseases and disorders disclosed herein include, but are not limited to, HIV infection with lymphoma, hemophilia A, hemophilia B, hypercholesterolemia, atherosclerotic cardiovascular disease, renal impairment, chronic hepatitis B, acute intermittent porphyria, atypical hemolytic uraemic syndrome, primary hyperoxaluria, hereditary transthyretin amyloidosis (hATTR), al -antitrypsin deficiency liver disease, hepatitis B, sickle cell disease, primary hyperoxaluria, ewing sarcoma, advanced gynecological cancer, stage III/IV ovarian cancer, pancreatic cancer, advanced solid tumors, hepatocellular carcinoma/liver cancer, lymphoma and leukemias, heart disease, heart failure, keloids, hypertrophic cicatrix, relapsed or re
  • the disease or disorder can be a cancer.
  • the cancer can be a solid tumor, a liquid tumor, or a combination thereof.
  • the nucleic acid complex herein described or a composition thereof can be administered to the cells, tissues and/or organs comprising a tumor using any suitable administration route.
  • the nucleic acid complex or a composition thereof can be administered to the cells, tissues and/or organs comprising a tumor via subcutaneous injection or intratumoral delivery.
  • the cancer can be selected from the group consisting of colon cancer, rectal cancer, renal-cell carcinoma, liver cancer, non-small cell carcinoma of the lung, cancer of the small intestine, cancer of the esophagus, melanoma, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, non -Hodgkin lymphoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, solid tumors of childhood, cancer of the bladder, cancer of the kidney or
  • the cancer can be a hematologic cancer, for example, chronic lymphocytic leukemia (CLL), acute leukemias, acute lymphoid leukemia (ALL), B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (T-ALL), chronic myelogenous leukemia (CML), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitf s lymphoma, diffuse large B cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin's lymphom
  • Non-limiting examples of cancers that can be prevented and/or treated using the nucleic acid complexes and compositions disclosed herein include: renal cancer; kidney cancer; glioblastoma multiforme; metastatic breast cancer; breast carcinoma; breast sarcoma; neurofibroma; neurofibromatosis; pediatric tumors; neuroblastoma; malignant melanoma; carcinomas of the epidermis; leukemias such as but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myclodysplastic syndrome, chronic leukemias such as but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as but not limited to
  • the cancer is myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, or papillary adenocarcinomas.
  • the disease or disorder can be a neurological disease or disorder.
  • Neurological diseases or disorders are diseases or disorders of the central and peripheral nervous system including the brain, spinal cord, cranial nerves, peripheral nerves, nerve roots, autonomic nervous system, neuromuscular junction, and muscles.
  • Neurological disorders can include epilepsy, Alzheimer’s disease and other dementias, cerebrovascular diseases including stroke, migraine and other headache disorders, multiple sclerosis, Parkinson's disease, neuroinfections, brain tumors, and traumatic disorders of the nervous system due to head trauma.
  • a disease or a disorder is a neurodegenerative disease or disorder.
  • Neurodegenerative diseases or disorders are a heterogeneous group of disorders that are characterized by the progressive degeneration of the structure and function of the central nervous system or peripheral nervous system.
  • neurodegenerative diseases are diseases marked by continuous and progressive deterioration of the function of neural cells which are not caused by any underlying trauma or infection.
  • Exemplary neurodegenerative diseases or disorders include, but are not limited to, Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and prion diseases.
  • a disease or a disorder is a disease or condition of the central nervous system (CNS).
  • CNS central nervous system
  • exemplary disease or a condition of the CNS include, but are not limited to, Adrenoleukodystrophy, Alzheimer disease, Amyotrophic lateral sclerosis, Angelman syndrome, Ataxia telangiectasia, Charcot-Marie-Tooth syndrome, Cockayne syndrome, Deafness, Duchenne muscular dystrophy, Epilepsy, Essential tremor, Fragile X syndrome, Friedreich's ataxia, Gaucher disease, Huntington disease, Lesch-Nyhan syndrome, Maple syrup urine disease, Menkes syndrome, Myotonic dystrophy, Narcolepsy, Neurofibromatosis, Niemann-Pick disease, Parkinson disease, Phenylketonuria, Prader-Willi syndrome, Refsum disease, Rett syndrome, Spinal muscular atrophy, Spinocerebellar ataxia, Tangier disease, Tay- Sachs disease, Tuberous sclerosis,
