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WO2025101204A1 - Ciblage intratumoral de pd-1 - Google Patents

Ciblage intratumoral de pd-1 Download PDF

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WO2025101204A1
WO2025101204A1 PCT/US2023/085041 US2023085041W WO2025101204A1 WO 2025101204 A1 WO2025101204 A1 WO 2025101204A1 US 2023085041 W US2023085041 W US 2023085041W WO 2025101204 A1 WO2025101204 A1 WO 2025101204A1
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nucleic acid
double stranded
modified
chemically
acid molecule
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Robert BITTERMAN
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Phio Pharmaceuticals Corp
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Phio Pharmaceuticals Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
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    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/55—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds
    • A61K47/551—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being also a pharmacologically or therapeutically active agent, i.e. the entire conjugate being a codrug, i.e. a dimer, oligomer or polymer of pharmacologically or therapeutically active compounds one of the codrug's components being a vitamin, e.g. niacinamide, vitamin B3, cobalamin, vitamin B12, folate, vitamin A or retinoic acid
    • A—HUMAN NECESSITIES
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/00—Structure or type of the nucleic acid
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Definitions

  • BACKGROUND Immune checkpoint-targeted monoclonal antibodies directed to PD-1 have expanded the therapeutic options for the treatment of multiple types of cancers. By blocking co- inhibitory receptors expressed by antitumor T cells, these antibodies can break the immune tolerance against tumor cells and allow the generation of durable cancer immunity. Benefits in overall survival over conventional therapies have been demonstrated for treated subjects. However, only a minority of subjects develop a tumor response with long-term survival benefits. Moreover, the systemic delivery of immunotherapies can be responsible for severe auto-immune toxicities. SUMMARY Described herein is an intratumoral immunotherapy approach in which a chemically- modified double stranded nucleic acid molecule targeting PD-1 is administered to a subject having a cutaneous malignancy.
  • aspects of the disclosure relate to methods for treating cutaneous squamous cell carcinoma (cSCC) in a subject having a tumor, the method comprising administering to the subject a chemically-modified double stranded nucleic acid molecule that is directed against a gene encoding PD-1, wherein the chemically-modified double stranded nucleic acid molecule is administered by intratumoral injection.
  • cSCC cutaneous squamous cell carcinoma
  • Basal cell carcinoma Squamous cell carcinoma, Malignant melanoma, Merkel cell carcinoma, Sebaceous carcinoma, Keratoacanthoma, Angiosarcoma, Malignant fibrous histiocytoma, Cutaneous B cell and T cell lymphoma, Leiomyosarcoma, Kaposi’s sarcoma, Dermatofibrosarcoma protuberans, Adnexal carcinoma, Porocarcinoma, Spindle cell tumor (sarcoma), Atypical fibroxanthoma, in situ squamous cell carcinoma (Bowen’s disease), or metastases of local presentation of other cancers (e.g.
  • Paget’s disease of the breast) in a subject having a tumor comprising administering to the subject a chemically-modified double stranded nucleic acid molecule that is directed against a gene encoding PD-1, wherein the chemically- modified double stranded nucleic acid molecule is administered by intratumoral injection.
  • the chemically-modified double stranded nucleic acid molecule is an isolated asymmetric double stranded nucleic acid molecule comprising a guide strand, with a minimal length of 16 nucleotides, and a passenger strand of 8-18 nucleotides, wherein the double stranded nucleic acid molecule has a double stranded region and a single stranded region, the single stranded region having 4-12 nucleotides in length and having at least three nucleotide backbone modifications.
  • the chemically-modified double stranded nucleic acid molecule is attached to a hydrophobic conjugate, optionally wherein the hydrophobic conjugate is cholesterol.
  • the chemically-modified double stranded nucleic acid molecule comprises at least 12 contiguous nucleotides of any one of SEQ ID NOs: 1-74.
  • the guide strand of the chemically-modified double stranded nucleic acid molecule comprises the sequence of SEQ ID NO: 52.
  • the passenger strand of the chemically-modified double stranded nucleic acid molecule comprises the sequence of SEQ ID NO: 51.
  • the chemically-modified double stranded nucleic acid molecule is administered at least twice, at least three times, at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, or at least ten times. In some embodiments, the chemically-modified double stranded nucleic acid molecule is administered at weekly intervals, twice a week, three times a week, or between 1- 3 times a week. In some embodiments, the chemically-modified double stranded nucleic acid molecule is administered at a dose of between 1 mg/ml – 25 mg/ml. In some embodiments, the volume of injection is between 0.1-10 ml per lesion.
  • the chemically-modified double stranded nucleic acid molecule is administered to the subject in advance of surgical resection.
  • one lesion is injected in the subject.
  • multiple lesions are injected in the subject.
  • FIG.1 provides a schematic showing a representative treatment regimen.
  • DETAILED DESCRIPTION In some aspects, the disclosure relates to compositions and methods for intratumoral immunotherapy.
  • PD-1 or “PD1” or “PDCD1” refers to Programmed Cell Death Protein 1, which is a cell surface receptor that functions to down-regulate the immune system and promote immune self-tolerance by suppressing T-cell-mediated inflammatory activity.
  • PDCD1 is encoded by a nucleic acid sequence represented by NCBI Reference Sequence Number NM_005018.2.
  • a chemically-modified double stranded nucleic acid molecule that is directed against a gene encoding PD-1 comprises or is directed to a sequence comprising at least 12 contiguous nucleotides of any one of SEQ ID NOs: 1-74 or a sequence within Tables 1 or 2.
  • Table 1 is incorporated by reference from U.S. Patent No. US 10,934,550, the entire contents of which are incorporated herein by reference.
  • Table 2 is incorporated by reference from US Patent Publication No. US2020/0215113, the entire contents of which are incorporated herein by reference.
  • Table 1 Representative PD-1 sequences
  • Table 2 Additional representative PD-1 sequences
  • An additional representative PD-1 sequence corresponds to PD 38, which comprises: P.mU.fU.fA.mC.mC.fA.mU.mU.mU.fA.fU.mU.fA*mU*mA*mA*mC*mG*mG.
  • the chemically-modified double stranded nucleic acid molecule comprises or consists of at least 12 nucleotides of any one of SEQ ID NOs: 41-74 within Table 2. In some embodiments, the chemically-modified double stranded nucleic acid molecule comprises or consists of SEQ ID NOs: 51 and/or 52. In some embodiments, the chemically-modified double stranded nucleic acid molecule corresponds to PD26 as depicted in Table 2.
  • the chemically-modified double stranded nucleic acid molecule comprises or consists of at least 12 nucleotides of SEQ ID NO: 75 and/or 76. In some embodiments, the chemically-modified double stranded nucleic acid molecule comprises or consists of SEQ ID NO: 75 and/or 76. In some embodiments, the chemically- modified double stranded nucleic acid molecule corresponds to PD38. Aspects of the disclosure relate to intratumoral injection of chemically-modified double stranded nucleic acid molecules described herein, such as PD26.
  • the chemically-modified double stranded nucleic acid molecules are injected into a tumor of a subject having: Basal cell carcinoma, Squamous cell carcinoma, Malignant melanoma, Merkel cell carcinoma, Sebaceous carcinoma, Keratoacanthoma, Angiosarcoma, Malignant fibrous histiocytoma, Cutaneous B cell and T cell lymphoma, Leiomyosarcoma, Kaposi’s sarcoma, Dermatofibrosarcoma protuberans, Adnexal carcinoma, Porocarcinoma, Spindle cell tumor (sarcoma), Atypical fibroxanthoma, in situ squamous cell carcinoma (Bowen’s disease) or metastases of local presentation of other cancers (e.g.
  • nucleic acid molecule includes but is not limited to: INTASYLTM, sd-rxRNA, rxRNAori, oligonucleotides, ASO, siRNA, shRNA, miRNA, ncRNA, cp- lasiRNA, aiRNA, single-stranded nucleic acid molecules, double-stranded nucleic acid molecules, RNA and DNA.
  • the nucleic acid molecule is a chemically-modified nucleic acid molecule, such as a chemically-modified oligonucleotide.
  • the nucleic acid molecule is double stranded. In some embodiments, the nucleic acid molecule is a chemically-modified double-stranded nucleic acid molecule. In some embodiments, chemically-modified double stranded nucleic acid molecules as described herein are INTASYLTM (also known as sd-rxRNA) molecules. Aspects of the disclosure relate to INTASYLTM molecules that target a gene encoding PD-1. In some embodiments, an INTASYLTM molecule described herein comprises or consists of, or is targeted to or directed against, a sequence set forth in Table 2, or a fragment thereof.
  • an “sd-rxRNA” or an “sd-rxRNA molecule” or an “INTASYLTM” or an “INTASYLTM molecule” or an INTASYL compound” refers to a self-delivering RNA molecule such as those described in, and incorporated by reference from, US Patent No.
  • an INTASYLTM refers to an isolated asymmetric double stranded nucleic acid molecule comprising a guide strand, with a minimal length of 16 nucleotides, and a passenger strand of 8-18 nucleotides in length, wherein the double stranded nucleic acid molecule has a double stranded region and a single stranded region, the single stranded region having 4-12 nucleotides in length and having at least three nucleotide backbone modifications.
  • the double stranded nucleic acid molecule has one end that is blunt or includes a one or two nucleotide overhang.
  • INTASYLTM molecules can be optimized through chemical modification, and in some instances through attachment of hydrophobic conjugates.
  • an INTASYLTM comprises an isolated double stranded nucleic acid molecule comprising a guide strand and a passenger strand, wherein the region of the molecule that is double stranded is from 8-15 nucleotides long, wherein the guide strand contains a single stranded region that is 4-12 nucleotides long, wherein the single stranded region of the guide strand contains 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphorothioate modifications, and wherein at least 40% of the nucleotides of the double stranded nucleic acid are modified.
  • INTASYLTM molecules are much more effectively taken up by cells compared to conventional siRNAs.
  • RNAi molecules are highly efficient in silencing of target gene expression and offer significant advantages over previously described RNAi molecules including high activity in the presence of serum, efficient self-delivery, compatibility with a wide variety of linkers, and reduced presence or complete absence of chemical modifications that are associated with toxicity.
  • duplex polynucleotides In contrast to single-stranded polynucleotides, duplex polynucleotides have traditionally been difficult to deliver to a cell as they have rigid structures and a large number of negative charges which makes membrane transfer difficult.
  • INTASYLTM molecules although partially double-stranded, are recognized in vivo as single-stranded and, as such, are capable of efficiently being delivered across cell membranes. As a result, INTASYLTM molecules are capable in many instances of self-delivery.
  • INTASYLTM molecules may be formulated in a manner similar to conventional RNAi agents or they may be delivered to the cell or subject alone (or with non-delivery type carriers) and allowed to self-deliver.
  • the double stranded nucleic acid molecules of the disclosure in some aspects have a combination of asymmetric structures including a double stranded region and a single stranded region of 5 nucleotides or longer, specific chemical modification patterns and are conjugated to lipophilic or hydrophobic molecules.
  • this class of RNAi like compounds have superior efficacy in vitro and in vivo.
  • the double stranded nucleic acid molecules of the disclosure comprise an asymmetric compound comprising a duplex region (required for efficient RISC entry of 8-15 bases long) and single stranded region of 4-12 nucleotides long.
  • the duplex region is 13 or 14 nucleotides long. A 6 or 7 nucleotide single stranded region is preferred in some embodiments.
  • the single stranded region of the double stranded nucleic acid molecules can comprise 2-12 phosphorothioate internucleotide linkages (referred to as phosphorothioate modifications). 6-8 phosphorothioate internucleotide linkages are preferred in some embodiments.
  • the double stranded nucleic acid molecules of the disclosure also include a chemical modification pattern, which provides stability and is compatible with RISC entry. In some embodiments, the combination of these elements has resulted in unexpected properties which are highly useful for delivery of RNAi reagents in vitro and in vivo.
  • the chemical modification pattern which provides stability and is compatible with RISC entry can include modifications to the sense, or passenger, strand as well as the antisense, or guide, strand.
  • the passenger strand can be modified with any chemical entities that confer stability and do not interfere with activity.
  • modifications include 2’ ribo modifications (O-methyl, 2’ F, 2 deoxy and others) and backbone modification like phosphorothioate modifications.
  • a preferred chemical modification pattern in the passenger strand can include O-methyl modification of C and U nucleotides within the passenger strand or alternatively the passenger strand may be completely O-methyl modified.
  • the guide strand for example, may also be modified by any chemical modification which confers stability without interfering with RISC entry.
  • a preferred chemical modification pattern in the guide strand can include the majority of C and U nucleotides being 2’ F modified and the 5’ end being phosphorylated.
  • Another preferred chemical modification pattern in the guide strand can include 2’O-methyl modification of position 1 and C/U in positions 11-18 and 5’ end chemical phosphorylation.
  • Yet another preferred chemical modification pattern in the guide strand can include 2’O-methyl modification of position 1 and C/U in positions 11-18 and 5’ end chemical phosphorylation and 2’F modification of C/U in positions 2-10.
  • the passenger strand and/or the guide strand contains at least one 5-methyl C or U modifications.
  • At least 30% of the nucleotides in the chemically-modified double stranded nucleic acid molecule are modified. For example, at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the chemically
  • 100% of the nucleotides in the chemically- modified double stranded nucleic acid molecule are modified.
  • the above-described chemical modification patterns of the oligonucleotides of the disclosure are well tolerated and actually improve efficacy of asymmetric RNAi compounds.
  • elimination of any of the described components (guide strand stabilization, phosphorothioate stretch, sense strand stabilization and hydrophobic conjugate) or increase in size in some instances results in sub-optimal efficacy and in some instances complete loss of efficacy.
  • the combination of elements results in development of a compound, which is fully active following passive delivery to cells such as HeLa cells, or T- cells.
  • the chemically-modified double stranded nucleic acid molecule can be further improved in some instances by improving the hydrophobicity of compounds using novel types of chemistries.
  • one chemistry is related to use of hydrophobic base modifications. Any base in any position might be modified, as long as modification results in an increase of the partition coefficient of the base.
