WO2013103693A1 - Complexes polynucléotidiques à entaille pour une interférence par arn multivalente - Google Patents

Complexes polynucléotidiques à entaille pour une interférence par arn multivalente Download PDF

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WO2013103693A1
WO2013103693A1 PCT/US2013/020109 US2013020109W WO2013103693A1 WO 2013103693 A1 WO2013103693 A1 WO 2013103693A1 US 2013020109 W US2013020109 W US 2013020109W WO 2013103693 A1 WO2013103693 A1 WO 2013103693A1
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polynucleotide
mol
target
region
lipid
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PCT/US2013/020109
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Todd M. Hauser
Amy C.H. Lee
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Protiva Biotherapeutics Inc.
Halo-Bio Rnai Therapeutics, Inc.
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Priority to AU2013202315A priority Critical patent/AU2013202315A1/en
Publication of WO2013103693A1 publication Critical patent/WO2013103693A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • 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 N.A.
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/51—Physical structure in polymeric form, e.g. multimers, concatemers
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/52—Physical structure branched
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/533—Physical structure partially self-complementary or closed having a mismatch or nick in at least one of the strands
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/12—Applications; Uses in screening processes in functional genomics, i.e. for the determination of gene function

Definitions

  • the present invention relates generally to precisely structured polynucleotide molecules, and methods of using the same for multivalent RNA interference and the treatment of disease.
  • RNA interference RNA interference
  • Antisense strategies for gene silencing have attracted much attention in recent years.
  • the underlying concept is simple yet, in principle, effective: antisense nucleic acids (NA) base pair with a target RNA resulting in inactivation of the targeted RNA.
  • NA antisense nucleic acids
  • Target RNA recognition by antisense RNA or DNA can be considered a hybridization reaction. Since the target is bound through sequence complementarily, this implies that an appropriate choice of antisense NA should ensure high specificity. Inactivation of the targeted RNA can occur via different pathways, dependent on the nature of the antisense NA (either modified or unmodified DNA or RNA, or a hybrid thereof) and on the properties of the biological system in which inhibition is to occur.
  • RNAi based gene suppression is a widely accepted method in which a sense and an antisense RNA form double-stranded RNA (dsRNA), e.g., as a long RNA duplex, a 19-24 nucleotide duplex, or as a short-hairpin dsRNA duplex (shRNA), which is involved in gene modulation by involving enzyme and/or protein complex machinery.
  • dsRNA double-stranded RNA
  • shRNA duplex short-hairpin dsRNA duplex
  • the long RNA duplex and the shRNA duplex are pre-cursors that are processed into small interfering RNA (siRNA) by the endoribonuclease described as Dicer.
  • siRNA or directly introduced siRNA is believed to join the protein complex RISC for guidance to a complementary gene, which is cleaved by the RISC/siRNA complex.
  • RNAi DNA antisense oligonucleotides exhibit only short-term effectiveness and are usually toxic at the doses required; similarly, the use of antisense RNAs has also proved ineffective due to stability problems. Also, the siRNA used in RNAi has proven to result in significant off-target suppression due to either strand guiding cleaving complexes potential involvement in endogenous regulatory pathways. Various methods have been employed in attempts to improve antisense stability by reducing nuclease sensitivity and chemical modifications to siRNA have been utilized.
  • RNAi multivalence examples include modifying the normal phosphodiester backbone, e.g., using phosphorothioates or methyl phosphonates, incorporating 2'-OMe-nucleotides, using peptide nucleic acids (PNAs) and using 3 '-terminal caps, such as 3'-aminopropyl modifications or 3 '-3' terminal linkages.
  • PNAs peptide nucleic acids
  • 3 '-terminal caps such as 3'-aminopropyl modifications or 3 '-3' terminal linkages.
  • these methods can be expensive and require additional steps.
  • the use of non-naturally occurring nucleotides and modifications precludes the ability to express the antisense or siRNA sequences in vivo, thereby requiring them to be synthesized and administered afterwards.
  • the siRNA duplex exhibits primary efficacy to a single gene and off-target to a secondary gene. This unintended effect is negative and is not a reliable RNAi multival
  • the present invention provides novel compositions and methods, which include precisely structured oligonucleotides that are useful in specifically regulating gene expression of one or more genes simultaneously when the nucleotide target site sequence of each is not identical to the other.
  • the present invention provides isolated, three-stranded, polynucleotide complexes, capable of silencing gene expression by RNA interference, wherein one of the three strands includes a nick that divides the nicked strand into two shorter portions. The nick inactivates the nicked strand which is thereby prevented from interacting with an off-target RNA molecule.
  • the present invention includes an isolated precisely structured three- stranded polynucleotide complex comprising a region or regions having a sequence complementary to a target gene or sequence at multiple sites or complementary to multiple genes at single sites.
  • the isolated precisely structured three-stranded polynucleotide complex comprises one or more regions having a sequence complementary to one or more regions within a target gene or sequence.
  • the isolated precisely structured three-stranded polynucleotide complex comprises two or more, e.g., three, regions having sequences complementary to the same region within a target gene or sequence.
  • Certain embodiments relate to polynucleotide complexes of at least three separate polynucleotides, comprising (a) a first polynucleotide comprising a target-specific region that is complementary to a first target sequence, a 5' region, and a 3' region; (b) a second polynucleotide comprising a target-specific region that is complementary to a second target sequence, a 5 ' region, and a 3 ' region; and (c) a third polynucleotide comprising a null region or a target-specific region that is complementary to a third target specific, a 5 ' region, and a 3' region, wherein each of the target-specific regions of the first, second, and third polynucleotides are complementary to a different target sequence or wherein two or more of the target-specific regions of the first, second, and third polynucleotides are complementary to the same target sequence, wherein the 5' region of the first polynucleot
  • the first polynucleotide, or the second polynucleotide or the third polynucleotide includes a nick that divides the nicked polynucleotide into two shorter polynucleotide portions.
  • each of the shorter polynucleotide portions is from 5 to 12 nucleotides in length.
  • the nick is located within a target-specific region, although the nick can be located outside a target specific region.
  • the first, second, and/or third polynucleotide comprises about 8-30 or about 15-30 nucleotides. In certain embodiments, the first, second, and/or third polynucleotide comprises about 8-25 or about 17-25 nucleotides. In certain embodiments, one or more of the self-complementary regions comprises about 5-10 nucleotide pairs. In certain embodiments, one or more of the self-complementary regions comprises about 7-8 nucleotide pairs.
  • each of said first, second, and third target sequences are present in the same gene, cDNA, mRNA, or microR A. In certain embodiments, at least two of said first, second, and third target sequences are present in different genes, cDNAs, mRNAs, or microRNAs.
  • all or a portion of the 5' and/or 3' region of each polynucleotide is also complementary to the target sequence for that polynucleotide.
  • one or more of the self-complementary regions comprises a 3' overhang.
  • the present invention further includes a composition comprising a physiologically acceptable carrier and a polynucleotide of the present invention.
  • the present invention further includes a lipid-nucleic acid particle comprising a polynucleotide complex of the present invention encapsulated within a lipid particle.
  • the present invention further includes a pharmaceutical composition comprising a lipid- nucleic acid particle of the present invention and a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a method for reducing the expression of a gene, comprising introducing a polynucleotide complex of the present invention into a cell.
  • the cell is plant, animal, protozoan, viral, bacterial, or fungal.
  • the cell is mammalian.
  • the polynucleotide complex is introduced directly into the cell, while in other embodiments, the polynucleotide complex is introduced extracellularly by a means sufficient to deliver the isolated polynucleotide into the cell.
  • the present invention includes a method for treating a disease, comprising introducing a polynucleotide complex of the present invention into a cell, wherein overexpression of the targeted gene is associated with the disease.
  • the disease is a cancer.
  • the present invention further provides a method of treating an infection in a patient, comprising introducing into the patient a polynucleotide complex of the present invention, wherein the isolated polynucleotide mediates entry, replication, integration, transmission, or maintenance of an infective agent.
  • the present invention provides a method for identifying a function of a gene, comprising introducing into a cell a polynucleotide complex of the present invention, wherein the polynucleotide complex inhibits expression of the gene, and determining the effect of the introduction of the polynucleotide complex on a characteristic of the cell, thereby determining the function of the targeted gene.
  • the method is performed using high throughput screening.
  • Figure 1 shows a polynucleotide complex of three separate polynucleotide molecules.
  • A indicates the region comprising sequence complementary to a site on a target gene (hatched);
  • B indicates the region comprising sequence complementary to a second site on the target gene or a site on a different gene (cross-hatched);
  • C indicates the region comprising sequence complementary to a third site on the target gene or a site on a different gene (filled in black).
  • the numbers 1, 2, and 3 indicate the 3' end of each oligonucleotide that guides gene silencing;
  • A loads in the direction of 1, (B) in the direction 2, and
  • C in the direction 3.
  • the 3' and 5' regions of each molecule, which hybridize to each other to form their respective self-complementary or double-stranded regions, are indicated by connecting bars.
  • Each polynucleotide comprises a two nucleotide 3' overhang.
  • Figure 2 shows a polynucleotide complex of three separate polynucleotide molecules wherein one of the polynucleotide molecules is nicked.
  • A indicates the region comprising sequence complementary to a site on a target gene (hatched);
  • B indicates the region comprising sequence complementary to a second site on the target gene or a site on a different gene (cross-hatched);
  • C indicates the region comprising sequence complementary to a third site on the target gene or a site on a different gene (filled in black).
  • the numbers 1, 2, and 3 indicate the 3' end of each oligonucleotide that guides gene silencing;
  • A loads in the direction of 1, (B) in the direction 2, and
  • C in the direction 3.
  • Each polynucleotide comprises a two nucleotide 3' overhang.
  • the polynucleotide that includes region (C) includes a nick (R) that divides region (C) into a first portion (PI) and a second portion (P2).
  • Figure 3 depicts a polynucleotide complex of the present invention having modified RNA bases.
  • L), (M), and (N) illustrate regions (defined by hashed lines) in which the Tm can be incrementally increased by the use of modified RNA (e.g., 2'-0-methyl RNA or 2'-fluoro RNA instead of 2' -OH RNA) to preference the annealing and/or the silencing order of ends 1, 2 or 3.
  • modified RNA e.g., 2'-0-methyl RNA or 2'-fluoro RNA instead of 2' -OH RNA
  • the polynucleotide complex includes a nick (R).
  • Figure 4 depicts a polynucleotide complex of the present invention wherein (O) illustrates a blunt-ended DNA strand that deactivates the silencing function of this strand.
  • the polynucleotide complex includes a nick (R).
  • Figure 5 depicts a polynucleotide complex of the present invention wherein (P) illustrates an end that can be utilized for conjugation of a delivery chemistry, ligand, antibody, or other payload or targeting molecule.
  • the oligonucleotide complex includes a nick (R).
  • the present invention provides novel compositions and methods for inhibiting the expression of a gene at two target sites, or for inhibiting the expression of multiple genes at one or two target sites, which sites are not of equivalent nucleotide sequences, in eukaryotes in vivo and in vitro.
