Dendritic conjugates for skin delivery of therapeutic oligonucleotides

Abstract

This disclosure provides compositions, systems, and methods for delivering therapeutic oligonucleotides to the skin. The oligonucleotides are conjugated to dendrons comprising hydrophilic end groups, phosphate groups, and / or hydrophobic chains.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 449,899, filed on March 3, 2023. The entirety of the patent applications referenced above is incorporated herein by reference.

[0002] This disclosure relates to dendritic conjugates for the skin delivery of therapeutic oligonucleotides. Specifically, compositions, systems, and methods for delivering oligonucleotide conjugates, comprising oligonucleotides and dendrons, to the skin are provided herein. [Background technology]

[0003] The interaction of therapeutic oligonucleotides with proteins in body fluids significantly affects their pharmacokinetic and pharmacodynamic properties (see Nguyen, VH & Lee, B.-J. “Protein corona: a new approach for nanomedicine design,” Int J Nanomedicine 12, 3137-3151 (2017), and Banker, MJ & Clark, TH “Plasma / serum protein binding determinations,” Curr Drug Metab 9, 854-859 (2008) (these are incorporated herein by reference)). Such interactions lead to the adhesion of proteins to oligonucleotides, altering the physical properties (charge, size, shape, surface chemistry) of therapeutic agents and influencing their in vivo distribution and behavior (Zhao, Z., Ukidve, A., Krishnan, V. & Mitragotri, S. “Effect of physicochemical and surface properties on in vivo fate of drug nanocarriers,” Advanced Drug Delivery Reviews 143, 3-21 (2019); Albanese, A., Tang, PS & Chan, WCW “The Effect of Nanoparticle Size, Shape, and Surface Chemistry on Biological Systems,” Annual Review of Biomedical Engineering 14, 1-16 (2012); and De Castro, CE et al. “The Protein Corona Conundrum: Exploring the Advantages and Drawbacks of its Presence around Amphiphilic Nanoparticles,” Bioconjugate Chemistry). See 31, 2638–2647 (2020) (these are incorporated herein by reference in their entirety).

[0004] The adhesion of biomolecules and proteins to the surface of therapeutic agents can offer several advantages (see Francia, V., Schiffelers, RM, Cullis, PR & Witzigmann, D. “The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy,” Bioconjugate Chemistry 31, 2046-2059 (2020) (the entire text is incorporated herein by reference)).

[0005] Strategic design of oligonucleotide conjugates is necessary because structure and binding affinity play a central role in cellular uptake and therapeutic efficacy.

[0006] Therefore, there remains a need for novel compositions, systems, and methods for effectively delivering therapeutic oligonucleotides. [Overview of the Initiative]

[0007] Compositions and methods for delivering oligonucleotide conjugates, which include oligonucleotides and dendrons, are provided herein. Oligonucleotide conjugates can efficiently knock down genes in the skin. Several different oligonucleotide conjugates, each containing different oligonucleotides and dendrons, exhibited skin delivery upon administration.

[0008] In one embodiment, the present disclosure provides a method for delivering an oligonucleotide conjugate to the skin of a subject, the method comprising administering the oligonucleotide conjugate to the subject, wherein the oligonucleotide conjugate comprises i) an oligonucleotide having 5' and 3' ends and complementarity to a target nucleic acid, and ii) a dendron bonded to the oligonucleotide and having terminal groups, phosphate groups, and / or hydrophobic chains, and the oligonucleotide conjugate is formulated for cutaneous administration.

[0009] In certain embodiments, the terminal group is a hydrophilic group containing hydroxide, amine, phosphate, sulfur, and / or sugar, amine, amide, ether, ester, N- or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C 1-24 a hydrophobic group containing an alkyl chain, and / or one or more aromatic rings.

[0010] In certain embodiments, the hydrophobic chain is saturated or unsaturated C 1-24 an alkyl group.

[0011] In certain embodiments, the dendron contains two branches.

[0012] In certain embodiments, the dendron contains four branches.

[0013] In certain embodiments, the dendron contains eight branches.

[0014] In certain embodiments, the dendron is attached to the 5'-end and / or 3'-end of the oligonucleotide.

[0015] In certain embodiments, the oligonucleotide contains an antisense oligonucleotide or siRNA.

[0016] In certain embodiments, the siRNA contains a sense strand and an antisense strand.

[0017] In certain embodiments, the dendron is attached to the 5'-end and / or 3'-end of the sense strand, or to the 5'-end and / or 3'-end of the antisense strand.

[0018] In certain embodiments, the dendron is attached to the 3'-end or 5'-end of the sense strand.