  • the preferred locations for modification chemistries are positions 4 and 5 of the pyrimidines. The major advantage of these positions is (a) ease of synthesis and (b) lack of interference with base-pairing and A form helix formation, which are essential for RISC complex loading and target recognition.
  • chemically-modified double stranded nucleic acid molecules where multiple deoxy Uridines are present without interfering with overall compound efficacy are used.
  • major improvement in tissue distribution and cellular uptake might be obtained by optimizing the structure of the hydrophobic conjugate.
  • the structure of sterol is modified to alter (increase/ decrease) C17 attached chain. This type of modification results in significant increase in cellular uptake and improvement of tissue uptake prosperities in vivo.
  • a chemically-modified double stranded nucleic acid molecule is a hydrophobically modified siRNA-antisense hybrid molecule, comprising a double- stranded region of about 13-22 base pairs, with or without a 3’- overhang on each of the sense and antisense strands, and a 3’ single-stranded tail on the antisense strand of about 2-9 nucleotides.
  • the chemically-modified double stranded nucleic acid molecule contains at least one 2’-O-Methyl modification, at least one 2’-Fluoro modification, and at least one phosphorothioate modification, as well as at least one hydrophobic modification selected from sterol, cholesterol, vitamin D, napthyl, isobutyl, benzyl, indol, tryptophane, phenyl, and the like hydrophobic modifiers.
  • a chemically-modified double stranded nucleic acid molecule comprises a plurality of such modifications.
  • aspects of the disclosure relate to isolated double stranded nucleic acid molecules comprising a guide (antisense) strand and a passenger (sense) strand.
  • double-stranded refers to one or more nucleic acid molecules in which at least a portion of the nucleomonomers are complementary and hydrogen bond to form a double-stranded region.
  • the length of the guide strand ranges from 16-29 nucleotides long.
  • the guide strand is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides long.
  • the guide strand has complementarity to a target gene.
  • Complementarity between the guide strand and the target gene may exist over any portion of the guide strand.
  • Complementarity as used herein may be perfect complementarity or less than perfect complementarity as long as the guide strand is sufficiently complementary to the target that it mediates RNAi.
  • complementarity refers to less than 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% mismatch between the guide strand and the target. Perfect complementarity refers to 100% complementarity.
  • siRNA sequences with insertions, deletions, and single point mutations relative to the target sequence have also been found to be effective for inhibition. Moreover, not all positions of a siRNA contribute equally to target recognition.
  • Mismatches in the center of the siRNA are most critical and essentially abolish target RNA cleavage. Mismatches upstream of the center or upstream of the cleavage site referencing the antisense strand are tolerated but significantly reduce target RNA cleavage. Mismatches downstream of the center or cleavage site referencing the antisense strand, preferably located near the 3' end of the antisense strand, e.g.1, 2, 3, 4, 5 or 6 nucleotides from the 3' end of the antisense strand, are tolerated and reduce target RNA cleavage only slightly.
  • the guide strand is at least 16 nucleotides in length and anchors the Argonaute protein in RISC.
  • the guide strand loads into RISC it has a defined seed region and target mRNA cleavage takes place across from position 10-11 of the guide strand.
  • the 5’ end of the guide strand is or is able to be phosphorylated.
  • the nucleic acid molecules described herein may be referred to as minimum trigger RNA.
  • the length of the passenger strand ranges from 8-15 nucleotides long.
  • the passenger strand is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long.
  • the passenger strand has complementarity to the guide strand. Complementarity between the passenger strand and the guide strand can exist over any portion of the passenger or guide strand. In some embodiments, there is 100% complementarity between the guide and passenger strands within the double stranded region of the molecule.
  • Aspects of the disclosure relate to double stranded nucleic acid molecules with minimal double stranded regions. In some embodiments the region of the molecule that is double stranded ranges from 8-15 nucleotides long. In certain embodiments, the region of the molecule that is double stranded is 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides long.
  • the double stranded region is 13 or 14 nucleotides long. In some embodiments, the region of the molecule that is double stranded is 13-22 nucleotides long. In certain embodiments, the region of the molecule that is double stranded is 16, 17, 18, 19, 20, 21 or 22 nucleotides long. There can be 100% complementarity between the guide and passenger strands, or there may be one or more mismatches between the guide and passenger strands. In some embodiments, on one end of the double stranded molecule, the molecule is either blunt-ended or has a one-nucleotide overhang. The single stranded region of the molecule is in some embodiments between 4-12 nucleotides long.
  • the single stranded region can be 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleotides long. However, in certain embodiments, the single stranded region can also be less than 4 or greater than 12 nucleotides long. In certain embodiments, the single stranded region is at least 6 or at least 7 nucleotides long. In some embodiments, the single stranded region is 2-9 nucleotides long, including 2 or 3 nucleotides long.
  • RNAi constructs associated with the disclosure can have a thermodynamic stability ( ⁇ G) of less than -13 kkal/mol. In some embodiments, the thermodynamic stability ( ⁇ G) is less than -20 kkal/mol.
  • a ( ⁇ G) value higher than -13 kkal/mol is compatible with aspects of the disclosure.
  • a molecule with a relatively higher ( ⁇ G) value may become active at a relatively higher concentration, while a molecule with a relatively lower ( ⁇ G) value may become active at a relatively lower concentration.
  • the ( ⁇ G) value may be higher than -9 kkcal/mol.
  • RNAi constructs associated with the disclosure containing minimal double stranded regions, are unexpected because molecules of almost identical design but lower thermodynamic stability have been demonstrated to be inactive (Rana et al 2004).
  • a stretch of 8-10 bp of dsRNA or dsDNA will be structurally recognized by protein components of RISC or co-factors of RISC.
  • the triggering compound there is a free energy requirement for the triggering compound that it may be either sensed by the protein components and/or stable enough to interact with such components so that it may be loaded into the Argonaute protein.
  • thermodynamic stability is increased through the use of LNA bases. In some embodiments, additional chemical modifications are introduced.
  • nucleotide length of the guide and/or passenger strand, and/or the number of phosphorothioate modifications in the guide and/or passenger strand can in some aspects influence potency of the RNA molecule, while replacing 2’-fluoro (2’F) modifications with 2’-O-methyl (2’OMe) modifications can in some aspects influence toxicity of the molecule.
  • reduction in 2’F content of a molecule is predicted to reduce toxicity of the molecule.
  • RNA molecules described herein have no 2’F modification and yet are characterized by equal efficacy in cellular uptake and tissue penetration. Such molecules represent a significant improvement over prior art, such as molecules described by Accell and Wolfrum, which are heavily modified with extensive use of 2’F.
  • a guide strand is approximately 18-19 nucleotides in length and has approximately 2-14 phosphate modifications.
  • a guide strand can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more than 14 nucleotides that are phosphate- modified.
  • the guide strand may contain one or more modifications that confer increased stability without interfering with RISC entry.
  • the phosphate modified nucleotides such as phosphorothioate modified nucleotides, can be at the 3’ end, 5’ end or spread throughout the guide strand.
  • the 3’ terminal 10 nucleotides of the guide strand contains 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphorothioate modified nucleotides.
  • the guide strand can also contain 2’F and/or 2’OMe modifications, which can be located throughout the molecule.
  • the nucleotide in position one of the guide strand is 2’OMe modified and/or phosphorylated.
  • C and U nucleotides within the guide strand can be 2’F modified.
  • C and U nucleotides in positions 2-10 of a 19 nt guide strand (or corresponding positions in a guide strand of a different length) can be 2’F modified.
  • C and U nucleotides within the guide strand can also be 2’OMe modified.
  • C and U nucleotides in positions 11-18 of a 19 nt guide strand can be 2’OMe modified.
  • the nucleotide at the most 3’ end of the guide strand is unmodified.
  • the majority of Cs and Us within the guide strand are 2’F modified and the 5’ end of the guide strand is phosphorylated.
  • position 1 and the Cs or Us in positions 11-18 are 2’OMe modified and the 5’ end of the guide strand is phosphorylated.
  • position 1 and the Cs or Us in positions 11-18 are 2’OMe modified, the 5’ end of the guide strand is phosphorylated, and the Cs or Us in position 2-10 are 2’F modified.
  • an optimal passenger strand is approximately 11-14 nucleotides in length.
  • the passenger strand may contain modifications that confer increased stability.
  • One or more nucleotides in the passenger strand can be 2’OMe modified.
  • one or more of the C and/or U nucleotides in the passenger strand is 2’OMe modified, or all of the C and U nucleotides in the passenger strand are 2’OMe modified. In certain embodiments, all of the nucleotides in the passenger strand are 2’OMe modified.
  • One or more of the nucleotides on the passenger strand can also be phosphate-modified such as phosphorothioate modified.
  • the passenger strand can also contain 2’ ribo, 2’F and 2 deoxy modifications or any combination of the above.
  • Chemical modification patterns on both the guide and passenger strand can be well tolerated and a combination of chemical modifications can lead to increased efficacy and self-delivery of RNA molecules.
  • Aspects of the disclosure relate to RNAi constructs that have extended single-stranded regions relative to double stranded regions, as compared to molecules that have been used previously for RNAi.
  • the single stranded region of the molecules may be modified to promote cellular uptake or gene silencing.
  • phosphorothioate modification of the single stranded region influences cellular uptake and/or gene silencing.
  • the region of the guide strand that is phosphorothioate modified can include nucleotides within both the single stranded and double stranded regions of the molecule.
  • the single stranded region includes 2-12 phosphorothioate modifications.
  • the single stranded region can include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphorothioate modifications.
  • the single stranded region contains 6-8 phosphorothioate modifications. Molecules associated with the disclosure are also optimized for cellular uptake.
  • the guide and/or passenger strands can be attached to a conjugate.
  • the conjugate is hydrophobic.
  • the hydrophobic conjugate can be a small molecule with a partition coefficient that is higher than 10.
  • the conjugate can be a sterol-type molecule such as cholesterol, or a molecule with an increased length polycarbon chain attached to C17, and the presence of a conjugate can influence the ability of an RNA molecule to be taken into a cell with or without a lipid transfection reagent.
  • the conjugate can be attached to the passenger or guide strand through a hydrophobic linker.
  • a hydrophobic linker is 5-12C in length, and/or is hydroxypyrrolidine- based.
  • a hydrophobic conjugate is attached to the passenger strand and the CU residues of either the passenger and/or guide strand are modified. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the CU residues on the passenger strand and/or the guide strand are modified.
  • molecules associated with the disclosure are self-delivering (sd). As used herein, “self- delivery” refers to the ability of a molecule to be delivered into a cell without the need for an additional delivery vehicle such as a transfection reagent. In certain embodiments, the polynucleotide contains 5'- and/or 3'-end overhangs.
  • the number and/or sequence of nucleotides overhang on one end of the polynucleotide may be the same or different from the other end of the polynucleotide.
  • one or more of the overhang nucleotides may contain chemical modification(s), such as phosphorothioate or 2’-OMe modification.
  • the polynucleotide is unmodified.
  • at least one nucleotide is modified.
  • the modification includes a 2’-H or 2’-modified ribose sugar at the 2nd nucleotide from the 5’-end of the guide sequence.
  • the “2nd nucleotide” is defined as the second nucleotide from the 5'-end of the polynucleotide.
  • “2’-modified ribose sugar” includes those ribose sugars that do not have a 2’-OH group.
  • “2’-modified ribose sugar” does not include 2’-deoxyribose (found in unmodified canonical DNA nucleotides).
  • the 2’-modified ribose sugar may be 2'-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, or combination thereof.
  • the 2’-modified nucleotides are pyrimidine nucleotides (e.g., C /U).
  • Examples of 2’-O-alkyl nucleotides include 2’-O-methyl nucleotides, or 2'-O-allyl nucleotides.
  • the chemically-modified double stranded nucleic acid molecule with the above-referenced 5'-end modification exhibits significantly (e.g., at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more) less “off-target” gene silencing when compared to similar constructs without the specified 5'-end modification, thus greatly improving the overall specificity of the RNAi reagent or therapeutics.
  • “off-target” gene silencing refers to unintended gene silencing due to, for example, spurious sequence homology between the antisense (guide) sequence and the unintended target mRNA sequence.
  • Certain guide strand modifications may further increase nuclease stability, and/or lower interferon induction, without significantly decreasing RNAi activity. Certain combinations of modifications may result in further unexpected advantages, as partly manifested by enhanced ability to inhibit target gene expression, enhanced serum stability, and/or increased target specificity, etc.
  • the guide strand comprises a 2’-O-methyl modified nucleotide at the 2 nd nucleotide on the 5’-end of the guide strand and no other modified nucleotides.
  • the modified RNAi constructs may have improved stability in serum and/or cerebral spinal fluid compared to an unmodified RNAi constructs having the same sequence.
  • the structure of the RNAi construct does not induce interferon response in primary cells, such as mammalian primary cells, including primary cells from human, mouse and other rodents, and other non-human mammals.
  • the RNAi construct may also be used to inhibit expression of a target gene in an invertebrate organism.
  • the 3’-end of the structure may be blocked by protective group(s).
  • protective groups such as inverted nucleotides, inverted abasic moieties, or amino-end modified nucleotides may be used.
  • Inverted nucleotides may comprise an inverted deoxynucleotide.
  • Inverted abasic moieties may comprise an inverted deoxyabasic moiety, such as a 3',3'-linked or 5',5'-linked deoxyabasic moiety.
  • the chemically-modified double stranded nucleic acid molecules of the disclosure are capable of inhibiting the synthesis of any target protein encoded by target gene(s).
  • the disclosure includes methods to inhibit expression of a target gene either in a cell in vitro, or in vivo.
  • the chemically-modified double stranded nucleic acid molecules of the disclosure are useful for treating a patient with a disease characterized by the overexpression of a target gene.
  • the target gene can be endogenous or exogenous (e.g., introduced into a cell by a virus or using recombinant DNA technology) to a cell.
  • Such methods may include introduction of RNA into a cell in an amount sufficient to inhibit expression of the target gene.
  • such an RNA molecule may have a guide strand that is complementary to the nucleotide sequence of the target gene, such that the composition inhibits expression of the target gene.
  • the disclosure also relates to vectors expressing the nucleic acids of the disclosure, and cells comprising such vectors or the nucleic acids.
  • the cell may be a mammalian cell in vivo or in culture, such as a human cell.