  • the present invention provides polynucleotide complexes comprising two, three, or more regions having sequences complementary to regions of one or more target genes, which are capable of targeting and reducing expression of the target genes.
  • the compositions and methods of the present invention may be used to inhibit the expression of a single target gene by targeting one site or multiple sites within the target gene or its expressed RNA. Alternatively, they may be used to target two or more different genes by targeting sites within two or more different genes or their expressed RNAs.
  • the present invention offers significant advantages over traditional siRNA molecules.
  • polynucleotide complexes of the present invention target two regions within a single target gene, they are capable of achieving greater inhibition of gene expression from the target gene, as compared to an RNAi agent that targets only one region within a target gene.
  • polynucleotide complexes of the present invention that target two different target genes may be used to inhibit the expression of multiple target genes associated with a disease or disorder using a single polynucleotide complex.
  • polynucleotide complexes of the present invention do not require the additional non-targeting strand present in conventional double-stranded RNAi agents, so they do not have off-target effects caused by the non-targeting strand. Further, the presence of a nick in one of the polynucleotide strands inactivates the nicked strand and prevents it from silencing an off-target gene. Accordingly, the polynucleotide complexes of the present invention offer surprising advantages over polynucleotide inhibitors of the prior art, including antisense RNA and RNA interference molecules, including increased potency and increased effectiveness against one or more target genes.
  • the present invention is also based upon the recognition of the polynucleotide structure guiding a protein complex for cleavage using only one, or two of the guide strands, which are complementary to one or two distinct nucleic sequences of the target genes.
  • This multivalent function results in a markedly broader and potent inhibition of a target gene or group of target genes than that of dsRNA, while utilizing many of the same endogenous mechanisms.
  • Certain embodiments of the present invention are also based upon the recognition of the polynucleotide structure directionally by presentation of the 3' overhangs and 5' phosphate resulting in a sense strand free complex, which contributes to greater specificity than that of dsRNA-based siRNA.
  • compositions of the present invention may be delivered to a cell or subject with an accompanying guarantee of specificity predicted by the single guide strand complementary to the target gene or multiple target genes.
  • the present invention includes polynucleotide complexes that comprise two or more targeting regions complementary to regions of one or more target genes (inclusive of coding and non-coding sequences), mRNAs, or microRNAs.
  • the polynucleotide complexes of the present invention may be referred to as multivalent RNAs (mv-RNAs), since they comprise at least two targeting regions complementary to one or more regions of one or more target genes.
  • mv-RNAs multivalent RNAs
  • the compositions and methods of the present invention may be used to inhibit or reduce expression of one or more target genes, either by targeting , one, two or more regions within a single target gene, or by targeting one or more regions within two or more target genes.
  • the polynucleotide complexes of the present invention include a polynucleotide strand that includes a nick, although it is also within the scope of the present invention for a polynucleotide strand to include more than one nick (e.g., 2, 3, 4, 5, 6, 7 or 8 nicks).
  • a nick refers to the absence of a covalent bond (e.g., a phosphodiester bond) linking adjacent subunits (e.g., nucleotides) within a polynucleotide strand.
  • a strand containing a single nick is composed of two adjacent portions, referred to as a first portion and a second portion, that are not directly, covalently, linked to each other, and that are held within the polynucleotide complex by non-covalent bonding (e.g., hydrogen bonding) to at least one other polynucleotide strand as described more fully herein.
  • the first and second portions each typically have a length of from 5 to 12 subunits (e.g., where the subunits are nucleotides).
  • the nick(s) inactivates the nicked strand so that the nicked strand cannot cause the R Ai-mediated suppression of a target gene.
  • polynucleotide complexes of the present invention comprise three or more separate oligonucleotides, each having a 5 ' and 3 ' end, with two or more of the oligonucleotides comprising a targeting region, which oligonucleotides hybridize to each other as described herein to form a complex.
  • Each of the strands is referred to herein as a "guide strand.”
  • Each guide strand comprises regions complementary to other guide strands.
  • Polynucleotide molecules of this aspect of the invention also include at least one nick.
  • the present invention provides polynucleotide complexes that comprise at least two guide strands which comprise regions that are complementary to different sequences within one or more target genes.
  • the polynucleotide complexes of the present invention comprise two, three or more separate polynucleotides each comprising one or more guide strands, which can hybridize to each other to form a complex.
  • Certain embodiments of the present invention are directed to polynucleotide complexes having at least three guide strands, two or more of which are partially or fully complementary to one or more target genes; and each having about 4 to about 12, about 5 to about 10, or preferably about 7 to about 8, nucleotides on either end that are complementary to each other (i.e., complementary to a region of another guide strand), allowing the formation of a polynucleotide complex (see, e.g., Figure 2).
  • each end of a guide strand may comprise nucleotides that are complementary to nucleotides at one end of another of the guide strands of the polynucleotide complex or molecule.
  • Certain embodiments may include polynucleotide complexes that comprise 4, 5, 6 or more individual polynucleotide molecules or guide strands.
  • a polynucleotide complex of the present invention comprises at least three separate polynucleotides, which include: (1) a first polynucleotide comprising a target- specific region that is complementary to a first target sequence, a 5' region, and a 3' region; (2) a second polynucleotide comprising a target-specific region that is complementary to a second target sequence, a 5' region, and a 3' region; and (3) a third polynucleotide comprising either a null region or a target-specific region that is complementary to a third target specific, a 5' region, and a 3' region, wherein each of the target-specific regions of the first, second, and third polynucleotides are complementary to a different target sequence or wherein two or more of the target-specific regions of the first, second, and third polynucleotides are complementary to the same target sequence, wherein the 5 ' region of the first polynucleotide is complementary
  • the first polynucleotide, the second polynucleotide or the third polynucleotide includes at least one nick (typically a single nick) that divides the nicked polynucleotide into at least two shorter polynucleotides (e.g., a first polynucleotide portion and a second polynucleotide portion).
  • a polynucleotide complex of the present invention comprises at least three separate oligonucleotides, each having a 5' end and a 3' end wherein one of the oligonucleotides is nicked.
  • Figure 1 depicts a polynucleotide complex that lacks a nick in one of the constituent oligonucleotides.
  • a region at the 5' end of the first oligonucleotide anneals to a region at the 3' end of the third oligonucleotide;
  • a region at the 5' end of the third oligonucleotide anneals to a region at the 3' end of the second oligonucleotide;
  • a region at the 5' end of the second oligonucleotide anneals to a region at the 3' end of the first oligonucleotide.
  • Each of the three oligonucleotides can target the same or different genes.
  • the ability to target up to three separate sequences on three separate genes is often a desirable property of trivalent mv-R A complexes, such as the complex shown in Figure 1
  • Such partially inactivated complexes are desirable, for example, where two functional targeting strands are sufficient to target one or two genes, and designing a third, inactive, strand presents significant problems, such as the difficulty of ensuring that the third strand does not interact with an off-target sequence thereby causing an undesirable inactivation of a gene that is essential for normal cellular function.
  • Figure 2 herein depicts the same complex that is depicted in Figure 1, except that the complex depicted in Figure 2 includes a nick (R) in region (C) of one strand, thereby dividing region (C) into a first portion (PI) and a second portion (P2).
  • the nick functionally inactivates the nicked strand which is no longer useful for targeting a gene or other nucleic acid molecule.
  • the nicked strand is highly unlikely to cause off-target gene silencing.
  • oligonucleotides are present in the complex shown in Figure 2, then they anneal to other oligonucleotides of the complex in a similar manner.
  • the regions at the ends of the oligonucleotides that anneal to each other may include the ultimate nucleotides at either or both the 5' and/or 3' ends. Where the regions of both the hybridizing 3' and 5' ends include the ultimate nucleotides of the oligonucleotides, the resulting double-stranded region is blunt- ended.
  • the region at the 3 ' end that anneals does not include the ultimate and/or penultimate nucleotides, resulting in a double-stranded region having a one or two nucleotide 3' overhang.
  • Figure 3 depicts a polynucleotide complex of the present invention having modified RNA bases.
  • L), (M), and (N) illustrate regions (defined by hashed lines) in which the Tm can be incrementally increased by the use of modified RNA (e.g., 2'-0-methyl RNA or 2'-fluoro RNA instead of 2'-OH RNA) to favor the annealing and/or the silencing order of ends 1 , 2 or 3.
  • the polynucleotide complex includes a nick (R).
  • Figure 4 depicts a polynucleotide complex of the present invention wherein (O) illustrates a blunt-ended DNA strand that deactivates the silencing function of this strand.
  • the polynucleotide complex includes a nick (R).
  • Figure 5 depicts a polynucleotide complex of the present invention wherein (P) illustrates an end that can be utilized for conjugation of a delivery chemistry, ligand, antibody, or other payload or targeting molecule.
  • the oligonucleotide complex includes a nick (R).
  • polynucleotides complexes of the present invention include isolated polynucleotides comprising three single-stranded regions, at least two of which are complementary to two or more target sequences, each target sequence located within one or more target genes, and comprising at least two or three self-complementary regions interconnecting the 5 ' or 3 ' ends of the single-stranded regions, by forming a double-stranded region, such as a stem-loop structure.
  • the polynucleotides may also be referred to herein as the oligonucleotides.
  • the polynucleotide complexes of the present invention comprise two or more regions of sequence complementary to a target gene.
  • these regions are complementary to the same target genes or genes, while in other embodiments, they are complementary to two or more different target genes or genes.
  • complementary refers to nucleotide sequences that are fully or partially complementary to each other, according to standard base pairing rules.
  • partially complementary refers to sequences that have less than full complementarity, but still have a sufficient number of complementary nucleotide pairs to support binding or hybridization within the stretch of nucleotides under physiological conditions.
  • the region of a guide strand complementary to a target gene may comprise one or more nucleotide mismatches as compared to the target gene.
  • the mismatched nucleotide(s) in the guide strand may be substituted with an unlocked (UNA) nucleic acid or a phosphoramidite nucleic acid (e.g. , rSpacer, Glen Research , Sterling, VA, USA), to allow base-pairing, e.g., Watson-Crick base pairing, of the mismatched nucleotide(s) to the target gene.
  • UNA unlocked nucleic acid
  • a phosphoramidite nucleic acid e.g., rSpacer, Glen Research , Sterling, VA, USA
  • self-complementary or “self-complementary region” may refer to a region of a first nucleotide molecule that binds to a region of a second or third nucleotide molecule to form a polynucleotide complex of the invention (i.e., an RNAi polynucleotide complex), wherein the complex is capable of RNAi interference activity against two or more target sites.
  • a polynucleotide complex of the invention i.e., an RNAi polynucleotide complex
  • a "self complementary region” comprises a "3' region” of a first polynucleotide molecule that is bound or hybridized to a "5' region” of a separate polynucleotide molecule, to form a polynucleotide complex.
  • These 3' and '5 regions are typically defined in relation to their respective target-specific region, in that the 5' regions are on the 5 ' end of the target-specific region and the 3 ' regions are on the 3 ' end of the target specific region.
  • one or both of these 3' and 5' regions not only hybridize to their corresponding 3' or 5' regions to form a self-complementary region, but may be designed to also contain full or partial complementarity their respective target sequence, thereby forming part of the target-specific region.