  • the CNS disease is a movement disorder, a memory disorder, addiction, attention deficit/hyperactivity disorder (ADHD), autism, bipolar disorder, depression, encephalitis, epilepsy/seizure, migraine, multiple sclerosis, a neurodegenerative disorder, a psychiatric disease, a neuroinflammatory disease, Alzheimer’s disease, Huntington's disease, Parkinson's disease, Tourette syndrome, dystonia, or a combination thereof.
  • the disease is a neuroinflammatory disease.
  • the neuroinflammatory disease is Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, or a combination thereof.
  • the disease or disorder can be a central nervous system (CNS) or peripheral nervous system (PNS) disease or condition.
  • the nucleic acid complex herein described or a composition thereof can be administered to the cells, tissues and/or organs of the CNS and/or PNS using any suitable administration route.
  • the nucleic acid complex or a composition thereof can be administered to the cells, tissues and/or organs of the CNS and/or PNS of a subject via intrathecal injection, intracerebroventricular injection, or intracerebral injection to penetrate the blood-brain barrier.
  • the cell(s), tissue(s), and/or organ(s) of the CNS and/or PNS comprises damaged or inflamed cell(s), tissue(s), or organ(s).
  • the cells(s), tissue(s), and/or organ(s) of the CNS and/or PNS comprise the brain, the white matter, the gray matter, the brainstem, the cerebellum, the diencephalon, the cerebrum, the spinal cord, the cranial nerve, dorsal root ganglia, cell(s) of any of the preceding, tissue(s) of any of the preceding, or a combination thereof.
  • the method herein described comprises administering a nucleic acid complex herein described to a subject in need thereof, allowing the nucleic acid complex to be distributed into one or more regions of the nervous system, thereby reducing the activity of the target RNA or protein expression from the target RNA in the one or more regions of the nervous system of the subject to treat the neurological disease or disorder.
  • administration of the nucleic acid complex allows the distribution of the nucleic acid complex to the one or more regions of the nervous systems comprising a central nervous system, a peripheral nervous system, or both.
  • administration of the nucleic acid complex allows the distribution of the nucleic acid complex to one or more regions of the nervous system comprising connective tissues/organs involving in the function and pathways between the central and peripheral nervous systems.
  • the one or more regions of the nervous system comprises dorsal root ganglion that carries sensory neural signals to the CNS from the PNS.
  • administration of the nucleic acid complex allows the distribution of the nucleic acid complex to one or more regions of the central nervous system.
  • the one or more regions of the central nervous system can comprise the brain, the white matter, the gray matter, the brainstem, the cerebellum, the diencephalon, the cerebrum, the spinal cord, the cranial nerve, or a combination thereof.
  • the one or more regions of the central nervous system comprise spinal cord, cerebrum (e.g., frontal lobe, parietal lobe, occipital lobe, temporal lobe, left hemisphere, and right hemisphere), cerebral cortex (e.g., prefrontal cortex, sensory cortex, visual cortex, auditory cortex, motor cortex), basal ganglia (e.g., striatum), thalamus, subthalamus, epithalamus, hypothalamus, amygdala, hippocampus (e.g, ventral hippocampus, dorsal hippocampus, and intermediate hippocampus), cerebellum, brain stem (e.g., midbrain, pons, medulla oblongata), left hemisphere, right hemisphere, corpus callosum, or a combination thereof.
  • cerebrum e.g., frontal lobe, parietal lobe, occipital lob
  • the one or more regions of the CNS comprises cerebral cortex, subicular cortex, hippocampus, corpus callosum, fornix, lateral ventricle, stria terminalis, caudate putamen, internal capsule, piriform cortex, globus pallidus, optic tract, amygdala, anterior commissure, ventral striatum, lateral olfactory tract, cerebellum, pons, medulla, middle cerebellar peduncle, or a combination thereof.
  • the administration of the nucleic acid complex to a subject in need thereof results in reduction or loss of expression of the target nucleic acid in the target cells.
  • the reduction of the target nucleic acid after the administration of the nucleic acid complex herein described is about, at least, or at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
  • the reduction occurs in one or more of the regions selected from the group consisting of: right cortex, prefrontal cortex, sensory cortex, visual cortex, striatum, dorsal hippocampus, ventral hippocampus, thalamus, cerebellum, midbrain, left hemisphere, right hemisphere, spinal cord upper, spinal cord lower, dorsal root ganglia, or a combination thereof.
  • the nucleic acid complex described herein is specific and potent while achieving safety and tolerability in the subject being treated.