  • compositions comprising the subject chemically- modified double stranded nucleic acid molecules, and a pharmaceutically acceptable carrier or diluent.
  • the method may be carried out in vitro, ex vivo, or in vivo, in, for example, mammalian cells in culture, such as a human cell in culture.
  • the target cells e.g., mammalian cell
  • a longer duplex polynucleotide including a first polynucleotide that ranges in size from about 16 to about 30 nucleotides; a second polynucleotide that ranges in size from about 26 to about 46 nucleotides, wherein the first polynucleotide (the antisense strand) is complementary to both the second polynucleotide (the sense strand) and a target gene, and wherein both polynucleotides form a duplex and wherein the first polynucleotide contains a single stranded region longer than 6 bases in length and is modified with alternative chemical modification pattern, and/or includes a conjugate moiety that facilitates cellular delivery.
  • between about 40% to about 90% of the nucleotides of the passenger strand between about 40% to about 90% of the nucleotides of the guide strand, and between about 40% to about 90% of the nucleotides of the single stranded region of the first polynucleotide are chemically modified nucleotides.
  • the chemically modified nucleotide in the polynucleotide duplex may be any chemically modified nucleotide known in the art, such as those discussed in detail above.
  • the chemically modified nucleotide is selected from the group consisting of 2’ F modified nucleotides ,2'-O-methyl modified and 2’deoxy nucleotides.
  • the chemically modified nucleotides results from “hydrophobic modifications” of the nucleotide base.
  • the chemically modified nucleotides are phosphorothioates.
  • chemically modified nucleotides are combination of phosphorothioates, 2’-O- methyl, 2’deoxy, hydrophobic modifications and phosphorothioates. As these groups of modifications refer to modification of the ribose ring, back bone and nucleotide, it is feasible that some modified nucleotides will carry a combination of all three modification types. In another embodiment, the chemical modification is not the same across the various regions of the duplex.
  • the first polynucleotide (the passenger strand), has a large number of diverse chemical modifications in various positions.
  • this polynucleotide up to 90% of nucleotides might be chemically modified and/or have mismatches introduced.
  • chemical modifications of the first or second polynucleotide include, but not limited to, 5’ position modification of Uridine and Cytosine (4-pyridyl, 2- pyridyl, indolyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc.), where the chemical modification might alter base pairing capabilities of a nucleotide.
  • 5’ position modification of Uridine and Cytosine (4-pyridyl, 2- pyridyl, indolyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), isobutyl, butyl, aminobenzyl; phenyl; naphthyl, etc.
  • the chemical modification might alter base pairing capabilities of a nucleotide.
  • the hydrophobic modifications are preferably positioned near the 5’ end of the guide strand, in other embodiments, they localized in the middle of the guides strand, in other embodiment they localized at the 3’ end of the guide strand and yet in another embodiment they are distributed thought the whole length of the polynucleotide.
  • the same type of patterns is applicable to the passenger strand of the duplex.
  • the other part of the molecule is a single stranded region. The single stranded region is expected to range from 7 to 40 nucleotides.
  • the single stranded region of the first polynucleotide contains modifications selected from the group consisting of between 40% and 90% hydrophobic base modifications, between 40%-90% phosphorothioates, between 40% -90% modification of the ribose moiety, and any combination of the preceding.
  • Efficiency of guide strand (first polynucleotide) loading into the RISC complex might be altered for heavily modified polynucleotides, so in one embodiment, the duplex polynucleotide includes a mismatch between nucleotide 9, 11, 12, 13, or 14 on the guide strand (first polynucleotide) and the opposite nucleotide on the sense strand (second polynucleotide) to promote efficient guide strand loading.
  • Double-stranded nucleic acid molecules of the disclosure may be formed by two separate complementary nucleic acid strands. Duplex formation can occur either inside or outside the cell containing the target gene.
  • the term “duplex” includes the region of the double-stranded nucleic acid molecule(s) that is (are) hydrogen bonded to a complementary sequence.
  • Double- stranded oligonucleotides of the disclosure may comprise a nucleotide sequence that is sense to a target gene and a complementary sequence that is antisense to the target gene.
  • the sense and antisense nucleotide sequences correspond to the target gene sequence, e.g., are identical or are sufficiently identical to effect target gene inhibition (e.g., are about at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical) to the target gene sequence.
  • the double-stranded oligonucleotide of the disclosure is double-stranded over its entire length, i.e., with no overhanging single-stranded sequence at either end of the molecule, i.e., is blunt-ended.
  • the individual nucleic acid molecules can be of different lengths.
  • a double-stranded oligonucleotide of the disclosure is not double-stranded over its entire length.
  • one of the molecules e.g., the first molecule comprising an antisense sequence
  • the second molecule hybridizing thereto leaving a portion of the molecule single-stranded.
  • a single nucleic acid molecule is used a portion of the molecule at either end can remain single-stranded.
  • a double-stranded nucleic acid molecule of the disclosure contains mismatches and/or loops or bulges, but is double-stranded over at least about 70% of the length of the oligonucleotide.
  • a double-stranded oligonucleotide of the disclosure is double-stranded over at least about 80% of the length of the oligonucleotide.
  • a double-stranded oligonucleotide of the disclosure is double-stranded over at least about 90%-95% of the length of the oligonucleotide.
  • a double-stranded oligonucleotide of the disclosure is double-stranded over at least about 96%-98% of the length of the oligonucleotide.
  • the double-stranded oligonucleotide of the disclosure contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • the nucleotides of the disclosure may be modified at various locations, including the sugar moiety, the phosphodiester linkage, and/or the base.
  • the base moiety of a nucleoside may be modified.
  • a pyrimidine base may be modified at the 2, 3, 4, 5, and/or 6 position of the pyrimidine ring.
  • the exocyclic amine of cytosine may be modified.
  • a purine base may also be modified.
  • a purine base may be modified at the 1, 2, 3, 6, 7, or 8 position.
  • the exocyclic amine of adenine may be modified.
  • a nitrogen atom in a ring of a base moiety may be substituted with another atom, such as carbon.
  • a modification to a base moiety may be any suitable modification. Examples of modifications are known to those of ordinary skill in the art.
  • the base modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles.
  • a pyrimidine may be modified at the 5 position.
  • the 5 position of a pyrimidine may be modified with an alkyl group, an alkynyl group, an alkenyl group, an acyl group, or substituted derivatives thereof.
  • the 5 position of a pyrimidine may be modified with a hydroxyl group or an alkoxyl group or substituted derivative thereof.
  • the N 4 position of a pyrimidine may be alkylated.
  • the pyrimidine 5-6 bond may be saturated, a nitrogen atom within the pyrimidine ring may be substituted with a carbon atom, and/or the O 2 and O 4 atoms may be substituted with sulfur atoms.
  • N 7 position and/or N 2 and/or N 3 position of a purine may be modified with an alkyl group or substituted derivative thereof.
  • a third ring may be fused to the purine bicyclic ring system and/or a nitrogen atom within the purine ring system may be substituted with a carbon atom.
  • Non-limiting examples of pyrimidines modified at the 5 position are disclosed in U.S. Patent 5591843, U.S. Patent 7,205,297, U.S. Patent 6,432,963, and U.S.
  • Patent 6,020,483; non-limiting examples of pyrimidines modified at the N 4 position are disclosed in U.S. Patent 5,580,731; non-limiting examples of purines modified at the 8 position are disclosed in U.S. Patent 6,355,787 and U.S. Patent 5,580,972; non-limiting examples of purines modified at the N 6 position are disclosed in U.S. Patent 4,853,386, U.S. Patent 5,789,416, and U.S. Patent 7,041,824; and non-limiting examples of purines modified at the 2 position are disclosed in U.S. Patent 4,201,860 and U.S. Patent 5,587,469, all of which are incorporated herein by reference.
  • Non-limiting examples of modified bases include N 4 ,N 4 -ethanocytosine, 7- deazaxanthosine, 7-deazaguanosine, 8-oxo-N 6 -methyladenine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, dihydrouracil, inosine, N 6 -isopentenyl-adenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N 6 -methyladenine, 7-methylguanine, 5-methylaminomethyl uracil, 5- methoxy aminomethyl-2-thiouracil, 5-methoxyura
  • the base moiety may be a heterocyclic base other than a purine or pyrimidine.
  • the heterocyclic base may be optionally modified and/or substituted.
  • Sugar moieties include natural, unmodified sugars, e.g., monosaccharide (such as pentose, e.g., ribose, deoxyribose), modified sugars and sugar analogs.
  • possible modifications of nucleomonomers, particularly of a sugar moiety include, for example, replacement of one or more of the hydroxyl groups with a halogen, a heteroatom, an aliphatic group, or the functionalization of the hydroxyl group as an ether, an amine, a thiol, or the like.
  • modified nucleomonomers are 2’-O-methyl nucleotides. Such 2’-O-methyl nucleotides may be referred to as “methylated,” and the corresponding nucleotides may be made from unmethylated nucleotides followed by alkylation or directly from methylated nucleotide reagents. Modified nucleomonomers may be used in combination with unmodified nucleomonomers. For example, an oligonucleotide of the disclosure may contain both methylated and unmethylated nucleomonomers. Some exemplary modified nucleomonomers include sugar- or backbone-modified ribonucleotides.
  • Modified ribonucleotides may contain a non-naturally occurring base (instead of a naturally occurring base), such as uridines or cytidines modified at the 5’- position, e.g., 5’-(2-amino)propyl uridine and 5’-bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza- adenosine; and N-alkylated nucleotides, e.g., N6-methyl adenosine.
  • uridines or cytidines modified at the 5’- position e.g., 5’-(2-amino)propyl uridine and 5’-bromo uridine
  • adenosines and guanosines modified at the 8-position e.g., 8-
  • sugar-modified ribonucleotides may have the 2’-OH group replaced by a H, alxoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH 2 , NHR, NR 2, ), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
  • Modified ribonucleotides may also have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, e.g., of phosphorothioate group. More generally, the various nucleotide modifications may be combined.
  • the antisense (guide) strand may be substantially identical to at least a portion of the target gene (or genes), at least with respect to the base pairing properties, the sequence need not be perfectly identical to be useful, e.g., to inhibit expression of a target gene's phenotype. Generally, higher homology can be used to compensate for the use of a shorter antisense gene. In some cases, the antisense strand generally will be substantially identical (although in antisense orientation) to the target gene.
  • the use of 2′-O-methyl modified RNA may also be beneficial in circumstances in which it is desirable to minimize cellular stress responses. RNA having 2′-O-methyl nucleomonomers may not be recognized by cellular machinery that is thought to recognize unmodified RNA.
  • RNAi e.g., siRNA
  • the sugar moiety can be a hexose and incorporated into an oligonucleotide as described (Augustyns, K., et al., Nucl. Acids. Res. 18:4711 (1992)).
  • Exemplary nucleomonomers can be found, e.g., in U.S. Pat. No.5,849,902, incorporated by reference herein. Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed., inside cover, and specific functional groups are generally defined as described therein.
  • a particular enantiomer of a compound of the present disclosure may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • the molecule contains a basic functional group, such as amino, or an acidic functional group, such as carboxyl, diastereomeric salts are formed with an appropriate optically-active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • oligonucleotides of the disclosure comprise 3′ and 5′ termini (except for circular oligonucleotides).
  • the 3′ and 5′ termini of an oligonucleotide can be substantially protected from nucleases e.g., by modifying the 3′ or 5′ linkages (e.g., U.S. Pat. No.5,849,902 and WO 98/13526).
  • oligonucleotides can be made resistant by the inclusion of a “blocking group.”
  • blocking group refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-O-CH2-CH2-O-) phosphate (PO3 2- ), hydrogen phosphonate, or phosphoramidite).
  • Blocking groups also include “end blocking groups” or “exonuclease blocking groups” which protect the 5′ and 3′ termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • Exemplary end-blocking groups include cap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3′-3′ or 5′-5′ end inversions (see, e.g., Ortiagao et al.1992. Antisense Res.
  • the 3′ terminal nucleomonomer can comprise a modified sugar moiety.
  • the 3′ terminal nucleomonomer comprises a 3′-O that can optionally be substituted by a blocking group that prevents 3′- exonuclease degradation of the oligonucleotide.
  • the 3′-hydroxyl can be esterified to a nucleotide through a 3′ ⁇ 3′ internucleotide linkage.
  • the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy.
  • the 3′ ⁇ 3′linked nucleotide at the 3′ terminus can be linked by a substitute linkage.
  • the 5′ most 3′ ⁇ 5′ linkage can be a modified linkage, e.g., a phosphorothioate or a P-alkyloxyphosphotriester linkage.
  • the two 5′ most 3′ ⁇ 5′ linkages are modified linkages.
  • the 5′ terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g., phosphate, phosphorothioate, or P- ethoxyphosphate.
  • a phosphorus containing moiety e.g., phosphate, phosphorothioate, or P- ethoxyphosphate.
  • protecting group it is meant that a particular functional moiety, e.g., O, S, or N, is temporarily blocked so that a reaction can be carried out selectively at another reactive site in a multifunctional compound.
  • a protecting group reacts selectively in good yield to give a protected substrate that is stable to the projected reactions; the protecting group should be selectively removable in good yield by readily available, preferably non-toxic reagents that do not attack the other functional groups; the protecting group forms an easily separable derivative (more preferably without the generation of new stereogenic centers); and the protecting group has a minimum of additional functionality to avoid further sites of reaction.
  • oxygen, sulfur, nitrogen, and carbon protecting groups may be utilized.