  • the target- specific region contains both a single-stranded region and self-complementary (i.e., double-stranded) region.
  • these "self-complementary regions” comprise about 5-12 nucleotide pairs, preferably 5-10 or 7-8 nucleotide pairs, including all integers in between.
  • each 3' region or 5' region comprises about 5-12 nucleotides, preferably 5-10 or 7-8 nucleotides, including all integers in between.
  • non-complementary indicates that in a particular stretch of nucleotides, there are no nucleotides within that align with a target to form A-T(U) or G-C hybridizations.
  • stretch of nucleotides indicates that in a stretch of nucleotides, there is at least one nucleotide pair that aligns with a target to form an A-T(U) or G-C hybridizations, but there are not a sufficient number of complementary nucleotide pairs to support binding within the stretch of nucleotides under physiological conditions.
  • isolated refers to a material that is at least partially free from components that normally accompany the material in the material's native state. Isolation connotes a degree of separation from an original source or surroundings. Isolated, as used herein, e.g., related to DNA, refers to a polynucleotide that is substantially away from other coding or non-coding sequences, and that the DNA molecule can contain large portions of unrelated coding DNA, such as large chromosomal fragments or other functional genes or polypeptide coding regions. Of course, this refers to the DNA molecule as originally isolated, and does not exclude genes or coding regions later added to the segment by the hand of man.
  • a polynucleotide complex of the present invention comprises RNA, DNA, or peptide nucleic acids, or a combination of any or all of these types of molecules.
  • a polynucleotide may comprise modified nucleic acids, or derivatives or analogs of nucleic acids.
  • nucleic acid modifications include, but are not limited to, biotin labeling, fluorescent labeling, amino modifiers introducing a primary amine into the polynucleotide, phosphate groups, deoxyuridine, halogenated nucleosides, phosphorothioates, 2'-0-Methyl RNA analogs, chimeric RNA analogs, wobble groups, universal bases, and deoxyinosine.
  • a "subunit" of a polynucleotide or oligonucleotide refers to one nucleotide (or nucleotide analog) unit.
  • the term may refer to the nucleotide unit with or without the attached intersubunit linkage, although, when referring to a "charged subunit", the charge typically resides within the intersubunit linkage ⁇ e.g., a phosphate or phosphorothioate linkage or a cationic linkage).
  • a given synthetic MV-RNA may utilize one or more different types of subunits and/or intersubunit linkages, mainly to alter its stability, Tm, RNase sensitivity, or other characteristics, as desired. For instance, certain embodiments may employ RNA subunits with one or more 2'-0-methyl RNA subunits.
  • the cyclic subunits of a polynucleotide or an oligonucleotide may be based on ribose or another pentose sugar or, in certain embodiments, alternate or modified groups.
  • modified oligonucleotide backbones include, without limitation, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5 ' linkages, 2 '-5 ' linked analogs of these, and those
  • PNAs peptide nucleic acids
  • LNAs locked nucleic acids
  • MOE 2'-methoxyethoxy oligonucleotides
  • the purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine.
  • bases such as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimel 15thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g. , 5-methylcytidine), 5-alkyluridines (e.g. , ribothymidine), 5-halouridine (e.g. , 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • modified bases in this aspect is meant nucleotide bases other than adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U), as illustrated above; such bases can be used at any position in the antisense molecule.
  • Ts and Us are interchangeable. For instance, with other antisense chemistries such as 2'-0-methyl antisense oligonucleotides that are more R A-like, the T bases may be shown as U.
  • PNAs peptide nucleic acid subunits.
  • PNAs are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone, consisting of N-(2-aminoethyl) glycine units to which pyrimidine or purine bases are attached.
  • PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm, Buchardt et al. 1993).
  • the backbone of PNAs is formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications (see structure below).
  • a backbone made entirely of PNAs is uncharged, resulting in PNA/DNA or PNA/RNA duplexes that exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases.
  • PNAs may be produced synthetically using any technique known in the art.
  • PNA is a DNA analog in which a polyamide backbone replaces the traditional phosphate ribose ring of DNA.
  • PNA is capable of sequence- specific binding in a helix form to DNA or RNA.
  • Characteristics of PNA include a high binding affinity to complementary DNA or RNA, a destabilizing effect caused by single-base mismatch, resistance to nucleases and proteases, hybridization with DNA or RNA independent of salt concentration and triplex formation with homopurine DNA.
  • PanageneTM has developed its proprietary Bts PNA monomers (Bts; benzothiazole-2-sulfonyl group) and proprietary oligomerisation process.
  • the PNA oligomerisation using Bts PNA monomers is composed of repetitive cycles of deprotection, coupling and capping.
  • Panagene's patents to this technology include US 6969766, US 7211668, US 7022851, US 7125994, US 7145006 and US 7179896.
  • Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.
  • LNAs locked nucleic acid subunits
  • the structures of LNAs are known in the art: for example, Wengel, et al., Chemical Communications (1998) 455; Tetrahedron (1998) 54, 3607, and Accounts of Chem. Research (1999) 32, 301); Obika, et al, Tetrahedron Letters (1997) 38, 8735; (1998) 39, 5401, and Bioorganic Medicinal Chemistry (2008)16, 9230.
  • Polynucleotides and oligonucleotides may incorporate one or more LNAs; in some cases, the compounds may be entirely composed of LNAs.
  • Methods for the synthesis of individual LNA nucleoside subunits and their incorporation into oligonucleotides are known in the art: U.S. Patents 7,572,582; 7,569,575; 7,084,125; 7,060,809; 7,053,207; 7,034,133; 6,794,499; and 6,670,461.
  • Typical intersubunit linkers include phosphodiester and phosphorothioate moieties; alternatively, non-phosphorous containing linkers may be employed.
  • One embodiment includes an LNA containing compound where each LNA subunit is separated by a R A or a DNA subunit (i.e., a deoxyribose nucleotide).
  • a DNA subunit i.e., a deoxyribose nucleotide.
  • Further exemplary compounds may be composed of alternating LNA and RNA or DNA subunits where the intersubunit linker is phosphorothioate.
  • Certain polynucleotides or oligonucleotides may comprise morpholino-based subunits bearing base-pairing moieties, joined by uncharged or substantially uncharged linkages.
  • morpholino oligomer or "PMO” (phosphoramidate- or phosphorodiamidate morpholino oligomer) refer to an oligonucleotide analog composed of morpholino subunit structures, where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged or cationic, joining the morpholino nitrogen of one subunit to a 5' exocyclic carbon of an adjacent subunit, and (ii) each morpholino ring bears a purine or pyrimidine or an equivalent base-pairing moiety effective to bind, by base specific hydrogen bonding, to a base in a polynucleotide.
  • the oxygen attached to phosphorus may be substituted with sulfur (thiophosphorodiamidate).
  • the 5' oxygen may be substituted with amino or lower alkyl substituted amino.
  • the pendant nitrogen attached to phosphorus may be unsubstituted, monosubstituted, or disubstituted with (optionally substituted) lower alkyl.
  • the purine or pyrimidine base pairing moiety is typically adenine, cytosine, guanine, uracil, thymine or inosine.
  • MV-RNA comprise at least one ligand tethered to an altered or non-natural nucleobase. Included are payload molecules and targeting molecules. A large number of compounds can function as the altered base. The structure of the altered base is important to the extent that the altered base should not substantially prevent binding of the oligonucleotide to its target, e.g., mRNA.
  • the altered base is difluorotolyl, nitropyrrolyl, nitroimidazolyl, nitroindolyl, napthalenyl, anthrancenyl, pyridinyl, quinolinyl, pyrenyl, or the divalent radical of any one of the non-natural nucleobases described herein.
  • the non-natural nucleobase is difluorotolyl, nitropyrrolyl, or nitroimidazolyl.
  • the non-natural nucleobase is difluorotolyl.
  • the ligand can be a steroid, bile acid, lipid, folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compound, polycyclic compound, crown ether, intercalator, cleaver molecule, protein-binding agent, or carbohydrate.
  • the ligand is a steroid or aromatic compound.
  • the ligand is cholesteryl.
  • the polynucleotide or oligonucleotide is tethered to a ligand for the purposes of improving cellular targeting and uptake.
  • a ligand for the purposes of improving cellular targeting and uptake.
  • an MV-RNA agent may be tethered to an antibody, or antigen binding fragment thereof.
  • an MV-RNA agent may be tethered to a specific ligand binding molecule, such as a polypeptide or polypeptide fragment that specifically binds a particular cell-surface receptor, or that more generally enhances cellular uptake, such as an arginine-rich peptide.
  • analog refers to a molecule, compound, or composition that retains the same structure and/or function (e.g., binding to a target) as a polynucleotide herein.
  • analogs include peptidomimetic and small and large organic or inorganic compounds.
  • an oligonucleotide of the present invention comprises a region that is complementary to a variant of a target gene sequence.
  • Polynucleotide complexes of the present invention comprise a sequence region, or two or more sequence regions, each of which is complementary, and in particular embodiments completely complementary, to a region of a target gene or polynucleotide sequences (or a variant thereof).
  • a target gene is a mammalian gene, e.g., a human gene, or a gene of a microorganism infecting a mammal, such as a virus.
  • a target gene is a therapeutic target.
  • a target gene may be a gene whose expression or overexpression is associated with a human disease or disorder. This may be a mutant gene or a wild type or normal gene.
  • Therapeutic target genes include, but are not limited to, oncogenes, growth factor genes, translocations associated with disease such as leukemias, inflammatory protein genes, transcription factor genes, growth factor receptor genes, anti- apoptotic genes, interleukins, sodium channel genes, potassium channel genes, such as, but not limited to the following genes or genes encoding the following proteins: apolipoprotein B (ApoB), apolipoprotein B-100 (ApoB-100), bcl family members, including bcl-2 and bcl-x, MLL-AF4, Huntington gene, AML-MT68 fusion gene, IK -B, Ahal, PCSK9, Eg5, transforming growth factor beta (TGFbeta), Navl .8, RhoA, HIF-lalpha, Nogo-L, Nogo-R, toll-like receptor 9 (ApoB), apolipoprotein B-100), bcl family members, including bcl-2 and bcl-
  • polynucleotide complexes of the present invention comprise guide strands or target-specific regions targeting two or more genes, e.g., two or more genes associated with a particular disease or disorder.
  • they may include guide strands complementary to interleukin-1 gene or mRNA and tumor necrosis factor gene or mRNA; complementary to interleukin-1 gene or mRNA and interleukin-12 gene or mRNA; or complementary to interleukin-1 gene or mRNA, interleukin-12 gene or mRNA and tumor necrosis factor gene or mRNA, for treatment of rheumatoid arthritis.
  • they include guide strands complementary to osteopontin gene or mRNA and TNF gene or mRNA.
  • therapeutic target genes include genes and mRNAs encoding viral proteins, such as human immunodeficiency virus (HIV) proteins, HTLV virus proteins, hepatitis C virus (HCV) proteins, Ebola virus proteins, JC virus proteins, herpes virus proteins, human polyoma virus proteins, influenza virus proteins, and Rous sarcoma virus proteins.