[0019] In certain embodiments, the antisense strand is about 15 to 25 nucleotides in length.

[0020] In certain embodiments, the sense strand comprises approximately 15 to 25 nucleotides in length.

[0021] In certain embodiments, the antisense strand is 20 nucleotides long, 21 nucleotides long, or 22 nucleotides long.

[0022] In certain embodiments, the sense strand is 15 nucleotides long, 16 nucleotides long, 18 nucleotides long, or 20 nucleotides long.

[0023] In certain embodiments, the siRNA includes a double-stranded region of 15 to 20 base pairs.

[0024] In certain embodiments, the siRNA includes a double-stranded region of 15, 16, 18, or 20 base pairs.

[0025] In certain embodiments, the siRNA includes at least one blunt end.

[0026] In certain embodiments, the siRNA includes at least one single-stranded nucleotide overhang.

[0027] In certain embodiments, the siRNA contains naturally occurring nucleotides.

[0028] In certain embodiments, the siRNA comprises at least one modified nucleotide.

[0029] In certain embodiments, the modified nucleotides include 2'-O-methyl modified nucleotides, 2'-deoxy-2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, debasalized nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural bases including nucleotides, or mixtures thereof.

[0030] In certain embodiments, the siRNA includes at least one modified nucleotide bond.

[0031] In certain embodiments, the modified nucleotide bond includes a phosphorothioate nucleotide bond.

[0032] In certain embodiments, the siRNA contains 4 to 16 phosphorothioate nucleotide interlinks.

[0033] In certain embodiments, the siRNA contains 8 to 13 phosphorothioate nucleotide interlinks.

[0034] In certain embodiments, the siRNA contains at least 80% chemically modified nucleotides.

[0035] In certain embodiments, the siRNA is fully chemically modified.

[0036] In certain embodiments, the sense strand contains one or more nucleotide mismatches between the antisense strand and the sense strand.

[0037] In certain embodiments, the antisense chain comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

[0038] In certain embodiments, the antisense chain comprises a 5' vinylphosphonate.

[0039] In certain embodiments, the nucleotides at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate bonds.

[0040] In certain embodiments, oligonucleotide conjugates are administered sequentially or simultaneously.

[0041] In certain embodiments, the oligonucleotide conjugate has the structure of formula I, [ka] In the formula, A is an oligonucleotide, and B, for each occurrence, independently comprises one or more hydrophobic chains, amines, amides, esters, N- or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, wherein the hydrophobic chains are saturated or unsaturated C 1-24 The alkyl chain contains, for each occurrence, independently, a hydrophilic group containing hydroxides, amines, phosphates, sulfur, and / or sugars, amines, amides, ethers, esters, N- or O-containing heterocycles, thiols, thioethers, and / or saturated or unsaturated C 1-24 It comprises a hydrophobic group containing an alkyl chain and / or one or more aromatic rings, where D is, for each occurrence independently, a branched unit containing one or more alkyl chains, amides, ethers, esters, and amines, the branched unit contains 2 to 4 branches, and m is, for each occurrence independently, 0 or 1.

[0042] In certain embodiments, the oligonucleotide conjugate has the structure of formula II. [ka]

[0043] In certain embodiments, the oligonucleotide conjugate has the structure of formula III, [ka]

[0044] In the formula, n is an integer between 1 and 24, independently for each occurrence.

[0045] In certain embodiments, C is OH, and n is independently 1, 6, or 12 for each occurrence.

[0046] In certain embodiments, the oligonucleotide conjugate has the structure of formula IV. [ka]

[0047] In certain embodiments, the oligonucleotide conjugate has the structure of formula V. [ka]

[0048] In certain embodiments, the oligonucleotide conjugate has the structure of formula VI, [ka] In the formula, n is an integer between 1 and 24, independently for each occurrence.

[0049] In certain embodiments, C is OH, and n is independently 1, 6, or 12 for each occurrence.

[0050] In certain embodiments, the oligonucleotide conjugate has the structure of formula VII. [ka]

[0051] In certain embodiments, the oligonucleotide conjugate formulation contains oligonucleotide conjugates in an amount of approximately 0.1 to 100 mg / kg body weight.

[0052] In certain embodiments, the oligonucleotide conjugate is administered intradermally.

[0053] In certain embodiments, the oligonucleotide conjugate has low toxicity.

[0054] In certain embodiments, subjects do not experience substantial changes in blood chemistry and complete blood count (CBC) after administration of the oligonucleotide conjugate. In certain embodiments, the changes in blood chemistry and CBC are approximately 10% or less compared to blood chemistry and CBC before administration of the oligonucleotide conjugate.