  • Hydroxyl protecting groups include methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4- methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydr
  • the protecting groups include methylene acetal, ethylidene acetal, 1-t-butylethylidene ketal, 1-phenylethylidene ketal, (4-methoxyphenyl)ethylidene acetal, 2,2,2-trichloroethylidene acetal, acetonide, cyclopentylidene ketal, cyclohexylidene ketal, cycloheptylidene ketal, benzylidene acetal, p- methoxybenzylidene acetal, 2,4-dimethoxybenzylidene ketal, 3,4-dimethoxybenzylidene acetal, 2-nitrobenzylidene acetal, methoxymethylene acetal, ethoxymethylene acetal, dimethoxymethylene ortho ester, 1-methoxyethylidene ortho ester,
  • Amino-protecting groups include methyl carbamate, ethyl carbamante, 9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1- dimethyl
  • protecting groups are detailed herein. However, it will be appreciated that the present disclosure is not intended to be limited to these protecting groups; rather, a variety of additional equivalent protecting groups can be readily identified using the above criteria and utilized in the method of the present disclosure. Additionally, a variety of protecting groups are described in Protective Groups in Organic Synthesis, Third Ed. Greene, T.W. and Wuts, P.G., Eds., John Wiley & Sons, New York: 1999, the entire contents of which are hereby incorporated by reference. It will be appreciated that the compounds, as described herein, may be substituted with any number of substituents or functional moieties.
  • substituted refers to the replacement of hydrogen radicals in a given structure with the radical of a specified substituent. When more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • substituted is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms.
  • this disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • Combinations of substituents and variables envisioned by this disclosure are preferably those that result in the formation of stable compounds useful in the treatment, for example, of infectious diseases or proliferative disorders.
  • the term “stable”, as used herein, preferably refers to compounds which possess stability sufficient to allow manufacture and which maintain the integrity of the compound for a sufficient period of time to be detected and preferably for a sufficient period of time to be useful for the purposes detailed herein.
  • aliphatic includes both saturated and unsaturated, straight chain (i.e., unbranched), branched, acyclic, cyclic, or polycyclic aliphatic hydrocarbons, which are optionally substituted with one or more functional groups.
  • aliphatic is intended herein to include, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties.
  • alkyl includes straight, branched and cyclic alkyl groups.
  • alkyl alkenyl
  • alkynyl an analogous convention applies to other generic terms such as “alkenyl,” “alkynyl,” and the like.
  • the terms “alkyl,” “alkenyl,” “alkynyl,” and the like encompass both substituted and unsubstituted groups.
  • “lower alkyl” is used to indicate those alkyl groups (cyclic, acyclic, substituted, unsubstituted, branched, or unbranched) having 1-6 carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-20 aliphatic carbon atoms.
  • the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-10 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-8 aliphatic carbon atoms. In still other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl, and alkynyl groups employed in the disclosure contain 1-4 carbon atoms.
  • Illustrative aliphatic groups thus include, but are not limited to, for example, methyl, ethyl, n-propyl, isopropyl, cyclopropyl, -CH 2 -cyclopropyl, vinyl, allyl, n-butyl, sec- butyl, isobutyl, tert-butyl, cyclobutyl, -CH2-cyclobutyl, n-pentyl, sec-pentyl, isopentyl, tert- pentyl, cyclopentyl, -CH2-cyclopentyl, n-hexyl, sec-hexyl, cyclohexyl, -CH2-cyclohexyl moieties and the like, which again, may bear one or more substituents.
  • Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1- yl, and the like.
  • Representative alkynyl groups include, but are not limited to, ethynyl, 2- propynyl (propargyl), 1-propynyl, and the like.
  • heteroaliphatic refers to aliphatic moieties that contain one or more oxygen, sulfur, nitrogen, phosphorus, or silicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moieties may be branched, unbranched, cyclic or acyclic and include saturated and unsaturated heterocycles such as morpholino, pyrrolidinyl, etc.
  • heteroaliphatic moieties are substituted by independent replacement of one or more of the hydrogen atoms thereon with one or more moieties including, but not limited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio; heteroalkylthio; heteroarylthio; -F; -Cl; -Br; -I; -OH; -NO2; -CN; -CF3; -CH2CF3; -CHCl2; -CH2OH; -CH2CH2OH; -CH2NH2; - CH2SO2CH3; -C(O)Rx; -CO2(Rx); -CON(Rx)2; -OC(O)Rx; -OCO2Rx; -OCON(Rx)2; -N
  • alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,
  • a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1- C 6 for straight chain, C 3 -C 6 for branched chain), and more preferably 4 or fewer.
  • preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C1-C6 includes alkyl groups containing 1 to 6 carbon atoms.
  • alkyl includes both “unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sul
  • Cycloalkyls can be further substituted, e.g., with the substituents described above.
  • An “alkylaryl” or an “arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • the term “alkyl” also includes the side chains of natural and unnatural amino acids.
  • n-alkyl means a straight chain (i.e., unbranched) unsubstituted alkyl group.
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
  • alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched- chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups.
  • alkenyl includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonen
  • a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2- C 6 for straight chain, C 3 -C 6 for branched chain).
  • cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.
  • alkenyl includes both “unsubstituted alkenyls” and “substituted alkenyls,” the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
  • alkynyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond.
  • alkynyl includes straight-chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched- chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups.
  • a straight chain or branched chain alkynyl group has 6 or fewer carbon atoms in its backbone (e.g., C 2 -C 6 for straight chain, C 3 -C 6 for branched chain).
  • C 2 -C 6 includes alkynyl groups containing 2 to 6 carbon atoms.
  • alkynyl includes both “unsubstituted alkynyls” and “substituted alkynyls,” the latter of which refers to alkynyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to five carbon atoms in its backbone structure.
  • Lower alkenyl and “lower alkynyl” have chain lengths of, for example, 2-5 carbon atoms.
  • alkoxy includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkoxy groups include methoxy, ethoxy, isopropyloxy, propoxy, butoxy, and pentoxy groups. Examples of substituted alkoxy groups include halogenated alkoxy groups.
  • the alkoxy groups can be substituted with independently selected groups such as alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulffiydryl, alkylthio, arylthio, thio
  • halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy, trichloromethoxy, etc.
  • heteroatom includes atoms of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
  • hydroxy or “hydroxyl” includes groups with an -OH or -O ⁇ (with an appropriate counterion).
  • halogen includes fluorine, bromine, chlorine, iodine, etc.
  • perhalogenated generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
  • substituted includes independently selected substituents which can be placed on the moiety and which allow the molecule to perform its intended function.
  • substituents include alkyl, alkenyl, alkynyl, aryl, (CR′R′′)0-3NR′R′′, (CR′R′′)0- 3CN, NO2, halogen, (CR′R′′)0-3C(halogen)3, (CR′R′′)0-3CH(halogen)2, (CR′R′′)0- 3 CH 2 (halogen), (CR′R′′) 0-3 CONR′R′′, (CR′R′′) 0-3 S(O) 1-2 NR′R′′, (CR′R′′) 0-3 CHO, (CR′R′′) 0- 3 O(CR′R′′) 0-3 H, (CR′R′′) 0-3 S(O) 0-2 R′, (CR′R′′) 0-3 O(CR′R′′) 0-3 H, (CR′R′′) 0-3 COR′, (CR
  • amine or “amino” includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • ether includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms.
  • alkoxyalkyl which refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
  • polynucleotide refers to a polymer of two or more nucleotides.
  • the polynucleotides can be DNA, RNA, or derivatives or modified versions thereof.
  • the polynucleotide may be single-stranded or double-stranded.
  • the polynucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc.
  • the polynucleotide may comprise a modified base moiety which is selected from the group including but not limited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7- methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine
  • the olynucleotide may compirse a modified sugar moiety (e.g., 2′- fluororibose, ribose, 2′-deoxyribose, 2′-O-methylcytidine, arabinose, and hexose), and/or a modified phosphate moiety (e.g., phosphorothioates and 5′ -N-phosphoramidite linkages).
  • a nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes.
  • RNA double- or single- stranded genomic and cDNA
  • RNA any synthetic and genetically manipulated polynucleotide
  • sense and antisense polynucleotides This includes single- and double-stranded molecules, i.e., DNA-DNA, DNA-RNA, and RNA-RNA hybrids, as well as “protein nucleic acids” (PNA) formed by conjugating bases to an amino acid backbone.
  • PNA protein nucleic acids
  • base includes the known purine and pyrimidine heterocyclic bases, deazapurines, and analogs (including heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., 1-alkyl-, 1-alkenyl-, heteroaromatic- and 1-alkynyl derivatives) and tautomers thereof.
  • purines include adenine, guanine, inosine, diaminopurine, and xanthine and analogs (e.g., 8-oxo-N 6 -methyladenine or 7-diazaxanthine) and derivatives thereof.
  • Pyrimidines include, for example, thymine, uracil, and cytosine, and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(1-propynyl)uracil, 5-(1- propynyl)cytosine and 4,4-ethanocytosine).
  • suitable bases include non- purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.
  • the nucleomonomers of an oligonucleotide of the disclosure are RNA nucleotides.
  • the nucleomonomers of an oligonucleotide of the disclosure are modified RNA nucleotides.
  • oligonucleotides contain modified RNA nucleotides.
  • the term “nucleoside” includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose. Examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides. Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene, “Protective Groups in Organic Synthesis”, 2 nd Ed., Wiley-Interscience, New York, 1999).
  • nucleotide includes nucleosides which further comprise a phosphate group or a phosphate analog.
  • the nucleic acid molecules may be associated with a hydrophobic moiety for targeting and/or delivery of the molecule to a cell.
  • the hydrophobic moiety is associated with the nucleic acid molecule through a linker.
  • the association is through non-covalent interactions.
  • the association is through a covalent bond. Any linker known in the art may be used to associate the nucleic acid with the hydrophobic moiety.
  • Linkers known in the art are described in published international PCT applications, WO 92/03464, WO 95/23162, WO 2008/021157, WO 2009/021157, WO 2009/134487, WO 2009/126933, U.S. Patent Application Publication 2005/0107325, U.S. Patent 5,414,077, U.S. Patent 5,419,966, U.S. Patent 5,512,667, U.S. Patent 5,646,126, and U.S. Patent 5,652,359, which are incorporated herein by reference.
  • the linker may be as simple as a covalent bond to a multi-atom linker.
  • the linker may be cyclic or acyclic.
  • the linker may be optionally substituted.
  • the linker is capable of being cleaved from the nucleic acid. In certain embodiments, the linker is capable of being hydrolyzed under physiological conditions. In certain embodiments, the linker is capable of being cleaved by an enzyme (e.g., an esterase or phosphodiesterase). In certain embodiments, the linker comprises a spacer element to separate the nucleic acid from the hydrophobic moiety. The spacer element may include one to thirty carbon or heteroatoms. In certain embodiments, the linker and/or spacer element comprises protonatable functional groups. Such protonatable functional groups may promote the endosomal escape of the nucleic acid molecule.
  • the protonatable functional groups may also aid in the delivery of the nucleic acid to a cell, for example, neutralizing the overall charge of the molecule.
  • the linker and/or spacer element is biologically inert (that is, it does not impart biological activity or function to the resulting nucleic acid molecule).
  • the nucleic acid molecule with a linker and hydrophobic moiety is of the formulae described herein.
  • the nucleic acid molecule is of the formula: wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid.
  • the molecule is of the formula: . In certain embodiments, the molecule is of the formula: . In certain embodiments, the molecule is of the formula: . In certain embodiments, the molecule is of the formula: . In certain embodiments, the molecule is of the formula: . In certain embodiments, X is N. In certain embodiments, X is CH. In certain embodiments, A is a bond. In certain embodiments, A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted or unsubstituted, branched or unbranched aliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched aliphatic.
  • A is acyclic, substituted, unbranched aliphatic. In certain embodiments, A is acyclic, substituted, unbranched alkyl. In certain embodiments, A is acyclic, substituted, unbranched C 1-20 alkyl. In certain embodiments, A is acyclic, substituted, unbranched C 1-12 alkyl. In certain embodiments, A is acyclic, substituted, unbranched C1-10 alkyl. In certain embodiments, A is acyclic, substituted, unbranched C1-8 alkyl. In certain embodiments, A is acyclic, substituted, unbranched C 1-6 alkyl.
  • A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted, branched or unbranched heteroaliphatic. In certain embodiments, A is acyclic, substituted, unbranched heteroaliphatic. In certain embodiments, A is of the formula: . In certain embodiments, A is of one of the formulae: . embodiments, A is of one of the formulae: .
  • A is of one of the formulae: . In certain embodiments, A is of the formula: . In certain embodiments, A is of the formula: . In certain embodiments, A is of the formula: . In certain embodiments, A is of the formula: wherein each occurrence of R is independently the side chain of a natural or unnatural amino acid; and n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula: . In certain embodiments, each occurrence of R is independently the side chain of a natural amino acid. In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive.
  • A is of the formula: wherein n is an integer between 1 and 20, inclusive. In certain embodiments, A is of the formula: . In certain embodiments, n is an integer between 1 and 15, inclusive. In certain embodiments, n is an integer between 1 and 10, inclusive. In certain embodiments, n is an integer between 1 and 5, inclusive. In certain embodiments, A is of the formula:
  • n is an integer between 1 and 20, inclusive.
  • A is of the formula: .
  • n is an integer between 1 and 15, inclusive.
  • n is an integer between 1 and 10, inclusive.
  • n is an integer between 1 and 5, inclusive.
  • the molecule is of the formula: wherein X, R 1 , R 2 , and R 3 are as defined herein; and A′ is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic.
  • A′ is of one of the formulae: .
  • A is of one of the formulae: .
  • A is of one of the formulae: . In certain embodiments, A is of the formula: . In certain embodiments, A is of the formula: . In certain embodiments, R 1 is a steroid. In certain embodiments, R 1 is a cholesterol. In certain embodiments, R 1 is a lipophilic vitamin. In certain embodiments, R 1 is a vitamin A. In certain embodiments, R 1 is a vitamin E. In certain embodiments, R 1 is of the formula:
  • R A is substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic.
  • R 1 is of the formula: . In certain embodiments, R 1 is of the formula: .
  • X is N or CH;
  • A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic;
  • R 1 is a hydrophobic moiety;
  • R 2 is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid.
  • the nucleic acid molecule is of the formula: wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid.
  • the nucleic acid molecule is of the formula: wherein X is N or CH; A is a bond; substituted or unsubstituted, cyclic or acyclic, branched or unbranched aliphatic; or substituted or unsubstituted, cyclic or acyclic, branched or unbranched heteroaliphatic; R 1 is a hydrophobic moiety; R 2 is hydrogen; an oxygen-protecting group; cyclic or acyclic, substituted or unsubstituted, branched or unbranched aliphatic; cyclic or acyclic, substituted or unsubstituted, branched or unbranched heteroaliphatic; substituted or unsubstituted, branched or unbranched acyl; substituted or unsubstituted, branched or unbranched aryl; substituted or unsubstituted, branched or unbranched heteroaryl; and R 3 is a nucleic acid.