  • the administration of the nucleic acid complex does not result in a significant increase or decrease (e.g.. plus or minus 5% of a base value or the difference is not statistically significant) in the body weight inflammatory markers blood chemistry and/or liver kidney pancreas enzymes in the subject with respect to the levels prior to the administration.
  • the administration of the nucleic acid complex does not induce unintended inflammatory responses.
  • the administration the nucleic acid complex does not result an elevated glial fibrillary acidic protein and/or ionized calcium-binding adapter molecule 1 ( ⁇ BA- 1) level of the subject with respect to the levels prior to the administration.
  • Kits an elevated glial fibrillary acidic protein and/or ionized calcium-binding adapter molecule 1 ( ⁇ BA- 1) level of the subject with respect to the levels prior to the administration.
  • kits comprising one or more compositions described herein, in suitable packaging such as in a container, pack, or dispenser, and may further comprise written material that can include instructions for use, discussion of clinical studies, listing of side effects, and the like.
  • Such kits can also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider.
  • information can be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
  • a kit can comprise one or more unit doses described herein.
  • compositions can be in the form of kits of parts.
  • a kit of parts one or more components of the compositions disclosed herein are provided independent of one another (e.g., constructs, excipients, and/or diluents are provided as separate compositions) and are then employed (e.g., by a user) to generate the compositions.
  • This example describes performing RNAi activity of various nucleic acid complex constructs described herein.
  • RNAi siRNA constructs shown in FIG. 4 can be tested for RNAi activity.
  • the sensor strand of the constructs can be designed to sense an input nucleic acid, such as a NPPA gene sequence encoding atrial natriuretic peptide (ANP).
  • ANPPA atrial natriuretic peptide
  • CASi siRNA constructs can be assembled by thermally annealing the passenger strand, the core strand and the sensor strand in lx phosphate buffer saline.
  • the RNAi activities of the CASi siRNA constructs can be measured using dual luciferase assays.
  • CASi siRNA constructs can be co-transfected into HCT 116 cells with dual luciferase vectors carrying a calcineurin gene target sequence (PPP3A), using lipofectamine 2000. After 48 hours, cells can be lysed and assayed for knockdown of the target gene by comparing the luminescence value of Renilla luciferase that carries the target sequence to Firefly luciferase that can be used as a reference control. Examples of methods and procedures of assembling CASi siRNA constructs, cell transfection, and dual luciferase assays are described in, for example, international application WO/2020/033938, the content of which is incorporated herein by reference in its entirety. It is expected that the RNA complexes described herein have RNAi activities.
  • T1 conditionally activated siRNA T1 CASi
  • the T1 CASi consists of the sensor strand, core strand, and the passenger strand (FIG. 5A).
  • the sensor strand is complementary to the guide strand of mir23a-3p, a microRNA with high expression in the brain tissue.
  • the core strand has two domains. The first domain is complementary to the sensor strand to allow formation of the sensor duplex. The second domain is complementary to both rodent and primate Huntingtin gene mRNA.
  • the passenger strand is complementary to the second domain of the core strand and base-pairs with the core strand to form the siRNA.
  • a palmitic acid ligand can be added to the sensor strand to enhance delivery. In some T1 CASi constructs, the palmitic acid is attached to the 3’ end of the sensor strand.
  • Table 1 provides sequence diagrams of exemplary T1 CASi constructs and strands.
  • the chemical formulas of the mir23 sensor strand (8 nt with palmitic acid), HTT passenger strand, and mir23-HTT core strands are shown in FIG. 5B, 5C and 5D, respectively.
  • the constructs were assembled by thermal annealing. Strands were mixed in PBS buffer at 1 : 1 : 1.1 ratio of passenger : core : sensor, then heated above 75 °C, and then cooled to room temperature. Non-denaturing PAGE gel was used to compare the assembled construct (lane 2 in FIG. 5E) with the individual strands (lane 3 in FIG. 5E: core strand; lane 4 in FIG. 5E: passenger strand) and two-stranded sub-assemblies (lane 1 in FIG. 5E: RNAi duplex). Presence of a single band (lane 2) with slower migration than single strands (lanes 3 and 4) and duplexes (lane 1) indicates assembly of the correct construct.