[0055] In certain embodiments, oligonucleotide conjugates exhibit reduced uptake by immune cells compared to unconjugated oligonucleotides or oligonucleotides conjugated with cholesterol or DCA. In certain embodiments, immune cells are selected from the group consisting of lymphocytes, leukocytes, neutrophils, eosinophils, dendritic cells, macrophages, B cells, and T cells.

[0056] In certain embodiments, the oligonucleotide has one of the sequences of sequence numbers 25 and 28.

[0057] In one embodiment, the present disclosure provides a method for providing treatment to a patient in need of treatment for a skin disease, disorder, or injury, comprising administering to the patient an oligonucleotide conjugate comprising i) an oligonucleotide having 5' and 3' ends and complementarity to a target nucleic acid, and ii) a dendron conjugated to the oligonucleotide and comprising a hydrophilic end group, a phosphate group, and / or a hydrophobic chain, wherein the oligonucleotide conjugate is formulated for cutaneous administration.

[0058] In certain embodiments, the skin disease, disorder, or injury is selected from the group consisting of acne, pemphigus, alopecia areata, psoriasis, atopic dermatitis, Raynaud's phenomenon, epidermolysis bullosa, rosacea, hidradenitis suppurativa, scleroderma, ichthyosis, vitiligo, congenital onychoplakia, and combinations thereof.

[0059] In certain embodiments, skin diseases, disorders, or injuries are associated with the JAK1 gene.

[0060] In certain embodiments, the oligonucleotide conjugate inhibits the expression of the JAK1 gene.

[0061] In certain embodiments, the oligonucleotide has one of the sequences of sequence numbers 25 and 28.

[0062] In one embodiment, the present disclosure provides a method for administering a therapeutically effective amount of an oligonucleotide conjugate to the skin of a subject, the method comprising administering the oligonucleotide conjugate to a subject, wherein the oligonucleotide conjugate comprises i) an oligonucleotide having 5' and 3' ends and complementarity to a target nucleic acid, and ii) a dendron bonded to the oligonucleotide and having a hydrophilic end group, a phosphate group, and / or a hydrophobic chain, and the oligonucleotide conjugate is formulated for cutaneous administration.

[0063] In one embodiment, the present disclosure provides a method for inhibiting the expression of the JAK1 gene in cells, the method comprising: (a) introducing into cells an oligonucleotide conjugate comprising i) an oligonucleotide having 5' and 3' ends and complementarity to a target nucleic acid, and ii) a dendron bound to the oligonucleotide and comprising a hydrophilic end group, a phosphate group, and / or a hydrophobic chain; and (b) maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the JAK1 gene, thereby inhibiting the expression of the JAK1 gene in cells. In one embodiment, the present disclosure provides an oligonucleotide conjugate for use in a patient requiring treatment of a skin disease, disorder, or injury, wherein the oligonucleotide conjugate is administered to the patient and formulated for skin administration, and the oligonucleotide conjugate comprises i) an oligonucleotide having 5' and 3' ends and complementarity to a target nucleic acid, and ii) a dendron bonded to the oligonucleotide and having a hydrophilic end group, a phosphate group, and / or a hydrophobic chain.

[0064] In one embodiment, the present disclosure provides a pharmaceutical composition for providing treatment to a patient requiring treatment for a skin disease, disorder, or injury, comprising: i) an oligonucleotide comprising a 5' and 3' terminus and complementarity to a target nucleic acid; and ii) an oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and comprising a hydrophilic terminus, a phosphate group, and / or a hydrophobic chain; and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is formulated for cutaneous administration.

[0065] In one embodiment, the present disclosure provides a topical formulation comprising: (a) an oligonucleotide comprising a 5' and 3' terminal and complementarity to a target nucleic acid; ii) a dendron bonded to the oligonucleotide and comprising a hydrophilic end group, a phosphate group, and / or a hydrophobic chain; (b) optionally a pharmaceutically acceptable carrier; and (c) optionally a pharmaceutically acceptable salt.

[0066] These and other aspects of the applicant's teachings are described herein.

[0067] This patent or application document includes at least one drawing drawn in color. A copy of this patent or patent application publication containing the color drawing(s) will be provided by the Patent Office upon request and payment of the required fees.