  • the nucleic acid molecule is of the formula: . In certain embodiments, the nucleic acid molecule is of the formula: . In certain embodiments, the nucleic acid molecule is of the formula: wherein R 3 is a nucleic acid. In certain embodiments, the nucleic acid molecule is of the formula: wherein R 3 is a nucleic acid; and n is an integer between 1 and 20, inclusive. In certain embodiments, the nucleic acid molecule is of the formula: .
  • the nucleic acid molecule is of the formula: In certain embodiments, the nucleic acid molecule is of the formula: In certain embodiments, the nucleic acid molecule is of the formula: In certain embodiments, the nucleic acid molecule is of the formula: As used herein, the term “substitute linkage” includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers.
  • Substitute linkages include phosphodiester analogs, e.g., phosphorothioate, phosphorodithioate, and P- ethyoxyphosphodiester, P-ethoxyphosphodiester, P-alkyloxyphosphotriester, methylphosphonate, and nonphosphorus containing linkages, e.g., acetals and amides.
  • Such substitute linkages are known in the art (e.g., Bjergarde et al.1991. Nucleic Acids Res. 19:5843; Caruthers et al.1991. Nucleosides Nucleotides.10:47).
  • non-hydrolizable linkages are preferred, such as phosphorothiate linkages.
  • oligonucleotides of the disclosure comprise hydrophobically modified nucleotides or “hydrophobic modifications.”
  • hydrophobic modifications refers to bases that are modified such that (1) overall hydrophobicity of the base is significantly increased, and/or (2) the base is still capable of forming close to regular Watson –Crick interaction.
  • base modifications include 5- position uridine and cytidine modifications such as phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H5OH); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; and naphthyl.
  • Other types of conjugates that can be attached to the end (3’ or 5’ end), a loop region, or any other parts of a chemically modified double stranded nucleic acid molecule include a sterol, sterol type molecule, peptide, small molecule, protein, etc.
  • a chemically modified double stranded nucleic acid molecule such as an INTASYLTM molecule, may contain more than one conjugate (same or different chemical nature).
  • the conjugate is cholesterol.
  • the first nucleotide relative to the 5’end of the guide strand has a 2'-O-methyl modification, optionally wherein the 2'-O-methyl modification is a 5P-2'O- methyl U modification, or a 5’ vinyl phosphonate 2’-O-methyl U modification.
  • Antisense (guide) sequences of the disclosure can be “chimeric oligonucleotides” which comprise an RNA-like and a DNA-like region.
  • the language “RNase H activating region” includes a region of an oligonucleotide, e.g., a chimeric oligonucleotide, that is capable of recruiting RNase H to cleave the target RNA strand to which the oligonucleotide binds.
  • the RNase activating region contains a minimal core (of at least about 3-5, typically between about 3-12, more typically, between about 5-12, and more preferably between about 5-10 contiguous nucleomonomers) of DNA or DNA-like nucleomonomers.
  • the RNase H activating region comprises about nine contiguous deoxyribose containing nucleomonomers.
  • non-activating region includes a region of an antisense sequence, e.g., a chimeric oligonucleotide, that does not recruit or activate RNase H.
  • a non- activating region does not comprise phosphorothioate DNA.
  • the oligonucleotides of the disclosure comprise at least one non-activating region.
  • the non- activating region can be stabilized against nucleases or can provide specificity for the target by being complementary to the target and forming hydrogen bonds with the target nucleic acid molecule, which is to be bound by the oligonucleotide.
  • at least a portion of the contiguous polynucleotides are linked by a substitute linkage, e.g., a phosphorothioate linkage.
  • nucleotides beyond the guide sequence (2’- modified or not) are linked by phosphorothioate linkages.
  • Such constructs tend to have improved pharmacokinetics due to their higher affinity for serum proteins.
  • the phosphorothioate linkages in the non-guide sequence portion of the polynucleotide generally do not interfere with guide strand activity, once the latter is loaded into RISC.
  • high levels of phosphorothioate modification can lead to improved delivery.
  • the guide and/or passenger strand is completely phosphorothioated.
  • Antisense (guide) sequences of the present disclosure may include “morpholino oligonucleotides.” Morpholino oligonucleotides are non-ionic and function by an RNase H- independent mechanism. Each of the 4 genetic bases (Adenine, Cytosine, Guanine, and Thymine/Uracil) of the morpholino oligonucleotides is linked to a 6-membered morpholine ring. Morpholino oligonucleotides are made by joining the 4 different subunit types by, e.g., non-ionic phosphorodiamidate inter-subunit linkages.
  • Morpholino oligonucleotides have many advantages including: complete resistance to nucleases (Antisense & Nucl. Acid Drug Dev.1996.6:267); predictable targeting (Biochemica Biophysica Acta.1999.1489:141); reliable activity in cells (Antisense & Nucl. Acid Drug Dev.1997.7:63); excellent sequence specificity (Antisense & Nucl. Acid Drug Dev.1997.7:151); minimal non-antisense activity (Biochemica Biophysica Acta.1999.1489:141); and simple osmotic or scrape delivery (Antisense & Nucl. Acid Drug Dev.1997.7:291).
  • Morpholino oligonucleotides are also preferred because of their non-toxicity at high doses.
  • a discussion of the preparation of morpholino oligonucleotides can be found in Antisense & Nucl. Acid Drug Dev.1997.7:187.
  • the chemical modifications described herein are believed to promote single stranded polynucleotide loading into the RISC.
  • Single stranded polynucleotides have been shown to be active in loading into RISC and inducing gene silencing. However, the level of activity for single stranded polynucleotides appears to be 2 to 4 orders of magnitude lower when compared to a duplex polynucleotide.
  • the present disclosure provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient loading of the polynucleotide into the RISC complex and (c) improve uptake of the single stranded nucleotide by the cell.
  • the chemical modification patterns may include a combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications.
  • the 5’ end of the single polynucleotide may be chemically phosphorylated.
  • the present disclosure provides a description of the chemical modification patterns, which improve functionality of RISC inhibiting polynucleotides.
  • Single stranded polynucleotides have been shown to inhibit activity of a preloaded RISC complex through the substrate competition mechanism.
  • antagomers the activity usually requires high concentration and in vivo delivery is not very effective.
  • the present disclosure provides a description of the chemical modification patterns, which may (a) significantly increase stability of the single stranded polynucleotide (b) promote efficient recognition of the polynucleotide by the RISC as a substrate and/or (c) improve uptake of the single stranded nucleotide by the cell.
  • the chemical modification patterns may include a combination of ribose, backbone, hydrophobic nucleoside and conjugate type of modifications.
  • the modifications provided by the present disclosure are applicable to all polynucleotides. This includes single stranded RISC entering polynucleotides, single stranded RISC inhibiting polynucleotides, conventional duplexed polynucleotides of variable length (15- 40 bp),asymmetric duplexed polynucleotides, and the like. Polynucleotides may be modified with wide variety of chemical modification patterns, including 5’ end, ribose, backbone and hydrophobic nucleoside modifications. Synthesis Oligonucleotides of the disclosure can be synthesized by any method known in the art, e.g., using enzymatic synthesis and/or chemical synthesis.
  • the oligonucleotides can be synthesized in vitro (e.g., using enzymatic synthesis and chemical synthesis) or in vivo (using recombinant DNA technology well known in the art).
  • chemical synthesis is used for modified polynucleotides.
  • Chemical synthesis of linear oligonucleotides is well known in the art and can be achieved by solution or solid phase techniques. Preferably, synthesis is by solid phase methods.
  • Oligonucleotides can be made by any of several different synthetic procedures including the phosphoramidite, phosphite triester, H-phosphonate, and phosphotriester methods, typically by automated synthesis methods.
  • Oligonucleotide synthesis protocols are well known in the art and can be found, e.g., in U.S. Pat. No.5,830,653; WO 98/13526; Stec et al.1984. J. Am. Chem. Soc.106:6077; Stec et al.1985. J. Org. Chem.50:3908; Stec et al. J. Chromatog.1985.326:263; LaPlanche et al. 1986. Nucl. Acid. Res.1986.14:9081; Fasman G. D., 1989. Practical Handbook of Biochemistry and Molecular Biology.1989. CRC Press, Boca Raton, Fla.; Lamone.1993. Biochem. Soc.
  • the phosphoramidite and phosphite triester method can produce oligonucleotides having 175 or more nucleotides, while the H-phosphonate method works well for oligonucleotides of less than 100 nucleotides.
  • modified bases are incorporated into the oligonucleotide, and particularly if modified phosphodiester linkages are used, then the synthetic procedures are altered as needed according to known procedures.
  • Uhlmann et al. (1990, Chemical Reviews 90:543-584) provide references and outline procedures for making oligonucleotides with modified bases and modified phosphodiester linkages.
  • Other exemplary methods for making oligonucleotides are taught in Sonveaux.1994.
  • oligonucleotide Synthesis is also taught in “Oligonucleotide Synthesis - A Practical Approach” (Gait, M. J. IRL Press at Oxford University Press.1984).
  • linear oligonucleotides of defined sequence including some sequences with modified nucleotides, are readily available from several commercial sources.
  • the oligonucleotides may be purified by polyacrylamide gel electrophoresis, or by any of a number of chromatographic methods, including gel chromatography and high pressure liquid chromatography.
  • oligonucleotides may be subjected to DNA sequencing by any of the known procedures, including Maxam and Gilbert sequencing, Sanger sequencing, capillary electrophoresis sequencing, the wandering spot sequencing procedure or by using selective chemical degradation of oligonucleotides bound to Hybond paper. Sequences of short oligonucleotides can also be analyzed by laser desorption mass spectroscopy or by fast atom bombardment (McNeal, et al., 1982, J. Am. Chem. Soc.104:976; Viari, et al., 1987, Biomed. Environ.
  • RNAi constructs or at least portions thereof are transcribed from expression vectors encoding the subject constructs. Any art recognized vectors may be use for this purpose.
  • RNAi constructs may be isolated and purified, before desired modifications (such as replacing an unmodified sense strand with a modified one, etc.) are carried out.
  • Delivery/Carrier e.g., the particular patterns of modifications on the passenger strand and/or guide strand of the double stranded nucleic acid molecules described herein (e.g., INTASYLTM molecule) may facilitate entry of the guide strand into the nucleus, where the guide strand mediates gene silencing (e.g., silencing of target genes, such as PD-1).
  • gene silencing e.g., silencing of target genes, such as PD-1).
  • the guide strand e.g., antisense strand of the nucleic acid molecule (e.g., INTASYLTM molecule) may dissociate from the passenger strand and enter into the nucleus as a single strand. Once in the nucleus the single stranded guide strand may associate with RNAse H or another ribonuclease and cleave the target (e.g., PD-1) (“Antisense mechanism of action”).
  • the target e.g., PD-1
  • the guide strand e.g., antisense strand of the nucleic acid molecule (e.g., INTASYLTM molecule) may associate with an Argonaute (Ago) protein in the cytoplasm or outside the nucleus, forming a loaded Ago complex.
  • This loaded Ago complex may translocate into the nucleus and then cleave the target (e.g., PD-1).
  • both strands e.g.
  • a duplex) of the nucleic acid molecule may enter the nucleus and the guide strand may associate with RNAse H, an Ago protein or another ribonuclease and cleaves the target (e.g., PD-1).
  • the sense strand of the double stranded molecules described herein e.g., INTASYLTM molecule sense strand
  • a passenger strand described herein is joined (e.g., covalently bound, non- covalently bound, conjugated, hybridized via a region of complementarity, etc.) to certain molecules (e.g., antisense oligonucleotides, ASO) for the purpose of targeting said other molecule to the nucleus of a cell.
  • the molecule joined to a sense strand described herein is a synthetic antisense oligonucleotide (ASO).
  • the sense strand joined to an anti-sense oligonucleotide is between 8-15 nucleotides long, chemically modified, and comprises a hydrophobic conjugate.
  • an ASO can be joined to a complementary passenger strand by hydrogen bonding.
  • the disclosure provides a method of delivering a nucleic acid molecule to a cell, the method comprising administering an isolated nucleic acid molecule to a cell, wherein the isolated nucleic acid comprises a sense strand which is complementary to an anti-sense oligonucleotide (ASO), wherein the sense strand is between 8-15 nucleotides in length, comprises at least two phosphorothioate modifications, at least 50% of the pyrimidines in the sense strand are modified, and wherein the molecule comprises a hydrophobic conjugate.
  • ASO anti-sense oligonucleotide
  • Oligonucleotides and oligonucleotide compositions are contacted with (i.e., brought into contact with, also referred to herein as administered or delivered to) and taken up by one or more cells or a cell lysate.
  • the term “cells” includes prokaryotic and eukaryotic cells, preferably vertebrate cells, and, more preferably, mammalian cells.
  • the oligonucleotide compositions of the disclosure are contacted with bacterial cells.
  • the oligonucleotide compositions of the disclosure are contacted with eukaryotic cells (e.g., plant cell, mammalian cell, arthropod cell, such as insect cell). In some embodiments, the oligonucleotide compositions of the disclosure are contacted with stem cells. In some embodiments, the oligonucleotide compositions of the disclosure are contacted with immune cells, such as T-cells (e.g., CD8+ T-cells). In some embodiments, the T-cells are T SCM or T CM T-cells. In a preferred embodiment, the oligonucleotide compositions of the disclosure are contacted with human cells.
  • eukaryotic cells e.g., plant cell, mammalian cell, arthropod cell, such as insect cell.
  • stem cells e.g., stem cells.
  • the oligonucleotide compositions of the disclosure are contacted with immune cells, such as T-cells (e.g., CD8
  • the oligonucleotide compositions of the disclosure are contacted with tumor cells through intratumoral injection.
  • Oligonucleotide compositions of the disclosure can be contacted with cells in vitro, e.g., in a test tube or culture dish, (and may or may not be introduced into a subject) or in vivo, e.g., in a subject such as a mammalian subject, or ex vivo.