  • the assembled constructs were concentrated to ⁇ 3 mM concentration in lx PBS, then delivered by intracerebral ventricular injection (ICV) into the right ventricle of wild type mice. Mice were sacrificed at 14, 30, or 90 days after injection. The brain was collected and dissected into nine regions: sensory cortex, visual cortex, prefrontal cortex, striatum, thalamus, dorsal hippocampus (hippocampus), ventral hippocampus, mesencephalon (midbrain), and cerebellum (FIG. 6). Tissue from each brain region was lysed and assayed for HTT mRNA expression by qPCR. Knockdown of HTT mRNA was determined by comparing mRNA levels in construct-treated mice with saline-treated mice.
  • FIG. 7 shows mRNA knockdown of the mir23-HTT construct with a 3’ terminal palmitic acid (bottom panel) and without a 3’ terminal palmitic acid (top panel) in different brain regions 14 days after injection.
  • the results demonstrate that mir23-HTT constructs with a 3’ terminal palmitic acid achieved a higher degree of HTT mRNA knockdown (lower amount of remaining HTT mRNA) across all brain regions compared to the mir23-HTT constructs without a 3’ terminal palmitic acid.
  • FIG. 8 depicts a diagram showing mRNA knockdown of mir23-HTT constructs having a standard 8 nucleotide toehold (with and without palmitic acid), an extended 12 nucleotide toehold, or 16 nucleotide toehold.
  • the data suggests toehold length and palmitic acid have different effects on mir23-HTT CASi activity in different brain regions.
  • CASi construct with a 8 nucleotide toehold and palmitic acid achieved overall best knockdown effects across the brain regions. Without the palmitic acid, increasing toehold length from 8 nt to 12 nt improved knockdown activity in cortex and hippocampus.
  • FIG. 9 shows mRNA knockdown of mir23-HTT construct (with a standard 8 nucleotide toehold and 3’ palmitic acid) at 14 days, 30 days, and 90 days after injection of a single 15 nmol dose (425 pg) by unilateral ICV in the right ventricle.
  • the data suggests a potent and durable knockdown by the mir23-HTT CASi constructs in all brain regions.
  • Statistically significant RNAi activity was observed in brain regions including prefrontal cortex, sensory cortex, visual cortex, striatum, dorsal hippocampus, ventral hippocampus, thalamus, midbrain, cerebellum, right hemisphere and left hemisphere regions of the brain.
  • FIG. 10 shows HTT mRNA knockdown of mir23-HTT construct in the spinal cord. The data demonstrates that about 80% target knockdown rate still remains in the spinal cord 30 or even 90 days after the injection.
  • FIG. 11 showing the HTT mRNA level in various brain regions 30 days after CASi administration.
  • 5 nM mir23-HTT CASi construct (8 nucleotide toehold, with palmitic acid) was administrated to the animals through a unilateral ICV injection.
  • 10 dorsal root ganglia (DRG) from mice were also collected and tested for HTT mRNA knockdown.
  • About 25% HTT mRNA knockdown was observed across the central nervous system and DRG. The results suggest that not only was statistically significant RNAi activity observed in the central nervous system, the CASi construct also effectively reached the peripheral nervous region from central administration and achieved comparable knockdown in the DRG compared with the central nervous system.
  • the mir23-HTT CASi construct (8 nucleotide toehold with palmitic acid) was also administered to mice via ICV injection at 15 nmol (425 pg) to test for CNS inflammation by measuring astrocyte (GFAP) and microglia (IBA-1) activation.
  • FIG. 12 shows measurement of GFAP mRNA and IBA-1 mRNA by qPCR in various brain regions of CASi treated animals (t) in comparison to saline treated animals (c) at 14 days after injection.
  • the mice treated with the CASi did not show elevated GFAP mRNA or IBA-1 mRNA compared with saline-treated mice, indicating that injection of the construct did not induce an unintended inflammatory response.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Epidemiology (AREA)
  • Psychiatry (AREA)
  • Hospice & Palliative Care (AREA)
  • Psychology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Saccharide Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Medicinal Preparation (AREA)
PCT/US2022/073430 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes Ceased WO2023283550A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CN202280060504.5A CN117916376A (zh) 2021-07-06 2022-07-05 信号可激活核酸复合物
CA3224950A CA3224950A1 (en) 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes
AU2022308843A AU2022308843A1 (en) 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes
KR1020247004152A KR20240095155A (ko) 2021-07-06 2022-07-05 신호 활성화가능한 핵산 복합체
IL309943A IL309943A (en) 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes
EP22838546.4A EP4367245A4 (en) 2021-07-06 2022-07-05 SIGNAL-ACTIVABILABLE NUCLEIC ACID COMPLEXES
JP2024500651A JP2024525647A (ja) 2021-07-06 2022-07-05 シグナルで活性化可能な核酸複合体
US18/575,487 US20250257353A1 (en) 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163218852P 2021-07-06 2021-07-06
US63/218,852 2021-07-06