[0068] The aspects, features, advantages, and benefits of the embodiments described herein will become apparent with respect to the following embodiments, examples, claims, and accompanying drawings for carrying out the invention. [Brief explanation of the drawing]

[0069] [Figure 1] A schematic diagram of an oligonucleotide conjugate is shown. Figure 1A shows a schematic diagram of a dendron attached to an siRNA. Figure 1B shows a schematic diagram of a dendritic (D)-siRNA containing a dendron having hydrophilic (OH) and hydrophobic (CH3) terminal groups. [Figure 2] Figure 2A shows the structures of D-siRNA, DCA-siRNA, and siRNA analyzed by high-performance liquid chromatography (HPLC) and size exclusion chromatography (SEC). Figure 2A shows the structures of dendritic siRNA, docosan siRNA, and unconjugated siRNA tested for hydrophobicity / retention time on HPLC, showing different retention times. Figure 2B shows the reverse-phase HPLC traces of D-siRNA or DCA-siRNA after injection, showing the difference in hydrophobicity between them. Figure 2C shows the results of assays in which D-siRNA, DCA-siRNA, and siRNA were injected in vivo via subcutaneous or intravenous injection in mice (n=2), and plasma was collected 1 hour or 15 minutes after injection. Plasma was subjected to size exclusion chromatography (SEC), with plasma proteins monitored at 280 nm and Cy3-labeled oligonucleotides monitored at 570 nm, as previously reported. [Figure 3]Images of skin samples administered with dendritic (D)-JAK1 inhibitory siRNA and DCA-JAK1 inhibitory siRNA are shown. Figure 3 also shows JAK1 mRNA expression in the epidermis and dermis after injection of PBS, DCA-siRNA NTC, Unc-siRNA HTT, DCA-siRNA HTT, DD-siRNA HTT, Unc-siRNA JAK1, DCA-siRNA JAK1, and DD-siRNA JAK1 (intradermal injection of 200 μM siRNA in 50 μL in an 8 mm punch of human skin (1:3 μg siRNA)). It was observed that D-siRNA penetrated and distributed deep into the skin, and that D-siRNA did not result in dermal silencing, hypothetically due to leakage. [Figure 4] Images of skin samples administered with unconjugated siRNA, as well as DCA, cholesterol, diol, and dendrimer-conjugated siRNA, are shown. The top row corresponds to images of skin samples taken immediately after injection, and the bottom row corresponds to images of skin samples taken 24 hours after injection. [Figure 5] This image shows siRNA accumulation in various cells after intradermal injection of PBS, Unc-siRNA, DCA-siRNA, and D-siRNA into human skin (injection of 200 μM siRNA (1:3 μg siRNA) in 50 μL in an 8 mm punch of human skin). MFI = mean fluorescence intensity, n = 3, and punch size = 8 mm. [Figure 6] Figure 6A shows the expression of CXCL-9, CXCL-10, and CXCL-11 mRNA in skin cells after administration of PBS, NTC siRNA, Unc-JAK1 siRNA, DCA-JACK1 siRNA, and D-JACK1 siRNA. Figure 6B shows a schematic diagram of intradermal injection after IFN stimulation, IFN stimulation, and the JAK1 downstream signaling pathway. Figure 6B shows the CXCL-9, CXCL-10, and CXCL-11 mRNA expression percentages compared to a control after intradermal injection of 200 μM siRNA in 50 μL (1:3 μg siRNA) in an 8 mm punch of human skin for 4 days after 24 hours of interferon-gamma stimulation. [Figure 7]Blood chemistry and complete blood count results are shown for mice (c56bl / 6, male, n=3) injected with 100 mg / kg of DCA-siRNA or D-siRNA. Blood was collected 24 hours after subcutaneous injection. [Figure 8] Flow cytometry analysis of spleen and bone marrow cells from mice (n=3) subcutaneously injected with Cy3-labeled Htt-D-siRNA or Chol-D-siRNA is shown. Figures 8A and 8B show frequency distribution histograms of Cy3 fluorescence intensity in different cells (left) and normalized geometric fluorescence intensity bar graphs in B) spleen and C) bone (right), with p values ​​showing statistically significant differences compared to PBS controls (two-way ANOVA). [Figure 9] This report evaluates the efficacy of RNAiMax (7-point concentration-response study) in HeLa cells using 5'-conjugated D-siRNA, 3'-conjugated D-siRNA, DCA siRNA (the DCA conjugate is positioned at the 3' end of the sense strand as it grows from a solid support), and unconjugated siRNA. After 72 hours, incubation samples were analyzed by a Quantigene bDNA assay. Data were normalized to housekeeping genes (Hprt) and presented as a percentage of untreated control cells. n=3. Mean ± standard deviation (SD). [Modes for carrying out the invention]

[0070] For clarity, it will be understood that the following discussion describes various aspects of the embodiments of the applicant's teachings. It should be noted that the specific embodiments are not intended to be exhaustive or to limit the broader embodiments discussed herein. An embodiment described in conjunction with a particular embodiment is not necessarily limited to that embodiment, but may be implemented in conjunction with any other embodiment(s).