  • viral proteins such as human immunodeficiency virus (HIV) proteins, HTLV virus proteins, hepatitis C virus (HCV) proteins, Ebola virus proteins, JC virus proteins, herpes virus proteins, human polyoma virus proteins, influenza virus proteins, and Rous sarcoma virus proteins.
  • polynucleotide complexes of the present invention include guide strands complementary to two or more genes or mRNAs expressed by a particular virus, e.g., two or more HIV protein genes or two or more herpes virus protein genes.
  • they include guide strands having complementary to two or more herpes simplex virus genes or mR As, e.g., the UL29 gene or m NA and the Nectin-1 gene or mRNA of HSV-2, to reduce HSV-2 expression, replication or activity.
  • the polynucleotide complexes having regions targeting two or more HSV-2 genes or mRNAs are present in a formulation for topical delivery.
  • polynucleotide complexes of the present invention comprise one, two, three or more guide strands or target-specific regions that target an apolipoprotein B (ApoB) gene or mRNA, e.g., the human ApoB gene or mRNA or the mouse ApoB gene or mRNA.
  • ApoB apolipoprotein B
  • polynucleotide complexes of the present invention comprise one, two, three or more guide strands or regions that target HIV genes.
  • selection of a sequence region complementary to a target gene (or gene) is based upon analysis of the chosen target sequence and determination of secondary structure, T m , binding energy, and relative stability and cell specificity. Such sequences may be selected based upon their relative inability to form dimers, hairpins, or other secondary structures that would reduce structural integrity of the polynucleotide or prohibit specific binding to the target gene in a host cell.
  • Preferred target regions of the target gene or mRNA may include those regions at or near the AUG translation initiation codon and those sequences that are substantially complementary to 5 ' regions of the gene or mRNA.
  • These secondary structure analyses and target site selection considerations can be performed, for example, using v.4 of the OLIGO primer analysis software and/or the BLASTN 2.0.5 algorithm software (Altschul et ah, Nucleic Acids Res. 1997, 25(17):3389-402) or Oligoengine Workstation 2.0.
  • target sites are preferentially not located within the 5' and 3' untranslated regions (UTRs) or regions near the start codon (within approximately 75 bases), since proteins that bind regulatory regions may interfere with the binding of the polynucleotide.
  • potential target sites may be compared to an appropriate genome database, such as BLASTN 2.0.5, available on the NCBI server at www.ncbi.nlm, and potential target sequences with significant homology to other coding sequences eliminated.
  • the target sites are located within the 5 ' or 3 ' untranslated region (UTRs).
  • the self-complementary region of the polynucleotide may be composed of a particular sequence found in the gene of the target.
  • the target gene may be of any species, including, for example, plant, animal (e.g. mammalian), protozoan, viral (e.g., HIV), bacterial or fungal.
  • the target gene sequence and the complementary region of the polynucleotide may be complete complements of each other, or they may be less than completely complementary, as long as the strands hybridize to each other under physiological conditions.
  • the polynucleotide complexes of the present invention comprise at least one, two, or three regions complementary to one or more target genes.
  • the region complementary to a target gene is 15 to 17 to 24 nucleotides in length, including integer values within these ranges.
  • This region may be at least 16 nucleotides in length, at least 17 nucleotides in length, at least 20 nucleotides in length, at least 24 nucleotides in length, between 15 and 24 nucleotides in length, between 16 and 24 nucleotides in length, or between 17 and 24 nucleotides in length, inclusive of the end values, including any integer value within these ranges.
  • the level of inhibition of target gene expression is at least 90%, at least 95%, at least 98%>, and at least 99% or is almost 100%, and hence the cell or organism will in effect have the phenotype equivalent to a so- called "knock out" of a gene.
  • This method of knocking down gene expression can be used therapeutically or for research (e.g. , to generate models of disease states, to examine the function of a gene, to assess whether an agent acts on a gene, to validate targets for drug discovery).
  • the polynucleotide complexes of the invention can be used to target and reduce or inhibit expression of genes (inclusive of coding and non-coding sequences), cDNAs, mRNAs, or microRNAs. In particular embodiments, their guide strands or targeting regions bind to mRNAs or microRNAs. Targeted sequences may be present in genes, cDNAs, mRNAs, or microRNAs.
  • the invention further provides arrays of the polynucleotide of the invention, including microarrays. Microarrays are miniaturized devices typically with dimensions in the micrometer to millimeter range for performing chemical and biochemical reactions and are particularly suited for embodiments of the invention.
  • Arrays may be constructed via microelectronic and/or microfabrication using essentially any and all techniques known and available in the semiconductor industry and/or in the biochemistry industry, provided that such techniques are amenable to and compatible with the deposition and/or screening of polynucleotide sequences.
  • Microarrays of the invention are particularly desirable for high throughput analysis of multiple polynucleotides.
  • a microarray typically is constructed with discrete region or spots that comprise the polynucleotide of the present invention, each spot comprising one or more the polynucleotide, preferably at positionally addressable locations on the array surface.
  • Arrays of the invention may be prepared by any method available in the art. For example, the light-directed chemical synthesis process developed by Affymetrix (see, U.S. Pat. Nos. 5,445,934 and 5,856,174) may be used to synthesize biomolecules on chip surfaces by combining solid-phase photochemical synthesis with photolithographic fabrication techniques.
  • the chemical deposition approach developed by Incyte Pharmaceutical uses pre- synthesized cDNA probes for directed deposition onto chip surfaces (see, e.g., U.S. Pat. No. 5,874,554).
  • the three or more guide strands of a polynucleotide complex of the present invention may be individually chemically synthesized and annealed to produce the polynucleotide complex.
  • the polynucleotides of the invention may be used for a variety of purposes, all generally related to their ability to inhibit or reduce expression of one or more target genes. Accordingly, the invention provides methods of reducing expression of one or more target genes comprising introducing a polynucleotide complex of the present invention into a cell comprising said one or more target genes.
  • the polynucleotide complex comprises one or more guide strands that collectively target the one or more target genes.
  • a polynucleotide of the invention is introduced into a cell that contains a target gene or a homo log, variant or ortholog thereof, targeted by either one or two of the guide strands or targeting regions.
  • polynucleotides of the present invention may be used to reduce expression indirectly.
  • a polynucleotide complex of the present invention may be used to reduce expression of a transactivator that drives expression of a second gene (i.e., the target gene), thereby reducing expression of the second gene.
  • a polynucleotide may be used to increase expression indirectly.
  • a polynucleotide complex of the present invention may be used to reduce expression of a transcriptional repressor that inhibits expression of a second gene, thereby increasing expression of the second gene.
  • a target gene is a gene derived from the cell into which a polynucleotide is to be introduced, an endogenous gene, an exogenous gene, a transgene, or a gene of a pathogen that is present in the cell after transfection thereof.
  • the method of this invention may cause partial or complete inhibition of the expression of the target gene.
  • the cell containing the target gene may be derived from or contained in any organism (e.g., plant, animal, protozoan, virus, bacterium, or fungus).
  • target genes include genes, mR As, and microR As.
  • Inhibition of the expression of the target gene can be verified by means including, but not limited to, observing or detecting an absence or observable decrease in the level of protein encoded by a target gene, an absence or observable decrease in the level of a gene product expressed from a target gene (e.g., mRNAO, and/or a phenotype associated with expression of the gene, using techniques known to a person skilled in the field of the present invention.
  • a target gene e.g., mRNAO, and/or a phenotype associated with expression of the gene
  • Examples of cell characteristics that may be examined to determine the effect caused by introduction of a polynucleotide complex of the present invention include, cell growth, apoptosis, cell cycle characteristics, cellular differentiation, and morphology.
  • a polynucleotide complex of the present invention may be directly introduced to the cell (i.e., intracellular ly), or introduced extracellularly into a cavity or interstitial space of an organism, e.g., a mammal, into the circulation of an organism, introduced orally, introduced by bathing an organism in a solution containing the polynucleotide, or by some other means sufficient to deliver the polynucleotide into the cell.
  • a vector engineered to express a polynucleotide may be introduced into a cell, wherein the vector expresses the polynucleotide, thereby introducing it into the cell.
  • Methods of transferring an expression vector into a cell are widely known and available in the art, including, e.g., transfection, lipofection, scrape-loading, electroporation, microinjection, infection, gene gun, and retrotransposition.
  • a suitable method of introducing a vector into a cell is readily determined by one of skill in the art based upon the type of vector and the type of cell, and teachings widely available in the art.
  • Infective agents may be introduced by a variety of means readily available in the art, including, e.g., nasal inhalation.
  • target cells of the invention are primary cells, cell lines, immortalized cells, or transformed cells.
  • a target cell may be a somatic cell or a germ cell.
  • the target cell may be a non-dividing cell, such as a neuron, or it may be capable of proliferating in vitro in suitable cell culture conditions.
  • Target cells may be normal cells, or they may be diseased cells, including those containing a known genetic mutation.
  • Eukaryotic target cells of the invention include mammalian cells, such as, for example, a human cell, a murine cell, a rodent cell, and a primate cell.
  • a target cell of the invention is a stem cell, which includes, for example, an embryonic stem cell, such as a murine embryonic stem cell.
  • the polynucleotide complexes and methods of the present invention may be used to treat any of a wide variety of diseases or disorders, including, but not limited to, inflammatory diseases, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, reproductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, musculoskeletal diseases, neurological diseases, neuromuscular diseases, metabolic diseases, sexually transmitted diseases, skin and connective tissue diseases, urological diseases, and infections.
  • diseases or disorders including, but not limited to, inflammatory diseases, cardiovascular diseases, nervous system diseases, tumors, demyelinating diseases, digestive system diseases, endocrine system diseases, reproductive system diseases, hemic and lymphatic diseases, immunological diseases, mental disorders, musculoskeletal diseases, neurological diseases, neuromuscular diseases, metabolic diseases, sexually transmitted diseases, skin and connective tissue diseases, urological diseases, and infections.
  • the methods are practiced on an animal, in particular embodiments, a mammal, and in certain embodiments, a human.
  • the present invention includes methods of using a polynucleotide complex of the present invention for the treatment or prevention of a disease associated with gene deregulation, overexpression, or mutation.
  • a polynucleotide complex of the present invention may be introduced into a cancerous cell or tumor and thereby inhibit expression of a gene required for or associated with maintenance of the carcinogenic/tumorigenic phenotype.
  • a target gene may be selected that is, e.g., required for initiation or maintenance of a disease/pathology. Treatment may include amelioration of any symptom associated with the disease or clinical indication associated with the pathology.
  • the polynucleotides of the present invention are used to treat diseases or disorders associated with gene mutation.
  • a polynucleotide is used to modulate expression of a mutated gene or allele.
  • the mutated gene is a target of the polynucleotide complex, which will comprise a region complementary to a region of the mutated gene. This region may include the mutation, but it is not required, as another region of the gene may also be targeted, resulting in decreased expression of the mutant gene or gene. In certain embodiments, this region comprises the mutation, and, in related embodiments, the polynucleotide complex specifically inhibits expression of the mutant gene or gene but not the wild type gene or gene.