Publications (1)

Publication Number Publication Date
WO2023283550A1 true WO2023283550A1 (en) 2023-01-12

Family

ID=84802044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2022/073430 Ceased WO2023283550A1 (en) 2021-07-06 2022-07-05 Signal activatable nucleic acid complexes

Country Status (9)

Country Link
US (1) US20250257353A1 (https=)
EP (1) EP4367245A4 (https=)
JP (1) JP2024525647A (https=)
KR (1) KR20240095155A (https=)
CN (1) CN117916376A (https=)
AU (1) AU2022308843A1 (https=)
CA (1) CA3224950A1 (https=)
IL (1) IL309943A (https=)
WO (1) WO2023283550A1 (https=)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116478410A (zh) * 2023-06-20 2023-07-25 觅投克(北京)生物医学技术有限公司 一种菊糖修饰的聚乙烯亚胺衍生物及其制备方法和应用
US11999954B2 (en) 2018-08-10 2024-06-04 Clty of Hope Programmable conditional SIRNAS and uses thereof
US12054711B2 (en) 2017-07-14 2024-08-06 City Of Hope Meta-stable oligonucleotides junctions for delivery of therapeutics
US12385041B2 (en) 2017-08-10 2025-08-12 City Of Hope Conditional-siRNAS and uses thereof in treating cardiac hypertrophy

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696285B1 (en) * 1998-08-25 2004-02-24 Lucent Technolgies Inc. Nanomachines fueled by nucleic acid strand exchange
US20100112556A1 (en) * 2008-11-03 2010-05-06 Sampson Jeffrey R Method for sample analysis using q probes
US20150004615A1 (en) * 2013-07-01 2015-01-01 California Institute Of Technology SMALL CONDITIONAL RNAs
US20160130581A1 (en) * 2011-06-23 2016-05-12 California Institute Of Technology Targeting domain and related signal activated molecular delivery

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9029524B2 (en) * 2007-12-10 2015-05-12 California Institute Of Technology Signal activated RNA interference
US8710199B2 (en) * 2010-06-23 2014-04-29 California Institute Of Technology Signal activated molecular delivery
US9725715B2 (en) * 2011-06-23 2017-08-08 California Institute Of Technology Signal activatable constructs and related components compositions methods and systems
WO2019033083A1 (en) * 2017-08-10 2019-02-14 City Of Hope CONDITIONAL RNAIDS AND THEIR USES IN THE TREATMENT OF ACUTE MYELOID LEUKEMIA
EP3665281A4 (en) * 2017-08-10 2021-05-05 City of Hope CONDITIONAL SIRNA AND ITS USE IN THE TREATMENT OF CARDIAC HYERTROPHY
CN113166750B (zh) * 2018-08-10 2025-02-18 希望之城 可编程的条件性sirna及其用途