  • oligonucleotides are administered topically or through electroporation.
  • Oligonucleotides are taken up by cells at a slow rate by endocytosis, but endocytosed oligonucleotides are generally sequestered and not available, e.g., for hybridization to a target nucleic acid molecule.
  • cellular uptake can be facilitated by electroporation or calcium phosphate precipitation.
  • electroporation or calcium phosphate precipitation are only useful for in vitro or ex vivo embodiments, are not convenient and, in some cases, are associated with cell toxicity.
  • delivery of oligonucleotides into cells can be enhanced by suitable art recognized methods including calcium phosphate, DMSO, glycerol or dextran, electroporation, or by transfection, e.g., using cationic, anionic, or neutral lipid compositions or liposomes using methods known in the art (see e.g., WO 90/14074; WO 91/16024; WO 91/17424; U.S. Pat. No.4,897,355; Bergan et al.1993. Nucleic Acids Research.21:3567).
  • Enhanced delivery of oligonucleotides can also be mediated by the use of vectors (See e.g., Shi, Y.2003.
  • the chemically modified double stranded nucleic acid molecules of the disclosure may be delivered by using various beta-glucan containing particles, referred to as GeRPs (glucan encapsulated RNA loaded particle), described in, and incorporated by reference from, US Provisional Application No.61/310,611, filed on March 4, 2010 and entitled “Formulations and Methods for Targeted Delivery to Phagocyte Cells.”
  • GeRPs beta-glucan containing particles
  • Such particles are also described in, and incorporated by reference from US Patent Publications US 2005/0281781 A1, and US 2010/0040656, and in PCT publications WO 2006/007372, and WO 2007/050643.
  • the chemically modified double stranded nucleic acid molecule may be hydrophobically modified and optionally may be associated with a lipid and/or amphiphilic peptide.
  • the beta-glucan particle is derived from yeast.
  • the payload trapping molecule is a polymer, such as those with a molecular weight of at least about 1000 Da, 10,000 Da, 50,000 Da, 100 kDa, 500 kDa, etc.
  • Preferred polymers include (without limitation) cationic polymers, chitosans, or PEI (polyethylenimine), etc.
  • Glucan particles can be derived from insoluble components of fungal cell walls such as yeast cell walls.
  • the yeast is Baker’s yeast.
  • Yeast-derived glucan molecules can include one or more of ß-(1,3)-Glucan, ß-(1,6)-Glucan, mannan and chitin.
  • a glucan particle comprises a hollow yeast cell wall whereby the particle maintains a three dimensional structure resembling a cell, within which it can complex with or encapsulate a molecule such as an RNA molecule.
  • glucan particles can be prepared by extraction of insoluble components from cell walls, for example by extracting Baker’s yeast (Fleischmann’s) with 1M NaOH/pH 4.0 H2O, followed by washing and drying. Methods of preparing yeast cell wall particles are discussed in, and incorporated by reference from U.S. Patents 4,810,646, 4,992,540, 5,082,936, 5,028,703, 5,032,401, 5,322,841, 5,401,727, 5,504,079, 5,607,677, 5,968,811, 6,242,594, 6,444,448, 6,476,003, US Patent Publications 2003/0216346, 2004/0014715 and 2010/0040656, and PCT published application WO02/12348.
  • Protocols for preparing glucan particles are also described in, and incorporated by reference from, the following references: Soto and Ostroff (2008), “Characterization of multilayered nanoparticles encapsulated in yeast cell wall particles for DNA delivery.” Bioconjug Chem 19(4):840-8; Soto and Ostroff (2007), “Oral Macrophage Mediated Gene Delivery System,” Nanotech, Volume 2, Chapter 5 (“Drug Delivery”), pages 378-381; and Li et al. (2007), “Yeast glucan particles activate murine resident macrophages to secrete proinflammatory cytokines via MyD88-and Syk kinase-dependent pathways.” Clinical Immunology 124(2):170-181. Glucan containing particles such as yeast cell wall particles can also be obtained commercially.
  • Nutricell MOS 55 from Biorigin (Sao Paolo, Brazil), SAF-Mannan (SAF Agri, Minneapolis, Minn.), Nutrex (Sensient Technologies, Milwaukee, Wis.), alkali-extracted particles such as those produced by Nutricepts (Nutricepts Inc., Burnsville, Minn.) and ASA Biotech, acid-extracted WGP particles from Biopolymer Engineering, and organic solvent-extracted particles such as Adjuvax TM from Alpha-beta Technology, Inc. (Worcester, Mass.) and microparticulate glucan from Novogen (Stamford, Conn.).
  • Glucan particles such as yeast cell wall particles can have varying levels of purity depending on the method of production and/or extraction. In some instances, particles are alkali-extracted, acid-extracted or organic solvent-extracted to remove intracellular components and/or the outer mannoprotein layer of the cell wall. Such protocols can produce particles that have a glucan (w/w) content in the range of 50% - 90%. In some instances, a particle of lower purity, meaning lower glucan w/w content may be preferred, while in other embodiments, a particle of higher purity, meaning higher glucan w/w content may be preferred. Glucan particles, such as yeast cell wall particles, can have a natural lipid content.
  • the particles can contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% or more than 20% w/w lipid.
  • the presence of natural lipids may assist in complexation or capture of RNA molecules.
  • Glucan containing particles typically have a diameter of approximately 2-4 microns, although particles with a diameter of less than 2 microns or greater than 4 microns are also compatible with aspects of the disclosure.
  • the RNA molecule(s) to be delivered can be complexed or “trapped” within the shell of the glucan particle.
  • the shell or RNA component of the particle can be labeled for visualization, as described in, and incorporated by reference from, Soto and Ostroff (2008) Bioconjug Chem 19:840. Methods of loading GeRPs are discussed further below.
  • the optimal protocol for uptake of oligonucleotides will depend upon a number of factors, the most crucial being the type of cells that are being used. Other factors that are important in uptake include, but are not limited to, the nature and concentration of the oligonucleotide, the confluence of the cells, the type of culture the cells are in (e.g., a suspension culture or plated) and the type of media in which the cells are grown.
  • compositions comprising RNAi constructs as described herein, and a pharmaceutically acceptable carrier or diluent.
  • the disclosure relates to immunogenic compositions comprising the RNAi constructs described herein, and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier includes appropriate solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, it can be used in the therapeutic compositions. Supplementary active ingredients can also be incorporated into the compositions.
  • oligonucleotides may be incorporated into liposomes or liposomes modified with polyethylene glycol or admixed with cationic lipids for parenteral administration. Incorporation of additional substances into the liposome, for example, antibodies reactive against membrane proteins found on specific target cells, can help target the oligonucleotides to specific cell types (e.g., immune cells, such as T-cells). Encapsulating agents entrap oligonucleotides within vesicles.
  • an oligonucleotide may be associated with a carrier or vehicle, e.g., liposomes or micelles, although other carriers could be used, as would be appreciated by one skilled in the art.
  • a carrier or vehicle e.g., liposomes or micelles
  • Liposomes are vesicles made of a lipid bilayer having a structure similar to biological membranes. Such carriers are used to facilitate the cellular uptake or targeting of the oligonucleotide, or improve the oligonucleotide's pharmacokinetic or toxicologic properties.
  • the oligonucleotides of the present disclosure may also be administered encapsulated in liposomes, pharmaceutical compositions wherein the active ingredient is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the oligonucleotides depending upon solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phopholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, or other materials of a hydrophobic nature.
  • the diameters of the liposomes generally range from about 15 nm to about 5 microns.
  • the use of liposomes as drug delivery vehicles offers several advantages. Liposomes increase intracellular stability, increase uptake efficiency and improve biological activity. Liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane.
  • liposomes can deliver nucleic acids to cells and that the nucleic acids remain biologically active.
  • a lipid delivery vehicle originally designed as a research tool such as Lipofectin or LIPOFECTAMINETM 2000, can deliver intact nucleic acid molecules to cells.
  • Specific advantages of using liposomes include the following: they are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues.
  • formulations associated with the disclosure might be selected for a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues.
  • Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids.
  • the use of well-validated mixtures of fatty acids and/or fat emulsions currently used in pharmacology for parenteral nutrition may be utilized.
  • Liposome based formulations are widely used for oligonucleotide delivery.
  • lipid or liposome formulations contain at least one positively charged lipid (cationic lipids).
  • the presence of this positively charged lipid is believed to be essential for obtaining a high degree of oligonucleotide loading and for enhancing liposome fusogenic properties.
  • Several methods have been performed and published to identify optimal positively charged lipid chemistries.
  • the commercially available liposome formulations containing cationic lipids are characterized by a high level of toxicity. In vivo limited therapeutic indexes have revealed that liposome formulations containing positive charged lipids are associated with toxicity (e.g., elevation in liver enzymes) at concentrations only slightly higher than concentration required to achieve RNA silencing.
  • Nucleic acids associated with the disclosure can be hydrophobically modified and can be encompassed within neutral nanotransporters. Further description of neutral nanotransporters is incorporated by reference from PCT Application PCT/US2009/005251, filed on September 22, 2009, and entitled “Neutral Nanotransporters.” Such particles enable quantitative oligonucleotide incorporation into non-charged lipid mixtures. The lack of toxic levels of cationic lipids in such neutral nanotransporter compositions is an important feature. As demonstrated in PCT/US2009/005251, oligonucleotides can effectively be incorporated into a lipid mixture that is free of cationic lipids and such a composition can effectively deliver a therapeutic oligonucleotide to a cell in a manner that it is functional.
  • a high level of activity was observed when the fatty mixture was composed of a phosphatidylcholine base fatty acid and a sterol such as a cholesterol.
  • one preferred formulation of neutral fatty mixture is composed of at least 20% of DOPC or DSPC and at least 20% of sterol such as cholesterol. Even as low as 1:5 lipid to oligonucleotide ratio was shown to be sufficient to get complete encapsulation of the oligonucleotide in a non-charged formulation.
  • the neutral nanotransporters compositions enable efficient loading of oligonucleotide into neutral fat formulation.
  • the composition includes an oligonucleotide that is modified in a manner such that the hydrophobicity of the molecule is increased (for example a hydrophobic molecule is attached (covalently or no-covalently) to a hydrophobic molecule on the oligonucleotide terminus or a non-terminal nucleotide, base, sugar, or backbone), the modified oligonucleotide being mixed with a neutral fat formulation (for example containing at least 25 % of cholesterol and 25% of DOPC or analogs thereof).
  • a cargo molecule, such as another lipid can also be included in the composition.
  • compositions where part of the formulation is built into the oligonucleotide itself, enables efficient encapsulation of oligonucleotide in neutral lipid particles.
  • stable particles ranging in size from 50 to 140 nm can be formed upon complexing of hydrophobic oligonucleotides with preferred formulations.
  • the formulation by itself typically does not form small particles, but rather, forms agglomerates, which are transformed into stable 50-120 nm particles upon addition of the hydrophobic modified oligonucleotide.
  • neutral nanotransporter compositions include a hydrophobic modified polynucleotide, a neutral fatty mixture, and optionally a cargo molecule.
  • a “hydrophobic modified polynucleotide” as used herein is a polynucleotide of the disclosure (e.g., sd-rxRNA) that has at least one modification that renders the polynucleotide more hydrophobic than the polynucleotide was prior to modification.
  • the modification may be achieved by attaching (covalently or non-covalently) a hydrophobic molecule to the polynucleotide.
  • the hydrophobic molecule is or includes a lipophilic group.
  • lipophilic group means a group that has a higher affinity for lipids than its affinity for water.
  • lipophilic groups include, but are not limited to, cholesterol, a cholesteryl or modified cholesteryl residue, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, such as steroids, vitamins, such as vitamin E, fatty acids either saturated or unsaturated, fatty acid esters, such as triglycerides, pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin, coumarin, fluorescein, rho
  • the cholesterol moiety may be reduced (e.g., as in cholestan) or may be substituted (e.g., by halogen).
  • a combination of different lipophilic groups in one molecule is also possible.
  • the hydrophobic molecule may be attached at various positions of the polynucleotide. As described above, the hydrophobic molecule may be linked to the terminal residue of the polynucleotide such as the 3’ of 5’-end of the polynucleotide. Alternatively, it may be linked to an internal nucleotide or a nucleotide on a branch of the polynucleotide. The hydrophobic molecule may be attached, for instance to a 2'-position of the nucleotide.
  • the hydrophobic molecule may also be linked to the heterocyclic base, the sugar or the backbone of a nucleotide of the polynucleotide.
  • the hydrophobic molecule may be connected to the polynucleotide by a linker moiety.
  • the linker moiety is a non-nucleotidic linker moiety.
  • Non-nucleotidic linkers are e.g. abasic residues (dSpacer), oligoethyleneglycol, such as triethyleneglycol (spacer 9) or hexaethylenegylcol (spacer 18), or alkane-diol, such as butanediol.
  • the spacer units are preferably linked by phosphodiester or phosphorothioate bonds.
  • the linker units may appear just once in the molecule or may be incorporated several times, e.g., via phosphodiester, phosphorothioate, methylphosphonate, or amide linkages.
  • Typical conjugation protocols involve the synthesis of polynucleotides bearing an aminolinker at one or more positions of the sequence, however, a linker is not required. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction may be performed either with the polynucleotide still bound to a solid support or following cleavage of the polynucleotide in solution phase.
  • the hydrophobic molecule is a sterol type conjugate, a PhytoSterol conjugate, cholesterol conjugate, sterol type conjugate with altered side chain length, fatty acid conjugate, any other hydrophobic group conjugate, and/or hydrophobic modifications of the internal nucleoside, which provide sufficient hydrophobicity to be incorporated into micelles.
  • sterols refers or steroid alcohols are a subgroup of steroids with a hydroxyl group at the 3-position of the A-ring.
  • sterols are amphipathic lipids synthesized from acetyl-coenzyme A via the HMG-CoA reductase pathway.
  • the overall molecule is quite flat.
  • the hydroxyl group on the A ring is polar.
  • the rest of the aliphatic chain is non-polar.
  • sterols are considered to have an 8 carbon chain at position 17.