[0071] This disclosure relates to dendritic conjugates for the delivery of therapeutic oligonucleotides to the skin. Specifically, this disclosure provides compositions, systems, and methods for the delivery of therapeutic oligonucleotides conjugated to dendrons. Oligonucleotide conjugates disclosed herein can be delivered to the skin upon administration.

[0072] The oligonucleotide conjugates described herein can facilitate the simple, efficient, and non-toxic delivery of oligonucleotides (e.g., siRNA, antisense oligonucleotides (ASOs), macro-RNAs) and promote potent silencing of therapeutic targets in skin cells in vivo.

[0073] Unless otherwise specified, the terms used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, as well as hybridization, are well-known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are carried out in accordance with conventional methods well-known in the art, and unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out in accordance with the manufacturer's specifications, as is commonly done in the art or as described herein. The terms used herein in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and pharmaceutical chemistry, as well as their experimental procedures and techniques, are well-known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, drug preparation, formulation, delivery, and patient treatment.

[0074] Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of any potential ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of “or” shall mean “and / or” unless otherwise indicated. The use of the term “including,” as well as other forms such as “include” and “included,” is not limited to these.

[0075] To make this disclosure easier to understand, certain terms are defined first.

[0076] definition In this specification, the use of the singular form includes the plural form unless otherwise specified. Where used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include multiple references. Furthermore, the use of the term “including,” as well as other forms such as “include,” “includes,” and “included,” is not limited.

[0077] Wherever an aspect is described using the term “comprising” in this specification, it should be understood that other similar aspects are also provided, which are described using the terms “consisting of” and / or “consisting essentially of.”

[0078] As described herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.

[0079] The terms “approximately” or “essentially include” refer to a value or composition that falls within the tolerance range for a particular value or composition as determined by those skilled in the art, and this depends in part on how the value or composition is measured or determined, i.e., on the limitations of the measuring system. Where a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of “approximately” or “essentially include” should be assumed to be within the tolerance range for that particular value or composition.

[0080] Where used herein, the term “and / or” should be interpreted as each of the two specified features or components being specifically disclosed, with or without the other. Therefore, where the term “and / or” is used herein in phrases such as “A and / or B,” it is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, where the term “and / or” is used in phrases such as “A, B, and / or C,” it is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0081] When used herein in the context of oligonucleotide sequences, "A" represents a nucleoside containing the base adenine (e.g., adenosine or a chemically modified derivative thereof), "G" represents a nucleoside containing the base guanine (e.g., guanosine or a chemically modified derivative thereof), "U" represents a nucleoside containing the base uracil (e.g., uridine or a chemically modified derivative thereof), and "C" represents a nucleoside containing the base adenine (e.g., cytidine or a chemically modified derivative thereof).

[0082] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently bonded to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also referred to as “rare” nucleosides). The term "nucleotide" refers to a nucleoside having one or more phosphate groups bonded to the sugar moiety by an ester bond. Exemplary nucleotides include nucleoside monophosphate, diphosphate, and triphosphate. The terms "polynucleotide" and "nucleic acid molecule" are used synonymously herein and refer to polymers of nucleotides linked together by phosphodiester or phosphorothioate bonds between the 5' and 3' carbon atoms.

[0083] The terms “RNA,” “RNA molecule,” or “ribonucleic acid molecule” refer to polymers of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The terms “DNA,” “DNA molecule,” or “deoxyribonucleic acid molecule” refer to polymers of deoxyribonucleotides. DNA and RNA can be synthesized spontaneously (e.g., by DNA replication or DNA transcription, respectively). RNA can be modified after transcription. DNA and RNA can also be synthesized chemically. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double-stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that identifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to mRNA.