  • Such a polynucleotide is particularly useful in situations, e.g., where one allele is mutated but another is not. However, in other embodiments, this sequence would not necessarily comprise the mutation and may, therefore, comprise only wild-type sequence. Such a polynucleotide is particularly useful in situations, e.g., where all alleles are mutated.
  • a variety of diseases and disorders are known in the art to be associated with or caused by gene mutation, and the invention encompasses the treatment of any such disease or disorder with a the polynucleotide.
  • a gene of a pathogen is targeted for inhibition.
  • the gene could cause immunosuppression of the host directly or be essential for replication of the pathogen, transmission of the pathogen, or maintenance of the infection.
  • the target gene may be a pathogen gene or host gene responsible for entry of a pathogen into its host, drug metabolism by the pathogen or host, replication or integration of the pathogen's genome, establishment or spread of an infection in the host, or assembly of the next generation of pathogen.
  • cells at risk for infection by a pathogen or already infected cells may be targeted for treatment by introduction of a the polynucleotide according to the invention.
  • HIV human immunodeficiency virus
  • polynucleotide complexes of the present invention that target one or more HIV proteins are used to treat or inhibit HIV infection or acquired immune deficiency syndrome (AIDS).
  • the present invention is used for the treatment or development of treatments for cancers of any type.
  • tumors that can be treated using the methods described herein include, but are not limited to, neuroblastomas, myelomas, prostate cancers, small cell lung cancer, colon cancer, ovarian cancer, non-small cell lung cancer, brain tumors, breast cancer, leukemias, lymphomas, and others.
  • polynucleotide complexes of the present invention that target apolipoprotein B are used to treat, reduce, or inhibit atherosclerosis or heart disease.
  • ApoB is the primary apolipoprotein of low-density lipoproteins (LDLs), which is responsible for carrying cholesterol to tissues.
  • LDLs low-density lipoproteins
  • ApoB on the LDL particle acts as a ligand for LDL receptors, and high levels of ApoB can lead to plaques that cause vascular disease (atherosclerosis), leading to heart disease.
  • compositions of the invention may be administered to a patient in any of a number of ways, including parenteral, intravenous, systemic, local, topical, oral, intratumoral, intramuscular, subcutaneous, intraperitoneal, inhalation, or any such method of delivery.
  • the compositions are administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly.
  • the liposomal compositions are administered by intravenous infusion or intraperitoneally by a bolus injection.
  • the polynucleotide complexes of the invention can, for example, be delivered to a living subject ⁇ e.g., a human being) using lipid particles wherein the polynucleotide complexes are enclosed within the lipid particles.
  • the lipid particles thereby protect the polynucleotide complexes from chemical or enzymatic degradation, and also facilitate transport of the polynucleotide complexes to a tissue or organ of interest.
  • the lipid particles typically include one or more of the cationic (amino) lipids or salts thereof.
  • the lipid particles of the invention further comprise one or more non- cationic lipids.
  • the lipid particles further comprise one or more conjugated lipids capable of reducing or inhibiting particle aggregation.
  • Lipid particles include, but are not limited to, lipid vesicles such as liposomes.
  • a lipid vesicle includes a structure having lipid-containing membranes enclosing an aqueous interior.
  • lipid vesicles are used to encapsulate nucleic acids within the lipid vesicles.
  • lipid vesicles are complexed with nucleic acids to form lipoplexes.
  • the lipid particles used in the practice of the present invention typically comprise a polynucleotide complex of the present invention, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles.
  • the polynucleotide complex is fully encapsulated within the lipid portion of the lipid particle such that the polynucleotide complex in the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease or protease.
  • the lipid particles described herein are substantially non-toxic to mammals such as humans.
  • the lipid particles typically have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm.
  • the lipid particles of the invention also typically have a lipid:polynucleotide complex ratio (mass/mass ratio) of from about 1 : 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 2: 1 to about 25: 1, from about 3 : 1 to about 20: 1, from about 5 : 1 to about 15 : 1 , or from about 5 : 1 to about 10:1.
  • the lipid particles of the invention are serum-stable nucleic acid- lipid particles (LNP) which comprise a polynucleotide complex of the present invention, a cationic lipid, a non-cationic lipid ⁇ e.g., mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles ⁇ e.g., one or more PEG-lipid and/or POZ-lipid conjugates).
  • the LNP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified polynucleotide complex.
  • Nucleic acid- lipid particles and their method of preparation are described in, e.g., U.S. Patent Nos.
  • the nucleic acid may be fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation.
  • a LNP comprising a polynucleotide complex is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation.
  • the polynucleotide complex in the LNP is not substantially degraded after exposure of the particle to a nuclease at 37°C for at least about 20, 30, 45, or 60 minutes. In certain other instances, the polynucleotide complex in the LNP is not substantially degraded after incubation of the particle in serum at 37°C for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, the polynucleotide complex is complexed with the lipid portion of the particle.
  • the nucleic acid-lipid particle compositions are substantially nontoxic to mammals such as humans.
  • nucleic acid e.g., polynucleotide complex
  • nucleic acid-lipid particle is not significantly degraded after exposure to serum or a nuclease assay that would significantly degrade free DNA or RNA.
  • a fully encapsulated system preferably less than about 25% of the nucleic acid in the particle is degraded in a treatment that would normally degrade 100% of free nucleic acid, more preferably less than about 10%, and most preferably less than about 5% of the nucleic acid in the particle is degraded.
  • “Fully encapsulated” also indicates that the nucleic acid-lipid particles are serum- stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.
  • full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid.
  • Specific dyes such as OliGreen ® and RiboGreen ® (Invitrogen Corp.; Carlsbad, CA) are available for the quantitative determination of plasmid DNA, single-stranded deoxyribonucleotides, and/or single- or double-stranded ribonucleotides.
  • Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent.
  • the present invention provides a nucleic acid- lipid particle (e.g. , LNP) composition comprising a plurality of nucleic acid-lipid particles.
  • the LNP composition comprises nucleic acid (e.g., polynucleotide complex of the invention) that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%), from about 60%> to about 100%), from about 70%> to about 100%), from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70%) to about 95%, from about 80%> to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about
  • the LNP composition comprises nucleic acid (e.g., polynucleotide complex) that is fully encapsulated within the lipid portion of the particles, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%), from about 50% to about 95%, from about 60% to about 95%, from about 70% to about 95%), from about 80% to about 95%, from about 85% to about 95%, from about 90% to about 95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about 90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 9
  • the proportions of the components can be varied and the delivery efficiency of a particular formulation can be measured using, e.g., an endosomal release parameter (ERP) assay.
  • the present invention provides a lipid particle (e.g., LNP) composition comprising a plurality of lipid particles described herein and an antioxidant.
  • the antioxidant in the lipid particle composition reduces, prevents, and/or inhibits the degradation of a cationic lipid present in the lipid particle.
  • the antioxidant in the lipid particle composition reduces, prevents, and/or inhibits the degradation of the nucleic acid payload, e.g., by reducing, preventing, and/or inhibiting the formation of adducts between the nucleic acid and the cationic lipid.
  • antioxidants include hydrophilic antioxidants such as chelating agents (e.g., metal chelators such as ethylenediaminetetraacetic acid (EDTA), citrate, and the like), lipophilic antioxidants (e.g., vitamin E isomers, polyphenols, and the like), salts thereof; and mixtures thereof.
  • the antioxidant is typically present in an amount sufficient to prevent, inhibit, and/or reduce the degradation of the cationic lipid and/or active agent present in the particle, e.g., at least about 20 mM EDTA or a salt thereof, or at least about 100 mM citrate or a salt thereof.
  • An antioxidant such as EDTA and/or citrate may be included at any step or at multiple steps in the lipid particle formation process described in Section VI (e.g., prior to, during, and/or after lipid particle formation).
  • cationic lipids or salts thereof which may also be included in the lipid particles of the present invention include, but are not limited to, 1 ,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l ,2-di-Y-linolenyloxy-N,N-dimethylaminopropane ( ⁇ -DLenDMA), 1 ,2- dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA), 1 ,2-dilinoleoyloxy-(N,N- dimethyl)-butyl-4-amine (C2-DLinDAP), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]- dioxolane (DLin-K-C2-
  • CpLinDMA N,N-dimethyl-3,4-dioleyloxybenzylamine
  • DOcarbDAP 1,2-N,N'- dioleylcarbamyl-3-dimethylaminopropane
  • DODAC 1,2-N,N'-dilinoleylcarbamyl-3- dimethylaminopropane
  • DODAC N,N-dioleyl-N,N-dimethylammonium chloride
  • DODAC N,N-dioleyloxy-N,N-dimethylammonium chloride
  • DODMA l,2-dioleyloxy-N,N-dimethylaminopropane
  • DSDMA 1,2-distearyloxy-N,N- dimethylaminopropane
  • DSDMA N-(l-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • DDAB N,N-distearyl-N
  • Additional cationic lipids or salts thereof which may be present in the lipid particles described herein include novel cationic lipids such as CP-LenMC3, CP-y-LenMC3, CP-MC3, CP-DLen-C2K-DMA, CP-yDLen-C2K-DMA, CP-C2K-DMA, CP-DODMA, CP- DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-yDLenDMA, analogs thereof, and combinations thereof.
  • novel cationic lipids such as CP-LenMC3, CP-y-LenMC3, CP-MC3, CP-DLen-C2K-DMA, CP-yDLen-C2K-DMA, CP-C2K-DMA, CP-DODMA, CP- DPetroDMA, CP-DLinDMA, CP-DLenDMA, CP-yDLenDMA, analogs thereof,
  • Additional cationic lipids or salts thereof which may be present in the lipid particles described herein include MC3 analogs such as LenMC3, y-LenMC3, MC3MC, MC2C, MC2MC, MC3 Thioester, MC3 Ether, MC4 Ether, MC3 Alkyne, MC3 Amide, Pan- MC3, Pan-MC4, Pan-MC5, and combinations thereof.
  • Additional cationic lipids or salts thereof which may be present in the lipid particles described herein include the novel cationic lipids described in International Patent Application No. PCT/CA2010/001029, entitled “Improved Cationic Lipids and Methods for the Delivery of Nucleic Acids,” filed June 30, 2010.
  • Additional cationic lipids or salts thereof which may be present in the lipid particles described herein include the cationic lipids described in U.S. Patent Publication No. 20090023673. The disclosures of each of these patent documents are herein incorporated by reference in their entirety for all purposes.
  • the additional cationic lipid forms a salt (preferably a crystalline salt) with one or more anions.
  • the additional cationic lipid is the oxalate (e.g., hemioxalate) salt thereof, which is preferably a crystalline salt.