  • the term “sterol type molecules”, refers to steroid alcohols, which are similar in structure to sterols. The main difference is the structure of the ring and number of carbons in a position 21 attached side chain.
  • the term “PhytoSterols” (also called plant sterols) are a group of steroid alcohols, phytochemicals naturally occurring in plants. There are more than 200 different known PhytoSterols
  • the term “Sterol side chain” refers to a chemical composition of a side chain attached at the position 17 of sterol-type molecule. In a standard definition sterols are limited to a 4 ring structure carrying a 8 carbon chain at position 17. In this disclosure, the sterol type molecules with side chain longer and shorter than conventional are described. The side chain may branched or contain double back bones.
  • sterols useful in the disclosure include cholesterols, as well as unique sterols in which position 17 has attached side chain of 2-7 or longer than 9 carbons.
  • the length of the polycarbon tail is varied between 5 and 9 carbons.
  • Such conjugates may have significantly better in vivo efficacy, in particular delivery to liver.
  • These types of molecules are expected to work at concentrations 5 to 9 fold lower then oligonucleotides conjugated to conventional cholesterols.
  • the polynucleotide may be bound to a protein, peptide or positively charged chemical that functions as the hydrophobic molecule.
  • the proteins may be selected from the group consisting of protamine, dsRNA binding domain, and arginine rich peptides.
  • Exemplary positively charged chemicals include spermine, spermidine, cadaverine, and putrescine.
  • hydrophobic molecule conjugates may demonstrate even higher efficacy when it is combined with optimal chemical modification patterns of the polynucleotide (as described herein in detail), containing but not limited to hydrophobic modifications, phosphorothioate modifications, and 2’ ribo modifications.
  • the sterol type molecule may be a naturally occurring PhytoSterols.
  • the polycarbon chain may be longer than 9 and may be linear, branched and/or contain double bonds.
  • Some PhytoSterols may demonstrate tissue preference and thus be used as a way to delivery RNAi specifically to particular tissues.
  • the hydrophobic modified polynucleotide is mixed with a neutral fatty mixture to form a micelle.
  • the neutral fatty acid mixture is a mixture of fats that has a net neutral or slightly net negative charge at or around physiological pH that can form a micelle with the hydrophobic modified polynucleotide.
  • the term “micelle” refers to a small nanoparticle formed by a mixture of non-charged fatty acids and phospholipids.
  • the neutral fatty mixture may include cationic lipids as long as they are present in an amount that does not cause toxicity.
  • the neutral fatty mixture is free of cationic lipids.
  • a mixture that is free of cationic lipids is one that has less than 1% and preferably 0% of the total lipid being cationic lipid.
  • the term “cationic lipid” includes lipids and synthetic lipids having a net positive charge at or around physiological pH.
  • anionic lipid includes lipids and synthetic lipids having a net negative charge at or around physiological pH.
  • the neutral fats bind to the oligonucleotides of the disclosure by a strong but non- covalent attraction (e.g., an electrostatic, van der Waals, pi-stacking, etc. interaction).
  • the neutral fat mixture may include formulations selected from a class of naturally occurring or chemically synthesized or modified saturated and unsaturated fatty acid residues.
  • Fatty acids might exist in a form of triglycerides, diglycerides or individual fatty acids.
  • the neutral fatty mixture is preferably a mixture of a choline based fatty acid and a sterol.
  • Choline based fatty acids include for instance, synthetic phosphocholine derivatives such as DDPC, DLPC, DMPC, DPPC, DSPC, DOPC, POPC, and DEPC.
  • DOPC (chemical registry number 4235-95-4) is dioleoylphosphatidylcholine (also known as dielaidoylphosphatidylcholine, dioleoyl-PC, dioleoylphosphocholine, dioleoyl-sn-glycero-3- phosphocholine, dioleylphosphatidylcholine).
  • DSPC (chemical registry number 816-94-4) is distearoylphosphatidylcholine (also known as 1,2-Distearoyl-sn-Glycero-3-phosphocholine).
  • the sterol in the neutral fatty mixture may be for instance cholesterol.
  • the neutral fatty mixture may be made up completely of a choline based fatty acid and a sterol or it may optionally include a cargo molecule.
  • the neutral fatty mixture may have at least 20% or 25% fatty acid and 20% or 25% sterol.
  • the term “Fatty acids” relates to conventional description of fatty acid. They may exist as individual entities or in a form of two-and triglycerides.
  • the term “fat emulsions” refers to safe fat formulations given intravenously to subjects who are unable to get enough fat in their diet. It is an emulsion of soy bean oil (or other naturally occurring oils) and egg phospholipids.
  • Fat emulsions are being used for formulation of some insoluble anesthetics.
  • fat emulsions might be part of commercially available preparations like Intralipid, Liposyn, Nutrilipid, modified commercial preparations, where they are enriched with particular fatty acids or fully de novo- formulated combinations of fatty acids and phospholipids.
  • the cells to be contacted with an oligonucleotide composition of the disclosure are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours.
  • the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days.
  • the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.
  • lipid or molecule can optionally be any other lipid or molecule.
  • a lipid or molecule is referred to herein as a cargo lipid or cargo molecule.
  • Cargo molecules include but are not limited to intralipid, small molecules, fusogenic peptides or lipids or other small molecules might be added to alter cellular uptake, endosomal release or tissue distribution properties. The ability to tolerate cargo molecules is important for modulation of properties of these particles, if such properties are desirable. For instance the presence of some tissue specific metabolites might drastically alter tissue distribution profiles. For example use of Intralipid type formulation enriched in shorter or longer fatty chains with various degrees of saturation affects tissue distribution profiles of these type of formulations (and their loads).
  • a cargo lipid useful according to the disclosure is a fusogenic lipid.
  • the zwiterionic lipid DOPE (chemical registry number 4004-5-1, 1,2-Dioleoyl- sn-Glycero-3-phosphoethanolamine) is a preferred cargo lipid.
  • Intralipid may be comprised of the following composition: 1000 mL contain: purified soybean oil 90 g, purified egg phospholipids 12 g, glycerol anhydrous 22 g, water for injection q.s. ad 1000 mL. pH is adjusted with sodium hydroxide to pH approximately 8. Energy content/L: 4.6 MJ (190 kcal). Osmolality (approx.): 300 mOsm/kg water.
  • fat emulsion is Liposyn that contains 5% safflower oil, 5% soybean oil, up to 1.2% egg phosphatides added as an emulsifier and 2.5% glycerin in water for injection. It may also contain sodium hydroxide for pH adjustment. pH 8.0 (6.0 - 9.0). Liposyn has an osmolarity of 276 m Osmol/liter (actual). Variation in the identity, amounts and ratios of cargo lipids affects the cellular uptake and tissue distribution characteristics of these compounds. For example, the length of lipid tails and level of saturability will affect differential uptake to liver, lung, fat and cardiomyocytes.
  • the fat emulsion is based on a mixture of lipids. Such lipids may include natural compounds, chemically synthesized compounds, purified fatty acids or any other lipids.
  • the composition of fat emulsion is entirely artificial.
  • the fat emulsion is more than 70% linoleic acid.
  • the fat emulsion is at least 1% of cardiolipin.
  • Linoleic acid is an unsaturated omega-6 fatty acid. It is a colorless liquid made of a carboxylic acid with an 18-carbon chain and two cis double bonds.
  • the alteration of the composition of the fat emulsion is used as a way to alter tissue distribution of hydrophobicly modified polynucleotides. This methodology provides for the specific delivery of the polynucleotides to particular tissues.
  • the fat emulsions of the cargo molecule contain more than 70% of Linoleic acid (C18H32O2) and/or cardiolipin.
  • Fat emulsions like intralipid have been used before as a delivery formulation for some non-water soluble drugs (such as Propofol, re-formulated as Diprivan).
  • Unique features of the present disclosure include (a) the concept of combining modified polynucleotides with the hydrophobic compound(s), so it can be incorporated in the fat micelles and (b) mixing it with the fat emulsions to provide a reversible carrier.
  • micelles After injection into a blood stream, micelles usually bind to serum proteins, including albumin, HDL, LDL and other. This binding is reversible and eventually the fat is absorbed by cells. The polynucleotide, incorporated as a part of the micelle will then be delivered closely to the surface of the cells.
  • oligonucleotides of the disclosure can be complexed with a complexing agent to increase cellular uptake of oligonucleotides.
  • a complexing agent includes cationic lipids. Cationic lipids can be used to deliver oligonucleotides to cells. However, as discussed above, formulations free in cationic lipids are preferred in some embodiments.
  • cationic lipid includes lipids and synthetic lipids having both polar and non-polar domains and which are capable of being positively charged at or around physiological pH and which bind to polyanions, such as nucleic acids, and facilitate the delivery of nucleic acids into cells.
  • cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides, or derivatives thereof.
  • Straight-chain and branched alkyl and alkenyl groups of cationic lipids can contain, e.g., from 1 to about 25 carbon atoms.
  • Preferred straight chain or branched alkyl or alkene groups have six or more carbon atoms.
  • Alicyclic groups include cholesterol and other steroid groups.
  • Cationic lipids can be prepared with a variety of counterions (anions) including, e.g., Cl ⁇ , Br ⁇ , I ⁇ , F ⁇ , acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • counterions e.g., Cl ⁇ , Br ⁇ , I ⁇ , F ⁇ , acetate, trifluoroacetate, sulfate, nitrite, and nitrate.
  • cationic lipids examples include polyethylenimine, polyamidoamine (PAMAM) starburst dendrimers, Lipofectin (a combination of DOTMA and DOPE), Lipofectase, LIPOFECTAMINETM (e.g., LIPOFECTAMINETM 2000), DOPE, Cytofectin (Gilead Sciences, Foster City, Calif.), and Eufectins (JBL, San Luis Obispo, Calif.).
  • Exemplary cationic liposomes can be made from N-[1-(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium chloride (DOTMA), N-[1 -(2,3-dioleoloxy)-propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP), 3 ⁇ -[N-(N′,N′- dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), 2,3,-dioleyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide; and dimethyldioctadecylammonium bromide (DDAB).
  • DOTMA N-[1-(
  • DOTMA cationic lipid N-(1-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride
  • Cationic lipids have been used in the art to deliver oligonucleotides to cells (see, e.g., U.S. Pat. Nos.5,855,910; 5,851,548; 5,830,430; 5,780,053; 5,767,099; Lewis et al.1996. Proc. Natl. Acad. Sci. USA 93:3176; Hope et al.1998. Molecular Membrane Biology 15:1).
  • Other lipid compositions which can be used to facilitate uptake of the instant oligonucleotides can be used in connection with the claimed methods.
  • other lipid compositions are also known in the art and include, e.g., those taught in U.S. Pat. No.
  • lipid compositions can further comprise agents, e.g., viral proteins to enhance lipid-mediated transfections of oligonucleotides (Kamata, et al., 1994. Nucl. Acids. Res.22:536).
  • oligonucleotides are contacted with cells as part of a composition comprising an oligonucleotide, a peptide, and a lipid as taught, e.g., in U.S. patent 5,736,392.
  • Improved lipids have also been described which are serum resistant (Lewis, et al., 1996.
  • Cationic lipids and other complexing agents act to increase the number of oligonucleotides carried into the cell through endocytosis.
  • N-substituted glycine oligonucleotides can be used to optimize uptake of oligonucleotides. Peptoids have been used to create cationic lipid-like compounds for transfection (Murphy, et al., 1998. Proc. Natl. Acad. Sci.95:1517). Peptoids can be synthesized using standard methods (e.g., Zuckermann, R. N., et al.1992. J. Am. Chem.
  • Combinations of cationic lipids and peptoids, liptoids can also be used to optimize uptake of the subject oligonucleotides (Hunag, et al., 1998. Chemistry and Biology.5:345). Liptoids can be synthesized by elaborating peptoid oligonucleotides and coupling the amino terminal submonomer to a lipid via its amino group (Hunag, et al., 1998. Chemistry and Biology. 5:345).
  • a composition for delivering oligonucleotides of the disclosure comprises a number of arginine, lysine, histidine or ornithine residues linked to a lipophilic moiety (see e.g., U.S. Pat. No.5,777,153).
  • a composition for delivering oligonucleotides of the disclosure comprises a peptide having from between about one to about four basic residues.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine (can also be considered non-polar
  • asparagine, glutamine, serine, threonine, tyrosine, cysteine nonpolar side chains
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • a majority or all of the other residues of the peptide can be selected from the non-basic amino acids, e.g., amino acids other than lysine, arginine, or histidine. Preferably a preponderance of neutral amino acids with long neutral side chains are used.
  • a composition for delivering oligonucleotides of the disclosure comprises a natural or synthetic polypeptide having one or more gamma carboxyglutamic acid residues, or ⁇ -Gla residues. These gamma carboxyglutamic acid residues may enable the polypeptide to bind to each other and to membrane surfaces.
  • a polypeptide having a series of ⁇ -Gla may be used as a general delivery modality that helps an RNAi construct to stick to whatever membrane to which it comes in contact. This may at least slow RNAi constructs from being cleared from the blood stream and enhance their chance of homing to the target.
  • the gamma carboxyglutamic acid residues may exist in natural proteins (for example, prothrombin has 10 ⁇ -Gla residues).
  • they can be introduced into the purified, recombinantly produced, or chemically synthesized polypeptides by carboxylation using, for example, a vitamin K-dependent carboxylase.
  • the gamma carboxyglutamic acid residues may be consecutive or non-consecutive, and the total number and location of such gamma carboxyglutamic acid residues in the polypeptide can be regulated / fine tuned to achieve different levels of "stickiness"of the polypeptide.
  • the cells to be contacted with an oligonucleotide composition of the disclosure are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 12 hours to about 24 hours.
  • the cells to be contacted with an oligonucleotide composition are contacted with a mixture comprising the oligonucleotide and a mixture comprising a lipid, e.g., one of the lipids or lipid compositions described supra for between about 1 and about five days.
  • the cells are contacted with a mixture comprising a lipid and the oligonucleotide for between about three days to as long as about 30 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about five to about 20 days.
  • a mixture comprising a lipid is left in contact with the cells for at least about seven to about 15 days.