[0084] As used herein, the term “small interfering RNA” ("siRNA") (also referred to in the art as “short interfering RNA”) means RNA (or RNA analogues) containing about 10 to 50 nucleotides (or nucleotide analogues) that can induce or mediate RNA interference. siRNA is a double-stranded molecule formed by a sense strand and an antisense strand, the sense strand and antisense strand being sufficiently complementary to each other to form the double-stranded molecule. In certain embodiments, siRNA contains about 15 to 30 nucleotides or nucleotide analogues, or about 16 to 25 nucleotides (or nucleotide analogues), or about 18 to 23 nucleotides (or nucleotide analogues), or about 19 to 22 nucleotides (or nucleotide analogues) (e.g., 19, 20, 21, or 22 nucleotides or nucleotide analogues). The term “short” siRNA means siRNA containing about 21 nucleotides (or nucleotide analogues), e.g., 19, 20, 21, or 22 nucleotides. The term "long" siRNA refers to siRNA containing approximately 24–25 nucleotides, e.g., 23, 24, 25, or 26 nucleotides. Short siRNA may contain fewer than 19 nucleotides, e.g., 16, 17, or 18 nucleotides, provided that the short siRNA retains its ability to mediate RNAi. Similarly, long siRNA may contain more than 26 nucleotides, provided that the long siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0085] The terms “nucleotide analog,” “modified nucleotide,” “modified nucleotide,” or “chemically modified nucleotide” refer to non-standard nucleotides, including ribonucleotides or deoxyribonucleotides that do not exist in nature. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of a nucleotide, but so as to retain the ability of the nucleotide analog to perform its intended function. Examples of nucleotide positions that can be derivatized include the 5-position (e.g., 5-(2-amino)propyluridine, 5-bromouridine, 5-propin). Examples include lysine (e.g., 5-propenyluridine), the 6th position (e.g., 6-(2-amino)propyluridine), and the 8th position of adenosine and / or guanosine (e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine). Nucleotide analogs include deazanucleotides (e.g., 7-deaza-adenosine), O- and N-modified (e.g., alkylated, e.g., N6-methyladenosine, or others known in the art) nucleotides, and other heterocyclic modified nucleotide analogs (such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug.10(4):297-310).

[0086] The nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH2, NHR, NR2, or COOR, where R is a substituted or unsubstituted C1-C6 alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications are described in U.S. Patents No. 5,858,988 and No. 6,291,438. In certain embodiments, the nucleotide analogs include 2'-O-methyl modifications. In certain embodiments, the nucleotide analogs include 2'-fluoro modifications.

[0087] The phosphate group of a nucleotide may also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., phosphorothioate), or by other substitutions that enable the nucleotide to perform its intended function (e.g., those described in Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5):317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Patent No. 5,684,143). The specific modifications referenced above (e.g., phosphate group modifications) reduce the hydrolysis rate of polynucleotides containing such analogs, for example, in vivo or in vitro.

[0088] The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified or altered nucleotide compared to the corresponding unmodified or unaltered RNA, but retains the same or similar properties or functions as the corresponding unmodified or unaltered RNA. As discussed above, oligonucleotides may be linked with bonds that reduce the hydrolysis rate of the RNA analog compared to RNA molecules with phosphodiester bonds. For example, the nucleotides of an analog may include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoramidate, and / or phosphorothioate bonds. Some RNA analogs may include sugars and / or skeletal-modified ribonucleotides and / or deoxyribonucleotides. Such alterations or alterations may further include the addition of non-nucleotide substances to the terminal(s) or internal(s) (one or more nucleotides) of the RNA. The RNA analog only needs to be sufficiently similar to the native RNA that has the ability to mediate RNA interference.

[0089] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs spontaneously within cells and removes foreign RNA (e.g., viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA, and these fragments induce degradation mechanisms in other similar RNA sequences. Alternatively, RNAi can be initiated by humans, for example, to silencing the expression of a target gene.

[0090] An RNAi agent (e.g., an RNA silencing agent) having a strand that is "sufficiently complementary to the target mRNA sequence in order to induce target-specific RNA interference (RNAi)" means that the strand has a sequence sufficient to cause the disruption of the target mRNA by the RNAi mechanism or process.

[0091] As used herein, “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to an RNA molecule that, if produced by recombinant technology, substantially contains no other cellular material or culture medium, or, if chemically synthesized, substantially contains no chemical precursors or other chemical substances.

[0092] As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms mediated by RNA molecules (e.g., RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), querying, co-repression, and translational repression) that result in inhibition or “silencing” of the expression of the corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

[0093] The term "in vitro" has the meaning recognized in the art and includes, for example, purified reagents or extracts, such as cell extracts. The term "in vivo" also has the meaning recognized in the art and includes, for example, living cells such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0094] As used herein, “target” refers to a specific nucleic acid sequence (e.g., a gene, mRNA, miRNA, etc.) to which an oligonucleotide conjugate or branched oligonucleotide of this disclosure binds and / or otherwise affects its expression. In certain embodiments, the target is expressed in the eye. In certain embodiments, the target is expressed in specific ocular cells. In other embodiments, the target is associated with a specific disease or disorder in the subject.