  • cationic lipids such as ⁇ -DLenDMA, C2-DLinDMA and C2-DLinDAP, as well as additional cationic lipids, is described in International Patent Application No. PCT/CA2010/001029, entitled “Improved Cationic Lipids and Methods for the Delivery of Nucleic Acids,” filed June 30, 2010, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DLin-K-DMA, as well as additional cationic lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA, DLin-K-C4- DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA, DLin-K- TMA.C1, DLin-K 2 -DMA, D-Lin-K-N-methylpiperzine, DLin-M-C2-DMA, DO-C-DAP, DMDAP, and DOTAP.C1, as well as additional cationic lipids, is described in PCT Publication No. WO 2010/042877, entitled "Improved Amino Lipids and Methods for the Delivery of Nucleic Acids," filed October 9, 2009, the disclosure of which is incorporated herein by reference in its entirety for all purposes.
  • cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, is described in PCT Publication No. WO 09/086558, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • cationic lipids can be used, such as, e.g., LIPOFECTIN ® (including DOTMA and DOPE, available from GIBCO/BRL); LIPOFECT AMINE ® (including DOSPA and DOPE, available from GIBCO/BRL); and TRANSFECTAM ® (including DOGS, available from Promega Corp.).
  • LIPOFECTIN ® including DOTMA and DOPE, available from GIBCO/BRL
  • LIPOFECT AMINE ® including DOSPA and DOPE, available from GIBCO/BRL
  • TRANSFECTAM ® including DOGS, available from Promega Corp.
  • the cationic lipid comprises from about 50 mol % to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol % to about 80 mol %, from about 50 mol % to about 75 mol %, from about 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, from about 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %, or from about 55 mol % to about 70 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cationic lipid comprises about 50 mol %, 51 mol %, 52 mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59 mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (or any fraction thereof) of the total lipid present in the particle.
  • the cationic lipid comprises from about 2 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol % to about 50 mol %, from about 20 mol % to about 50 mol %, from about 20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, or about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the percentage of cationic lipid present in the lipid particles of the invention is a target amount, and that the actual amount of cationic lipid present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the non-cationic lipids used in the lipid particles of the invention can be any of a variety of neutral uncharged, zwitterionic, or anionic lipids capable of producing a stable complex.
  • Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyl
  • acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.
  • non-cationic lipids include sterols such as cholesterol and derivatives thereof.
  • cholesterol derivatives include polar analogues such as 5 -cholestanol, 5P-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5 -cholestanone, 5P-cholestanone, and cholesteryl decanoate; and mixtures thereof.
  • the cholesterol derivative is a polar analogue such as cholesteryl-(4'-hydroxy)-butyl ether.
  • cholesteryl-(2'-hydroxy)-ethyl ether is described in PCT Publication No. WO 09/127060, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the non-cationic lipid present in the lipid particles ⁇ e.g., LNP comprises or consists of a mixture of one or more phospholipids and cholesterol or a derivative thereof.
  • the non-cationic lipid present in the lipid particles ⁇ e.g., LNP comprises or consists of one or more phospholipids, e.g., a cholesterol-free lipid particle formulation.
  • the non-cationic lipid present in the lipid particles ⁇ e.g., LNP) comprises or consists of cholesterol or a derivative thereof, e.g., a phospholipid- free lipid particle formulation.
  • non-cationic lipids suitable for use in the present invention include nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
  • nonphosphorous containing lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate,
  • the non-cationic lipid comprises from about 10 mol % to about 60 mol %, from about 20 mol % to about 55 mol %, from about 20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, from about 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %, from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 35 mol % to about 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %, 43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or range therein
  • the lipid particles contain a mixture of phospholipid and cholesterol or a cholesterol derivative
  • the mixture may comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the phospholipid component in the mixture may comprise from about 2 mol % to about 20 mol %, from about 2 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol % to about 15 mol %, or from about 4 mol % to about 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the phospholipid component in the mixture comprises from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 27 mol % to about 37 mol %, from about 25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol component in the mixture comprises from about 25 mol % to about 35 mol %, from about 27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, from about 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %, from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • the cholesterol or derivative thereof may comprise up to about 25 mol %, 30 mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % of the total lipid present in the particle.
  • the cholesterol or derivative thereof in the phospholipid-free lipid particle formulation may comprise from about 25 mol % to about 45 mol %, from about 25 mol % to about 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 31 mol % to about 39 mol %, from about 32 mol % to about 38 mol %, from about 33 mol % to about 37 mol %, from about 35 mol % to about 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34 mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (or any fraction thereof or range therein) of the total mol %,
  • the non-cationic lipid comprises from about 5 mol % to about 90 mol %, from about 10 mol % to about 85 mol %, from about 20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), or about 60 mol % (e.g., phospholipid and cholesterol or derivative thereof) (or any fraction thereof or range therein) of the total lipid present in the particle.
  • non-cationic lipids suitable for use in the lipid particles of the present invention are described, for example, in PCT Publication No. WO 09/127060, the disclosure of which is herein incorporated by reference in its entirety for all purposes. It should be understood that the percentage of non-cationic lipid present in the lipid particles of the invention is a target amount, and that the actual amount of non-cationic lipid present in the formulation may vary, for example, by ⁇ 5 mol %.
  • the lipid particles of the invention may further comprise a lipid conjugate.
  • the conjugated lipid is useful in that it prevents the aggregation of particles.
  • Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates, cationic-polymer-lipid conjugates (CPLs), and mixtures thereof.
  • the particles comprise either a PEG- lipid conjugate or an ATTA-lipid conjugate together with a CPL.
  • the lipid conjugate is a PEG-lipid.
  • PEG-lipids include, but are not limited to, PEG coupled to dialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No. WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in, e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEG coupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG conjugated to ceramides as described in, e.g., U.S. Patent No. 5,885,613, PEG conjugated to cholesterol or a derivative thereof, and mixtures thereof.
  • PEG-DAA dialkyloxypropyls
  • PEG-DAG diacylglycerol
  • PEG-PE PEG coupled to phospholipids
  • PEG conjugated to ceramides as described in, e.g., U.S. Patent No. 5,885,613,
  • PEG-lipids suitable for use in the invention include, without limitation, mPEG2000-l,2-di-O-alkyl-s/?3- carbomoylglyceride (PEG-C-DOMG).
  • PEG-C-DOMG mPEG2000-l,2-di-O-alkyl-s/?3- carbomoylglyceride
  • PEG-lipid conjugates include, without limitation, 1 - [8 ' -( 1 ,2-dimyristoyl-3 -propanoxy)-carboxamido-3 ' ,6 ' -dioxaoctanyl] carbamoyl- o methyl-poly(ethylene glycol) (2KPEG-DMG).
  • 2KPEG-DMG 1 - [8 ' -( 1 ,2-dimyristoyl-3 -propanoxy)-carboxamido-3 ' ,6 ' -dioxaoctanyl] carbamoyl- o methyl-poly(ethylene glycol)
  • PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co.
  • MePEG-OH monomethoxypolyethylene glycol
  • MePEG-S monomethoxypolyethylene glycol- succinate
  • MePEG-S- NHS monomethoxypolyethylene glycol-succinimidyl succinate
  • MePEG-NH 2 monomethoxypolyethylene glycol-amine
  • MePEG-TRES monomethoxypolyethylene glycol-tresylate
  • MePEG-IM monomethoxypolyethylene glycol-imidazolyl-carbonyl
  • PEGs such as those described in U.S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present invention.
  • mPEG (20 KDa) amine e.g., mPEG (20 KDa) amine
  • monomethoxypolyethylenegly col-acetic acid MePEG-CH 2 COOH
  • PEG-DAA conjugates e.g., PEG-DAA conjugates.
  • the PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.
  • the PEG can be optionally substituted by an alkyl, alkoxy, acyl, or aryl group.
  • the PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety.
  • Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties.
  • the linker moiety is a non-ester containing linker moiety.
  • non-ester containing linker moiety refers to a linker moiety that does not contain a carboxylic ester bond (-OC(O)-).
  • Suitable non-ester containing linker moieties include, but are not limited to, amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulphide (-S-S-), ether (-0-), succinyl (- (0)CCH 2 CH 2 C(0)-), succinamidyl (-NHC(0)CH 2 CH 2 C(0)NH-), ether, disulphide, as well as combinations thereof (such as a linker containing both a carbamate linker moiety and an amido linker moiety).
  • a carbamate linker is used to couple the PEG to the lipid.
  • an ester containing linker moiety is used to couple the PEG to the lipid.
  • Suitable ester containing linker moieties include, e.g., carbonate (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), sulfonate esters, and combinations thereof.
  • Phosphatidylethanolamines having a variety of acyl chain groups of varying chain lengths and degrees of saturation can be conjugated to PEG to form the lipid conjugate.
  • Such phosphatidylethanolamines are commercially available, or can be isolated or synthesized using conventional techniques known to those of skilled in the art.
  • Phosphatidylethanolamines containing saturated or unsaturated fatty acids with carbon chain lengths in the range of C 10 to C 2 o are preferred.
  • Phosphatidylethanolamines with mono- or diunsaturated fatty acids and mixtures of saturated and unsaturated fatty acids can also be used.
  • Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl- phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).
  • DMPE dimyristoyl- phosphatidylethanolamine
  • DPPE dipalmitoyl-phosphatidylethanolamine
  • DOPE dioleoylphosphatidylethanolamine
  • DSPE distearoyl-phosphatidylethanolamine
  • the term “ATTA” or "polyamide” includes, without limitation, compounds described in U.S. Patent Nos. 6,320,017 and 6,586,559, the disclosures of which are herein incorporated by reference in their entirety for all purposes. These compounds include a compound having the formula:
  • R is a member selected from the group consisting of hydrogen, alkyl and acyl
  • R 1 is a member selected from the group consisting of hydrogen and alkyl; or optionally, R and R 1 and the nitrogen to which they are bound form an azido moiety
  • R 2 is a member of the group selected from hydrogen, optionally substituted alkyl, optionally substituted aryl and a side chain of an amino acid
  • R 3 is a member selected from the group consisting of hydrogen, halogen, hydroxy, alkoxy, mercapto, hydrazino, amino and NR 4 R 5 , wherein R 4 and R 5 are independently hydrogen or alkyl
  • n is 4 to 80
  • m is 2 to 6
  • p is 1 to 4
  • q is 0 or 1.
  • diacylglycerol or “DAG” includes a compound having 2 fatty acyl chains, R 1 and R 2 , both of which have independently between 2 and 30 carbons bonded to the 1- and 2- position of glycerol by ester linkages.
  • the acyl groups can be saturated or have varying degrees of unsaturation. Suitable acyl groups include, but are not limited to, lauroyl (C 12 ), myristoyl (C14), palmitoyl (Ci 6 ), stearoyl (Cig), and icosoyl (C20).
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristoyl ⁇ i.e., dimyristoyl), R 1 and R 2 are both stearoyl ⁇ i.e., distearoyl), etc.
  • Diacylglycerols have the following general formula:
  • dialkyloxypropyl includes a compound having 2 alkyl chains, R 1 and R 2 , both of which have independently between 2 and 30 carbons.
  • the alkyl groups can be saturated or have varying degrees of unsaturation.
  • Dialkyloxypropyls have the following general formula:
  • the PEG-lipid is a PEG-DAA conjugate having the following formula:
  • R 1 and R 2 are independently selected and are long-chain alkyl groups having from about 10 to about 22 carbon atoms; PEG is a polyethyleneglycol; and L is a non-ester containing linker moiety or an ester containing linker moiety as described above.