  • an oligonucleotide composition can be contacted with cells in the presence of a lipid such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as described herein.
  • a lipid such as cytofectin CS or GSV (available from Glen Research; Sterling, Va.), GS3815, GS2888 for prolonged incubation periods as described herein.
  • the incubation of the cells with the mixture comprising a lipid and an oligonucleotide composition does not reduce the viability of the cells.
  • the cells are substantially viable.
  • the cells are between at least about 70% and at least about 100% viable.
  • the cells are between at least about 80% and at least about 95% viable.
  • the cells are between at least about 85% and at least about 90% viable.
  • oligonucleotides are modified by attaching a peptide sequence that transports the oligonucleotide into a cell, referred to herein as a “transporting peptide.”
  • the composition includes an oligonucleotide which is complementary to a target nucleic acid molecule encoding the protein, and a covalently attached transporting peptide.
  • transporting peptide includes an amino acid sequence that facilitates the transport of an oligonucleotide into a cell.
  • Exemplary peptides which facilitate the transport of the moieties to which they are linked into cells are known in the art, and include, e.g., HIV TAT transcription factor, lactoferrin, Herpes VP22 protein, and fibroblast growth factor 2 (Pooga et al.1998. Nature Biotechnology.16:857; and Derossi et al.1998. Trends in Cell Biology.8:84; Elliott and O'Hare.1997. Cell 88:223). Oligonucleotides can be attached to the transporting peptide using known techniques, e.g., ( Prochiantz, A.1996. Curr. Opin. Neurobiol.6:629; Derossi et al.1998.
  • oligonucleotides bearing an activated thiol group are linked via that thiol group to a cysteine present in a transport peptide (e.g., to the cysteine present in the ⁇ turn between the second and the third helix of the antennapedia homeodomain as taught, e.g., in Derossi et al.1998. Trends Cell Biol.8:84; Prochiantz.1996.
  • a Boc-Cys-(Npys)OH group can be coupled to the transport peptide as the last (N-terminal) amino acid and an oligonucleotide bearing an SH group can be coupled to the peptide (Troy et al.1996. J. Neurosci.16:253).
  • a linking group can be attached to a nucleomonomer and the transporting peptide can be covalently attached to the linker.
  • a linker can function as both an attachment site for a transporting peptide and can provide stability against nucleases.
  • linkers examples include substituted or unsubstituted C 1 -C 20 alkyl chains, C 2 -C 20 alkenyl chains, C 2 -C 20 alkynyl chains, peptides, and heteroatoms (e.g., S, O, NH, etc.).
  • Other exemplary linkers include bifinctional crosslinking agents such as sulfosuccinimidyl-4-(maleimidophenyl)-butyrate (SMPB) (see, e.g., Smith et al. Biochem J 1991.276: 417-2).
  • SMPB sulfosuccinimidyl-4-(maleimidophenyl)-butyrate
  • oligonucleotides of the disclosure are synthesized as molecular conjugates which utilize receptor-mediated endocytotic mechanisms for delivering genes into cells (see, e.g., Bunnell et al.1992. Somatic Cell and Molecular Genetics.18:559, and the references cited therein).
  • Other carriers for in vitro and/or in vivo delivery of RNAi reagents are known in the art, and can be used to deliver the subject RNAi constructs (e.g., to a host cell, such as a T- cell). See, for example, U.S.
  • Therapeutic methods Examples of subjects include mammals, e.g., humans and other primates; cows, pigs, horses, and farming (agricultural) animals; dogs, cats, and other domesticated pets; mice, rats, and transgenic non-human animals.
  • the formulations of the present disclosure can be administered to a patient in a variety of forms adapted to the chosen route of administration, e.g., intratumorally, parenterally, orally, or intraperitoneally.
  • Parenteral administration which is preferred, includes administration by the following routes: intravenous; intramuscular; interstitially; intraarterially; subcutaneous; intra ocular; intranodal; intratumorally; intrasynovial; trans epithelial, including transdermal; pulmonary via inhalation; ophthalmic; sublingual and buccal; topically, including ophthalmic; dermal; ocular; rectal; and nasal inhalation via insufflation.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension include, for example, sodium carboxymethyl cellulose, sorbitol, or dextran, optionally, the suspension may also contain stabilizers.
  • the oligonucleotides of the disclosure can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligonucleotides may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included in the disclosure.
  • Drug delivery vehicles can be chosen e.g., for in vitro, for systemic administration. These vehicles can be designed to serve as a slow release reservoir or to deliver their contents directly to the target cell. An advantage of using some direct delivery drug vehicles is that multiple molecules are delivered per uptake. Such vehicles have been shown to increase the circulation half-life of drugs that would otherwise be rapidly cleared from the blood stream.
  • an active amount of an oligonucleotide of the present disclosure is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • an active amount of an oligonucleotide may vary according to factors such as the type of cell, the oligonucleotide used, and for in vivo uses the disease state, age, sex, and weight of the individual, and the ability of the oligonucleotide to elicit a desired response in the individual.
  • oligonucleotides within the cell is dependent upon the rates of uptake and efflux or degradation. Decreasing the degree of degradation prolongs the intracellular half-life of the oligonucleotide.
  • chemically-modified oligonucleotides e.g., with modification of the phosphate backbone, may require different dosing.
  • the exact dosage of a chemically-modified double stranded nucleic acid molecule and number of doses administered will depend upon the data generated experimentally and in clinical trials. Several factors such as the desired effect, the delivery vehicle, disease indication, and the route of administration, will affect the dosage. Dosages can be readily determined by one of ordinary skill in the art and formulated into the subject pharmaceutical compositions.
  • the duration of treatment will extend at least through the course of the disease symptoms.
  • Dosage regimens may be adjusted to provide the optimum therapeutic response.
  • the chemically-modified double stranded nucleic acid molecule may be repeatedly administered, e.g., several doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • One of ordinary skill in the art will readily be able to determine appropriate doses and schedules of administration of the subject chemically-modified double stranded nucleic acid molecules, whether they are to be administered to cells or to subjects.
  • Administration of chemically-modified double stranded nucleic acid molecules, such as through intratumoral injection can be optimized through testing of dosing regimens.
  • a single administration is sufficient.
  • the chemically-modified double stranded nucleic acid molecule can be administered in a slow-release formulation or device, as would be familiar to one of ordinary skill in the art.
  • the chemically-modified double stranded nucleic acid molecule is administered multiple times. In some instances it is administered daily, bi- weekly, weekly, every two weeks, every three weeks, monthly, every two months, every three months, every four months, every five months, every six months or less frequently than every six months. In some instances, it is administered multiple times per day, week, month and/or year.
  • aspects of the disclosure relate to administering chemically-modified double stranded nucleic acid molecules to a subject.
  • the subject is a patient and administering the chemically-modified double stranded nucleic acid molecule involves administering the chemically-modified double stranded nucleic acid molecule in a doctor’s office.
  • more than one chemically-modified double stranded nucleic acid molecule is administered simultaneously.
  • compositions may be administered that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 different chemically- modified double stranded nucleic acid molecules.
  • a composition comprises 2 or 3 different chemically-modified double stranded nucleic acid molecules.
  • one or more anticancer agents is administered to a subject in combination with one or more chemically-modified double stranded nucleic acid molecules as described by the disclosure.
  • An “anticancer agent” can be a small molecule, nucleic acid, protein, peptide, polypeptide (e.g., antibody, antibody fragment, etc.), or any combination of the foregoing.
  • an anticancer agent is administered to the subject prior to administration of the chemically-modified double stranded nucleic acid molecule. In some embodiments, an anticancer agent is administered to a subject after administration of the chemically-modified double stranded nucleic acid molecule. In some embodiments, an anticancer agent is administered concurrently (e.g., at the same time as) with a chemically- modified double stranded nucleic acid molecule.
  • anticancer agents include but are not limited to Abitrexate (Methotrexate), Abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), Ado- Trastuzumab Emtansine, Adriamycin PFS (Doxorubicin Hydrochloride), Adriamycin RDF (Doxorubicin Hydrochloride), Adrucil (Fluorouracil), Afinitor (Everolimus), Anastrozole, Aredia (Pamidronate Disodium), Arimidex (Anastrozole), Aromasin (Exemestane), CapecitabineClafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Docetaxel, Doxorubicin Hydrochloride, Efudex (Fluorouracil), Ellence (Epirubicin Hydrochloride), Epirubicin Hydrochloride, Everolimus, Exemestane, Farest
  • ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. Moreover, for the purposes of the present disclosure, the term “a” or “an” entity refers to one or more of that entity; for example, “a protein” or “a nucleic acid molecule” refers to one or more of those compounds or at least one compound.
  • a compound “selected from the group consisting of” refers to one or more of the compounds in the list that follows, including mixtures (i.e., combinations) of two or more of the compounds.
  • an isolated, or biologically pure, protein or nucleic acid molecule is a compound that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the compound has been purified.
  • An isolated compound of the present disclosure can be obtained from its natural source, can be produced using molecular biology techniques or can be produced by chemical synthesis. Compositions and methods described herein are further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference. EXAMPLES Example 1. Phase 1b Study for INTASYLTM targeting PD-1 A Phase1b, open-label non-randomized, single-arm dose-ranging study is being conducted.
  • This study is designed to evaluate the safety and tolerability of neoadjuvant intratumoral INTASYLTM targeting PD-1 in adult ( ⁇ 18 years) subjects with cutaneous squamous cell carcinoma, melanoma, or Merkel cell carcinoma.
  • the study treatment consists of IT injections of an INTASYLTM targeting PD-1 administered four times at weekly intervals, into a single tumor lesion.
  • the investigational product is formulated in a phosphate buffered saline solution.
  • the INTASYLTM targeting PD-1 corresponds to “PD 26” in Table 2, comprising SEQ ID NOs: 51 and 52 and is a self-delivering RNAi molecule targeting Programmed Cell Death Protein 1 (PD-1).
  • PD 26 is designed to decrease expression of the immune checkpoint PD-1. Both in vitro and in vivo pharmacology data have demonstrated that PD 26 can silence the expression of PD-1 in human T cells in a potent and durable manner, thereby increasing the function of these T cells and resulting in potent anti-tumoral effects. Intratumoral administration of immune check point inhibitors may be an effective way to overcome the systemic side effects associated with systemic administration of immune checkpoint inhibiting antibodies.
  • a high concentration of immunostimulatory products can be achieved in situ, while using small amounts of drugs.
  • Local delivery of immunotherapies allows multiple combination therapies, while preventing significant systemic toxicities exposure and off- target toxicities.
  • the purpose of this study is to evaluate the safety and tolerability of neoadjuvant use of intratumoral (IT) PD 26 in cutaneous squamous cell carcinoma (cSCC), melanoma, or Merkel cell carcinoma, to determine the pharmacokinetic (PK) profile of PD 26 after IT injection, to observe pathologic and immunologic tumor responses, and to determine the recommended dose or dose range for later clinical studies of PD 26.
  • IT intratumoral
  • cSCC cutaneous squamous cell carcinoma
  • melanoma melanoma
  • Merkel cell carcinoma pharmacokinetic profile of PD 26 after IT injection, to observe pathologic and immunologic tumor responses, and to determine the recommended dose or dose range for later clinical studies of PD 26.
  • At least one lesion is to be both resectable and accessible for IT injections and evaluable by RECIST 1.115. There will be no control group, and the study will be open label. Subjects will receive IT injections of PD 26 to one study lesion once weekly, 4 times over a 3-week period prior to surgical excision, which is to occur 5 weeks after the initial injection. Other lesions will not be injected but may be excised at the same time as the study lesion as per standard of care. Post tumor excision, subjects will be followed-up for approximately 11 weeks (or 13 weeks after the final injection of PD 26). A subject will be considered to have completed the study upon completion of the EOS visit procedures (approximately 11 weeks post excision follow-up).
  • Escalating dose concentrations of PD 26 (targeted at 1.14 mg/mL, 2.39 mg/mL, 5.01 mg/mL, 10.50 mg/mL and 22.00 mg/mL, though intermediate doses (1.71 mg/mL, 3.59 mg/mL, 7.54 mg/mL and 15.75 mg/mL) will be utilized based on DLT observations) are to be tested in a serial fashion in cohorts of 3 subjects each, as per a traditional 3+3 design. Enrollment of subjects within the same dose level cohort can occur in parallel. Dose escalation will occur following review and confirmation by the study-specific Safety Monitoring Committee (SMC), who will review cumulative safety data.
  • SMC Study-specific Safety Monitoring Committee
  • Enrollment of each subsequent dose cohort will be based on DLT observations during the observation period (FIG.1).
  • the study includes safety endpoints as well as endpoints designed to assess potential clinical meaningful effect.
  • the safety of IT PD 26 will be evaluated by assessment of the incidence, severity, onset and duration, seriousness, and relatedness of all treatment-emergent adverse events response (TEAEs) and laboratory abnormalities.
  • Clinical effect is assessed by pathological and tumor response to neoadjuvant therapy as assessed by standardized criteria to measure the response of a tumor to treatment, namely RECIST (version 1.1 and iRECIST version, adapted for use with IT therapy).
  • Plasma PK parameters of PD 26 will also be assessed.
  • PD 26 dosing syringes To prepare PD 26 dosing syringes, doses up to 22.00 mg/mL, PD 26 will be diluted with 0.9% NaCl (saline) prior to IT dosing to obtain the target dose (concentration and volume). PD 26 dosing syringes will be prepared using aseptic technique. The recommended volume of PD 26 to be injected into the study lesion is dependent on the size of the lesion as measured prior to injection and should be determined according to Table 3. Table 3: Intratumoral Injection Volume by Lesion Size * Up to 0.5 mL is allowed for tumors that based on morphology or location may otherwise not receive a uniform dose delivery. EQUIVALENTS Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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Abstract

Des aspects de la divulgation concernent des méthodes de traitement de troubles tels que le carcinome épidermoïde cutané (cSCC) chez un sujet ayant une tumeur par administration intratumorale de molécules d'acide nucléique double brin chimiquement modifiées dirigées contre un gène codant PD-1.
PCT/US2023/085041 2023-11-08 2023-12-20 Ciblage intratumoral de pd-1 Pending WO2025101204A1 (fr)

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