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6696285B1 (en) * 1998-08-25 2004-02-24 Lucent Technolgies Inc. Nanomachines fueled by nucleic acid strand exchange
US20100112556A1 (en) * 2008-11-03 2010-05-06 Sampson Jeffrey R Method for sample analysis using q probes
US20160130581A1 (en) * 2011-06-23 2016-05-12 California Institute Of Technology Targeting domain and related signal activated molecular delivery
US20150004615A1 (en) * 2013-07-01 2015-01-01 California Institute Of Technology SMALL CONDITIONAL RNAs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4367245A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US12054711B2 (en) 2017-07-14 2024-08-06 City Of Hope Meta-stable oligonucleotides junctions for delivery of therapeutics
US12385041B2 (en) 2017-08-10 2025-08-12 City Of Hope Conditional-siRNAS and uses thereof in treating cardiac hypertrophy
US11999954B2 (en) 2018-08-10 2024-06-04 Clty of Hope Programmable conditional SIRNAS and uses thereof
CN116478410A (zh) * 2023-06-20 2023-07-25 觅投克(北京)生物医学技术有限公司 一种菊糖修饰的聚乙烯亚胺衍生物及其制备方法和应用
CN116478410B (zh) * 2023-06-20 2023-09-12 觅投克(北京)生物医学技术有限公司 一种菊糖修饰的聚乙烯亚胺衍生物及其制备方法和应用

Also Published As

Publication number Publication date
EP4367245A4 (en) 2025-07-02
IL309943A (en) 2024-03-01
CA3224950A1 (en) 2023-01-12
EP4367245A1 (en) 2024-05-15
JP2024525647A (ja) 2024-07-12
CN117916376A (zh) 2024-04-19
KR20240095155A (ko) 2024-06-25
AU2022308843A1 (en) 2024-02-01
US20250257353A1 (en) 2025-08-14

Similar Documents

Publication Publication Date Title
US20250257353A1 (en) Signal activatable nucleic acid complexes
US20250188453A1 (en) Conditionally activatable nucleic acid complexes
JP6453275B2 (ja) 非対称性干渉ｒｎａの組成物およびその使用
CN107980062B (zh) 用于靶向亨廷汀mRNA的寡核苷酸化合物
EP2801615B1 (en) High-efficiency nanoparticle-type double-helical oligo-rna structure and method for preparing same
JP2022528487A (ja) Ｃ９ｏｒｆ７２のオリゴヌクレオチドベースの調節
Khatri et al. In vivo delivery aspects of miRNA, shRNA and siRNA
WO2011109698A1 (en) Formulations and methods for targeted delivery to phagocyte cells
US20240409933A1 (en) Condition-activatable nucleic acid constructs and their uses for treating neurological diseases
KR102701681B1 (ko) 이중가닥 올리고뉴클레오티드 및 이를 포함하는 코로나바이러스감염증-19〔covid-19〕치료용 조성물
WO2023283548A1 (en) Signal activatable nucleic acid constructs with wobble base pairings
WO2023230465A1 (en) Antisense oligonucleotides
CN118414428A (zh) 条件可激活核酸构建体及其用于治疗神经疾病的用途
JP7208911B2 (ja) 核酸分子発現の調節
WO2023168202A2 (en) Certain dux4 inhibitors and methods of use thereof
JP2026511049A (ja) 治療用オリゴヌクレオチドの脳送達のための樹状コンジュゲート
Akaneya Silencing the Brain by RNA Interference: A Promising Approach
HK1148535A (en) Composition of asymmetric rna duplex as microrna mimetic or inhibitor

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22838546

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 3224950

Country of ref document: CA

Ref document number: 309943

Country of ref document: IL

ENP Entry into the national phase

Ref document number: 2024500651

Country of ref document: JP

Kind code of ref document: A

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112024000248

Country of ref document: BR

WWE Wipo information: entry into national phase

Ref document number: 2022308843

Country of ref document: AU

Ref document number: 807343

Country of ref document: NZ

Ref document number: AU2022308843

Country of ref document: AU

ENP Entry into the national phase

Ref document number: 2022308843

Country of ref document: AU

Date of ref document: 20220705

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 202427007831

Country of ref document: IN

Ref document number: 1020247004152

Country of ref document: KR

WWE Wipo information: entry into national phase

Ref document number: 2022838546

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022838546

Country of ref document: EP

Effective date: 20240206

WWE Wipo information: entry into national phase

Ref document number: 11202400015W

Country of ref document: SG

WWE Wipo information: entry into national phase

Ref document number: 202280060504.5

Country of ref document: CN

ENP Entry into the national phase

Ref document number: 112024000248

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20240105

WWP Wipo information: published in national office

Ref document number: 18575487

Country of ref document: US