  • the long- chain alkyl groups can be saturated or unsaturated. Suitable alkyl groups include, but are not limited to, decyl (Cio), lauryl (C12), myristyl (C14), palmityl (Ci 6 ), stearyl (Cig), and icosyl (C 20 ).
  • R 1 and R 2 are the same, i.e., R 1 and R 2 are both myristyl ⁇ i.e., dimyristyl), R 1 and R 2 are both stearyl (i.e., distearyl), etc.
  • the PEG has an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG has an average molecular weight of from about 750 daltons to about 5,000 daltons ⁇ e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG has an average molecular weight of about 2,000 daltons or about 750 daltons.
  • the PEG can be optionally substituted with alkyl, alkoxy, acyl, or aryl groups.
  • the terminal hydroxyl group is substituted with a methoxy or methyl group.
  • "L" is a non-ester containing linker moiety. Suitable non-ester containing linkers include, but are not limited to, an amido linker moiety, an amino linker moiety, a carbonyl linker moiety, a carbamate linker moiety, a urea linker moiety, an ether linker moiety, a disulphide linker moiety, a succinamidyl linker moiety, and combinations thereof.
  • the non-ester containing linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAA conjugate).
  • the non- ester containing linker moiety is an amido linker moiety (i.e., a PEG- ⁇ -DAA conjugate).
  • the non-ester containing linker moiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).
  • the PEG-lipid conjugate is selected from:
  • the PEG-DAA conjugates are synthesized using standard techniques and reagents known to those of skill in the art. It will be recognized that the PEG-DAA conjugates will contain various amide, amine, ether, thio, carbamate, and urea linkages. Those of skill in the art will recognize that methods and reagents for forming these bonds are well known and readily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley 1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); and Furniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed. (Longman 1989).
  • the PEG-DAA conjugate is a PEG-didecyloxypropyl (C 10 ) conjugate, a PEG- dilauryloxypropyl (C 12 ) conjugate, a PEG-dimyristyloxypropyl (C 14 ) conjugate, a PEG- dipalmityloxypropyl (C 16 ) conjugate, or a PEG-distearyloxypropyl (C 18 ) conjugate.
  • the PEG preferably has an average molecular weight of about 750 or about 2,000 daltons.
  • the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the "2000” denotes the average molecular weight of the PEG, the “C” denotes a carbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl.
  • the PEG-lipid conjugate comprises PEG750-C- DMA, wherein the "750” denotes the average molecular weight of the PEG, the "C” denotes a carbamate linker moiety, and the "DMA” denotes dimyristyloxypropyl.
  • the terminal hydroxyl group of the PEG is substituted with a methyl group.
  • hydrophilic polymers can be used in place of PEG.
  • suitable polymers include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.
  • the lipid particles (e.g., LNP) of the present invention can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al, Bioconj. Chem., 11 :433-437 (2000); U.S. Patent No. 6,852,334; PCT Publication No. WO 00/62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes).
  • PEG poly(ethylene glycol)
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, from about 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol %, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol % to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % (or
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol % to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5 mol % to about 12 mol %, or about 2 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • PEG-lipid comprises from about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20 mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % to about 18 mol %, from about 2 mol % to about 15 mol %, from about 4
  • the lipid conjugate (e.g., PEG-lipid) comprises from about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (or any fraction thereof or range therein) of the total lipid present in the particle.
  • PEG-lipid comprises from about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6 mol %
  • lipid conjugates suitable for use in the lipid particles of the present invention are described in, e.g., PCT Publication No. WO 09/127060, and PCT Publication No. WO 2010/006282, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • the percentage of lipid conjugate (e.g., PEG-lipid) present in the lipid particles of the invention is a target amount, and that the actual amount of lipid conjugate present in the formulation may vary, for example, by ⁇ 2 mol %.
  • concentration of the lipid conjugate can be varied depending on the lipid conjugate employed and the rate at which the lipid particle is to become fusogenic.
  • the rate at which the lipid conjugate exchanges out of the lipid particle can be controlled, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the alkyl groups on the PEG-DAA conjugate.
  • the rate at which the lipid particle becomes fusogenic can be varied, for example, by varying the concentration of the lipid conjugate, by varying the molecular weight of the PEG, or by varying the chain length and degree of saturation of the alkyl groups on the PEG-DAA conjugate.
  • other variables including, for example, H, temperature, ionic strength, etc. can be used to vary and/or control the rate at which the lipid particle becomes fusogenic.
  • the lipid particles of the present invention e.g., LNP, in which an active agent or therapeutic agent such as an interfering RNA ⁇ e.g., siRNA) is entrapped within the lipid portion of the particle and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method, a direct dilution process, and an in-line dilution process.
  • an active agent or therapeutic agent such as an interfering RNA ⁇ e.g., siRNA
  • the non-cationic lipids are egg sphingomyelin (ESM), distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), l-palmitoyl-2- oleoyl-phosphatidylcholine (POPC), dipalmitoyl-phosphatidylcholine (DPPC), monomethyl- phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, 14:0 PE (1,2-dimyristoyl- phosphatidylethanolamine (DMPE)), 16:0 PE (1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE (1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18: 1 PE (1,2-dioleoyl- phosphatidylethanolamine (DOPE)), 18: 1 trans PE (1,2-dielaidoyl-phosphatidy
  • the present invention provides nucleic acid-lipid particles ⁇ e.g., LNP) produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid ⁇ e.g., interfering RNA) in a first reservoir, providing an organic lipid solution in a second reservoir (wherein the lipids present in the organic lipid solution are solubilized in an organic solvent, e.g., a lower alkanol such as ethanol), and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a lipid vesicle ⁇ e.g., liposome) encapsulating the nucleic acid within the lipid vesicle.
  • a continuous mixing method e.g., a process that includes providing an aqueous solution comprising a nucleic acid ⁇ e.g., interfering RNA) in a first
  • the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a nucleic acid- lipid particle.
  • the buffer solution i.e., aqueous solution
  • the nucleic acid-lipid particles formed using the continuous mixing method typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 n
  • the present invention provides nucleic acid-lipid particles (e.g., LNP) produced via a direct dilution process that includes forming a lipid vesicle (e.g., liposome) solution and immediately and directly introducing the lipid vesicle solution into a collection vessel containing a controlled amount of dilution buffer.
  • the collection vessel includes one or more elements configured to stir the contents of the collection vessel to facilitate dilution.
  • the amount of dilution buffer present in the collection vessel is substantially equal to the volume of lipid vesicle solution introduced thereto.
  • a lipid vesicle solution in 45% ethanol when introduced into the collection vessel containing an equal volume of dilution buffer will advantageously yield smaller particles.
  • the present invention provides nucleic acid-lipid particles (e.g., LNP) produced via an in-line dilution process in which a third reservoir containing dilution buffer is fluidly coupled to a second mixing region.
  • the lipid vesicle (e.g., liposome) solution formed in a first mixing region is immediately and directly mixed with dilution buffer in the second mixing region.
  • the second mixing region includes a T-connector arranged so that the lipid vesicle solution and the dilution buffer flows meet as opposing 180° flows; however, connectors providing shallower angles can be used, e.g., from about 27° to about 180° (e.g., about 90°).
  • a pump mechanism delivers a controllable flow of buffer to the second mixing region.
  • the flow rate of dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of lipid vesicle solution introduced thereto from the first mixing region.
  • This embodiment advantageously allows for more control of the flow of dilution buffer mixing with the lipid vesicle solution in the second mixing region, and therefore also the concentration of lipid vesicle solution in buffer throughout the second mixing process.
  • control of the dilution buffer flow rate advantageously allows for small particle size formation at reduced concentrations.
  • the nucleic acid-lipid particles formed using the direct dilution and in-line dilution processes typically have a size of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 1 10 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120 nm, 1 10 nm, 100 nm, 90
  • the particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.
  • the lipid particles of the invention e.g., LNP
  • the sizing may be conducted in order to achieve a desired size range and relatively narrow distribution of particle sizes.
  • Extrusion of the particles through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing particle sizes to a relatively well- defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved.
  • the particles may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in size.
  • the nucleic acids present in the particles are precondensed as described in, e.g., U.S. Patent Application No. 09/744, 103, the disclosure of which is herein incorporated by reference in its entirety for all purposes.
  • the methods may further comprise adding non-lipid polycations which are useful to effect the lipofection of cells using the present compositions.
  • suitable non-lipid polycations include, hexadimethrine bromide (sold under the brand name POLYBRENE®, from Aldrich Chemical Co., Milwaukee, Wisconsin, USA) or other salts of hexadimethrine.
  • suitable polycations include, for example, salts of poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine, polyallylamine, and polyethyleneimine. Addition of these salts is preferably after the particles have been formed.
  • the nucleic acid to lipid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02 to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about 0.08.
  • the ratio of the starting materials (input) also falls within this range.
  • the particle preparation uses about 400 ⁇ g nucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratio of about 0.01 to about 0.08 and, more preferably, about 0.04, which corresponds to 1.25 mg of total lipid per 50 ⁇ g of nucleic acid.
  • the particle has a nucleic acid:lipid mass ratio of about 0.08.
  • the lipid to nucleic acid ratios (mass/mass ratios) in a formed nucleic acid-lipid particle will range from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25 (25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) to about 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5 (5:1) to about 10 (10:1), or about
  • the conjugated lipid may further include a CPL.
  • CPL-containing LNP A variety of general methods for making LNP-CPLs (CPL-containing LNP) are discussed herein. Two general techniques include the "post-insertion” technique, that is, insertion of a CPL into, for example, a pre-formed LNP, and the "standard” technique, wherein the CPL is included in the lipid mixture during, for example, the LNP formation steps.
  • the post-insertion technique results in LNP having CPLs mainly in the external face of the LNP bilayer membrane, whereas standard techniques provide LNP having CPLs on both internal and external faces.
  • the method is especially useful for vesicles made from phospholipids (which can contain cholesterol) and also for vesicles containing PEG-lipids (such as PEG-DAAs and PEG- DAGs).
  • PEG-lipids such as PEG-DAAs and PEG- DAGs.
  • Methods of making LNP-CPLs are taught, for example, in U.S. Patent Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO 00/62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes.
  • Pharmaceutical Formulations are taught, for example, in U.S. Patent Nos. 5,705,385; 6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent Publication No. 20020072121; and PCT Publication No. WO
  • compositions of the invention may be formulated as pharmaceutical compositions suitable for delivery to a subject.
  • the pharmaceutical compositions of the invention will often further comprise one or more buffers (e.g., neutral buffered saline or phosphate buffered saline), carbohydrates (e.g., glucose, mannose, sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, bacteriostats, chelating agents such as EDTA or glutathione, adjuvants (e.g., aluminum hydroxide), solutes that render the formulation isotonic, hypotonic or weakly hypertonic with the blood of a recipient, suspending agents, thickening agents and/or preservatives.