[0095] As used herein, the term “target gene” is a gene whose expression is substantially inhibited or “silenced.” This silencing can be achieved by RNA silencing, for example, by cleaving the mRNA of the target gene or by translational repression of the target gene. The term “non-target gene” is a gene whose expression is not substantially silenced. In one embodiment, the polynucleotide sequences of the target gene and the non-target gene (e.g., the mRNA encoded by the target gene and the non-target gene) may differ by one or more nucleotides. In another embodiment, the target gene and the non-target gene may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target gene and the non-target gene may share less than 100% sequence identity. In another embodiment, the non-target gene may be a homolog of the target gene (e.g., an ortholog or paralog).

[0096] As used herein, the term “RNA silencing agent” refers to RNA capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, RNA silencing agents can prevent the complete processing of mRNA molecules (e.g., complete translation and / or expression) through post-transcriptional silencing mechanisms. Examples of RNA silencing agents include small (<50 b.p.), non-coding RNA molecules, such as double-stranded RNAs containing paired strands, and precursor RNAs capable of generating such small non-coding RNAs. Exemplary RNA silencing agents include siRNA, miRNA, siRNA-like double-stranded RNAs, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as their precursors. In one embodiment, RNA silencing agents can induce RNA interference. In another embodiment, RNA silencing agents can mediate translational repression.

[0097] As used herein, the term “rare nucleotide” refers to naturally occurring nucleotides that are rarely found, such as naturally occurring deoxyribonucleotides or ribonucleotides (for example, naturally occurring ribonucleotides other than guanosine, adenosine, cytosine, or uridine). Examples of rare nucleotides include, but are not limited to, inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0098] The term "engineered" indicates that a precursor or molecule is not found in nature, in that all or part of the nucleic acid sequence of the precursor or molecule is created or selected by humans, such as an engineered RNA precursor or engineered nucleic acid molecule. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by intracellular mechanisms. Therefore, an RNA precursor produced intracellularly from a transgene containing an engineered nucleic acid molecule is an engineered RNA precursor.

[0099] As used herein, the term “microRNA” (“miRNA”) is also known in the art as “small temporal RNA” (“stRNA”) and refers to a small (e.g., 10 to 50 nucleotides) RNA that is genetically encoded (e.g., by a virus, mammal, or plant genome) and can induce or mediate RNA silencing. “miRNA disorder” refers to a disease or disorder characterized by abnormal expression or activity of miRNA.

[0100] As used herein, the term “dual-function oligonucleotide” refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting moiety, L is the binding moiety, and μ is the miRNA recruitment moiety. As used herein, the terms “mRNA targeting moiety,” “targeting moiety,” “mRNA targeting portion,” or “targeting portion” refer to a domain, moiety, or region of a dual-function oligonucleotide that is sufficiently large and sufficiently complementary to the moiety or region of mRNA selected or targeted for silencing (i.e., the moiety has a sequence sufficient to capture the target mRNA).

[0101] As used herein, the terms “linking moiety” or “linking portion” refer to a domain, part, or region of an RNA silencing agent that covalently binds or links to mRNA.

[0102] As used herein, the term “antisense strand” of an RNA silencing agent, such as siRNA, refers to a strand substantially complementary to a section of approximately 10–50 nucleotides, e.g., approximately 15–30, 16–25, 18–23, or 19–22 nucleotides, of the mRNA of the gene targeted for silencing. The antisense strand, or first strand, has a sequence sufficiently complementary to the desired target mRNA sequence to induce target-specific silencing, such complementarity is sufficient to cause, for example, the disruption of the desired target mRNA by an RNAi mechanism or process (RNAi interference), or to cause translational repression of the desired target mRNA.

[0103] The terms “sense strand” or “second strand” of an RNA silencing agent, such as siRNA or other RNA silencing agents, refer to a strand that is complementary to the antisense strand or first strand. The antisense strand and sense strand may also be referred to as the first strand or second strand, where the first strand or second strand is complementary to the target sequence, and each second strand or first strand is complementary to the first strand or second strand. A miRNA double-stranded intermediate or siRNA-like double-stranded complex includes a miRNA strand that is sufficiently complementary to a section of approximately 10–50 nucleotides of mRNA of the gene targeted for silencing, and a miRNA* strand that is sufficiently complementary to form a double-stranded complex with the miRNA strand.

[0104] As used herein, the term “guide strand” refers to a strand of RNA silencing agent that enters the RISC complex and induces cleavage of the target mRNA, such as a double-stranded siRNA or an antisense strand of an siRNA sequence.