  • buffers e.g., neutral buffered saline or phosphate buffered saline
  • carbohydrates e.g., glucose, mannose, sucrose, dextrose or dextrans
  • the amount of the oligonucleotides administered to a patient can be readily determined by a physician based upon a variety of factors, including, e.g., the disease and the level of the oligonucleotides expressed from the vector being used (in cases where a vector is administered).
  • the amount administered per dose is typically selected to be above the minimal therapeutic dose but below a toxic dose.
  • the choice of amount per dose will depend on a number of factors, such as the medical history of the patient, the use of other therapies, and the nature of the disease.
  • the amount administered may be adjusted throughout treatment, depending on the patient's response to treatment and the presence or severity of any treatment-associated side effects.
  • the invention further includes a method of identifying gene function in an organism comprising the use of a polynucleotide complex of the present invention to inhibit the activity of a target gene of previously unknown function.
  • functional genomics envisions determining the function of uncharacterized genes by employing the invention to reduce the amount and/or alter the timing of target gene activity.
  • the invention may be used in determining potential targets for pharmaceutics, understanding normal and pathological events associated with development, determining signaling pathways responsible for postnatal development/aging, and the like.
  • the increasing speed of acquiring nucleotide sequence information from genomic and expressed gene sources including total sequences for the yeast, D. melanogaster, and C.
  • elegans genomes can be coupled with the invention to determine gene function in an organism (e.g., nematode).
  • an organism e.g., nematode
  • the preference of different organisms to use particular codons, searching sequence databases for related gene products, correlating the linkage map of genetic traits with the physical map from which the nucleotide sequences are derived, and artificial intelligence methods may be used to define putative open reading frames from the nucleotide sequences acquired in such sequencing projects.
  • a polynucleotide of the present invention is used to inhibit gene expression based upon a partial sequence available from an expressed sequence tag (EST), e.g., in order to determine the gene's function or biological activity. Functional alterations in growth, development, metabolism, disease resistance, or other biological processes would be indicative of the normal role of the EST's gene product.
  • EST expressed sequence tag
  • a polynucleotide can be introduced into an intact cell/organism containing the target gene in high throughput screening (HTS).
  • HTS high throughput screening
  • solutions containing the polynucleotide that are capable of inhibiting different expressed genes can be placed into individual wells positioned on a microtiter plate as an ordered array, and intact cells/organisms in each well can be assayed for any changes or modifications in behavior or development due to inhibition of target gene activity.
  • the function of the target gene can be assayed from the effects it has on the cell/organism when gene activity is inhibited.
  • the polynucleotides of the invention are used for chemocogenomic screening, i.e., testing compounds for their ability to reverse a disease modeled by the reduction of gene expression using a polynucleotide of the invention.
  • a characteristic of an organism is determined to be genetically linked to a polymorphism through RFLP or QTL analysis
  • the present invention can be used to gain insight regarding whether that genetic polymorphism might be directly responsible for the characteristic. For example, a fragment defining the genetic polymorphism or sequences in the vicinity of such a genetic polymorphism can be amplified to produce an R A, a polynucleotide can be introduced to the organism, and whether an alteration in the characteristic is correlated with inhibition can be determined.
  • the present invention is also useful in allowing the inhibition of essential genes. Such genes may be required for cell or organism viability at only particular stages of development or cellular compartments.
  • the functional equivalent of conditional mutations may be produced by inhibiting activity of the target gene when or where it is not required for viability.
  • the invention allows addition of a the polynucleotide at specific times of development and locations in the organism without introducing permanent mutations into the target genome.
  • the invention contemplates the use of inducible or conditional vectors that express a the polynucleotide only when desired.
  • the present invention also relates to a method of validating whether a gene product is a target for drug discovery or development.
  • a polynucleotide that targets the gene that corresponds to the gene for degradation is introduced into a cell or organism. The cell or organism is maintained under conditions in which degradation of the gene occurs, resulting in decreased expression of the gene. Whether decreased expression of the gene has an effect on the cell or organism is determined. If decreased expression of the gene has an effect, then the gene product is a target for drug discovery or development.
  • the polynucleotide complexes of the present invention comprise a novel and unique set of functional sequences, arranged in a manner so as to adopt a secondary structure containing one or more double-stranded regions (sometimes adjoined by stem-loop or loop structures), which imparts the advantages of the polynucleotide.
  • the present invention includes methods of designing the polynucleotide complexes of the present invention. Such methods typically involve appropriate selection of the various sequence components of the polynucleotide complexes.
  • the terms "primary strand”, “secondary strand”, and “key strand” refer to the various guide strands present within a polynucleotide complex of the present invention.
  • the basic design of the polynucleotide complex is as follows: DESIGN MOTIFS:
  • the polynucleotide is designed as follows: II. (secondary strand)(UU)(UU)(key strand)(UU)(primary strand) III. (secondary strand)(UU)(loop or stem-loop)(key strand)(UU)(loop or stem- loop)(primary strand)(UU)
  • target gene sequences start with one or more target gene sequences. For each gene, build a list of PRIMARY target sequences 17-24 nucleotide motifs that meet criteria of G/C content, specificity, and poly- A or poly-G free. For each, find also a SECONDARY and KEY strand.
  • the target sequence on the SECONDARY gene is the alignment start, minus the length of the motif, plus SEED SIZE to alignment start, plus SEED SIZE.
  • the SECONDARY strand is the reverse compliment.
  • SEED A as base 1 through SEED SIZE of the PRIMARY strand
  • SEED B as bases at motif length minus SEED SIZE to motif length of the SECONDARY strand.
  • Set a MID SECTION as characters "
  • Set key alignment sequence as SEED A, MID SECTION, SEED B.
  • the KEY strand is the reverse compliment.
  • CONSTRUCT OPTIONAL POLYNUCLEOTIDE g. Build candidate Stem A & B with (4-24) nucleotides that have melting temperature dominant to equal length region of target. Stem strands have A-T, G-C complementarity to each other. Length and composition depend upon which endoribonuclease is chosen for preprocessing of the stem- loop structure. h. Build candidate Stem C & D with (4-24) nucleotides that have melting temperature dominant to equal length region of target. Stem strands have A-T, G-C complementarity to each other, but no complementarity to Stem A & B. Length and composition depend upon which endoribonuclease is chosen for pre-processing of the stem-loop structure.
  • loop candidates with (4-12) A-T rich nucleotides into loop A & B. Length and composition depend upon which endoribonuclease is chosen for pre-processing of the stem- loop structure. Tetraloops as described are suggested for longer stems processed by R ase III or Pacl RNase III endoribonucleases as drawn in. Larger loops are suggested for preventing RNase III or Pacl processing and placed onto shorter stems.
  • j. Form a contiguous sequence for each motif candidate.
  • k Fold candidate sequence using software with desired parameters.
  • the programs of the present invention may further use input regarding the genomic sequence of the organism containing the target gene, e.g., public or private databases, as well as additional programs that predict secondary structure and/or hybridization characteristics of particular sequences, in order to ensure that the polynucleotide adopts the correct secondary structure and does not hybridize to non-target genes.
  • the present invention is based, in part, upon the surprising discovery that the polynucleotide complexes, as described herein, are extremely effective in reducing target gene expression of one or more genes.
  • the polynucleotide offer significant advantages over previously described antisense RNAs, including increased potency, and increased effectiveness to multiple target genes.
  • the polynucleotide of the invention offer additional advantages over traditional dsRNA molecules used for siRNA, since the use of the polynucleotide substantially eliminates the off-target suppression associated with dsRNA molecules and offers multivalent RNAi. It is understood that the compositions and methods of the present invention may be used to target a variety of different target genes.
  • target gene may refer to a gene, an mR A, or a microRNA. Accordingly, target sequences provided herein may be depicted as either DNA sequences or RNA sequences. One of skill the art will appreciate that the compositions of the present invention may include regions complementary to either the DNA or RNA sequences provided herein. Thus, where either a DNA or RNA target sequence is provided, it is understood that the corresponding RNA or DNA target sequence, respectively, may also be targeted.
  • This Example describes a method for making a polynucleotide complex of the present invention.
  • Step A Anneal oligonucleotides A and B.
  • oligos A and B withK ⁇ L of 10 X Annealing Buffer (e.g., lOOmM Tris-HCL, pH 7.5, 1M NaCl, lOmM EDTA).
  • 10 X Annealing Buffer e.g., lOOmM Tris-HCL, pH 7.5, 1M NaCl, lOmM EDTA.
  • Step B Add oligonucleotide C.
  • Step C Reduce Scaffolding.
  • Step 1 mix oligonucleotide strands in correct ratios in the annealing buffer described in Example 1.
  • the oligonucleotide strands are two un-nicked strands, and the first and second portions of the nicked oligonucleotide strand.
  • Step 2 heat the mixture of oligonucleotides prepared in Step 1 at 95°C for 2 minutes.
  • Step 3 cool the heated mixture prepared in Step 2 to ambient temperature within a 37°C chamber.
  • RNA interference RNA interference
  • Double stranded RNA induces specific developmental defects in zebrafish embyos. Biochem. Biophys. Res. Commun. 263, 156-161.
  • RNA interference can target pre-gene. Consequences for gene expression in Caenorhabiditis elegans operon. Genetics. 153, 1245-1256. 10. Fire, A. (1999) RNA-triggered gene silencing. Trends Genet. 15, 358-363.
  • RNAi Double stranded RNA directs the ATP dependent cleavage of gene at 21 to 23 nucleotide intervals. Cell. 101, 25-33.
  • RNA interference is mediated by 21 and 22 nucleotide RNAs. Genes and Dev. 15, 188-200.
  • RNA tetraloop structure forms the recognition site for Saccharomyces cerevisiae RNase III. [EMBO J. 2001].

Abstract

La présente invention concerne des complexes d'acides nucléiques multivalents de molécules d'acides nucléiques ayant au moins deux régions spécifiques d'une cible, dans lesquels les régions spécifiques d'une cible sont complémentaires à un gène cible unique au niveau de plus d'un site nucléotidique distinct et/ou dans lesquels les régions cibles sont complémentaires à au moins un gène cible ou une séquence cible. Le brin oligonucléotidique du complexe est entaillé et, par conséquent, inactivé. L'invention concerne également des procédés d'utilisation des complexes pour le traitement d'une variété de maladies et d'infections.
PCT/US2013/020109 2012-01-04 2013-01-03 Complexes polynucléotidiques à entaille pour une interférence par arn multivalente WO2013103693A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100209487A1 (en) * 2006-10-18 2010-08-19 Nastech Pharmaceutical Company Inc. Nicked or gapped nucleic acid molecules and uses thereof
WO2010141511A2 (fr) * 2009-06-01 2010-12-09 Halo-Bio Rnai Therapeutics, Inc. Polynucléotides pour interférence arn multivalente, compositions et procédés pour les utiliser

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100209487A1 (en) * 2006-10-18 2010-08-19 Nastech Pharmaceutical Company Inc. Nicked or gapped nucleic acid molecules and uses thereof
WO2010141511A2 (fr) * 2009-06-01 2010-12-09 Halo-Bio Rnai Therapeutics, Inc. Polynucléotides pour interférence arn multivalente, compositions et procédés pour les utiliser

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