[0105] As used herein, the term “asymmetry” refers to an imbalance in binding strength or base-pairing strength between the ends of an RNA silencing agent (e.g., between a terminal nucleotide on the first strand or stem portion and a terminal nucleotide on the opposite second strand or stem portion), such as the asymmetry of the double-stranded region of the RNA silencing agent (e.g., the stem of shRNA), resulting in the 5' end of one strand of the double being more frequently in a transient unpaired state (e.g., single-stranded state) than the 5' end of the complementary strand. This structural difference determines that one strand of the double is preferentially incorporated into the RISC complex. The strand whose 5' end is less tightly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0106] As used herein, the terms “bond strength” or “base pair strength” refer to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide double strand (e.g., an siRNA double strand), primarily due to H bonds between such nucleotides (or nucleotide analogs), van der Waals interactions, etc.

[0107] As used herein, “5' end” refers to the 5' terminal nucleotides, such as the 5' end of an antisense strand, e.g., 1 to about 5 nucleotides at the 5' end of an antisense strand. As used herein, “3' end” refers to a region complementary to the 5' terminal nucleotides of a complementary antisense strand, such as the 3' end of a sense strand, e.g., a region of 1 to about 5 nucleotides.

[0108] As used herein, the term “destabilized nucleotide” refers to a first nucleotide or nucleotide analog that can form a base pair with a second nucleotide or nucleotide analog, resulting in a base pair with a lower binding strength than a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilized nucleotide can form a mismatched base pair with the second nucleotide. In other embodiments, the destabilized nucleotide can form a fluctuating base pair with the second nucleotide. In yet another embodiment, the destabilized nucleotide can form an ambiguous base pair with the second nucleotide.

[0109] As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposite strands of an oligonucleotide double helix (e.g., a double helix formed by the strand of an RNA silencing agent and the target mRNA sequence), primarily due to H bonds between such nucleotides (or nucleotide analogs), van der Waals interactions, etc. As used herein, the terms “bond strength” or “base pair strength” refer to the strength of a base pair.

[0110] As used herein, the term “mismatched base pair” refers to a base pair consisting of a non-complementary or non-Watson-Crick base pair, such as a base pair that is not a typical complementary G:C, A:T, or A:U base pair. As used herein, the term “ambiguous base pair” (also known as indiscriminate base pair) refers to a base pair formed by a universal nucleotide.

[0111] As used herein, the term “universal nucleotide” (also known as “neutral nucleotide”) includes nucleotides (e.g., certain destabilized nucleotides) that have a base (“universal base” or “neutral base”) that does not significantly distinguish the bases on a complementary polynucleotide when forming base pairs. Universal nucleotides are primarily hydrophobic molecules that can efficiently assemble through stacking interactions to form antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA). The base moiety of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0112] As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has a sequence (e.g., in the antisense strand, mRNA targeting portion, or miRNA recruiting portion) sufficient to bind to the desired target and induce RNA silencing of the target mRNA.

[0113] As used herein, the term “translational repression” refers to the selective inhibition of mRNA translation. Spontaneous translational repression proceeds via miRNA cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur spontaneously or can be initiated by humans, for example, to silence the expression of a target gene.

[0114] As used herein, the term “toxicity” refers to the wide variety of adverse effects resulting from the use of the oligonucleotide conjugates of this disclosure, whether at therapeutic or non-therapeutic doses. Toxicity may be measured in a subject administered with the oligonucleotide conjugate by measuring one or more biological parameters of the subject. For example, but not limited to, a subject may have blood chemistry and complete blood count (CBC) determined before and after administration of the oligonucleotide conjugate. Changes in the blood chemistry of the CBC may indicate toxicity. Blood chemistry includes, but is not limited to, measurements of liver enzymes (e.g., alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP)), albumin, and / or amylase. An additional measurement of toxicity may be the uptake of the oligonucleotide conjugates of this disclosure into immune cells, with higher uptake being considered an indication of increased toxicity. Such uptake into immune cells may result in increased production of inflammatory cytokines.

[0115] Various methodologies of this disclosure include a step of comparing values, levels, features, properties, etc., with a “preferred control” (hereinafter synonymously referred to as “preferred control”). A “preferred control” or “appropriate control” is any control or standard that is useful for comparison purposes and is familiar to those skilled in the art. In one embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined before performing the RNAi methodology as described herein. For example, transcription rate, mRNA level, translation rate, protein level, biological activity, cellular properties or characteristics, genotype, phenotype, etc., may be determined before introducing the RNA silencing agent of this disclosure into cells or organisms. In another embodiment, a “preferred control” or “appropriate control” is a value, level, feature, property, etc., determined in, for example, a cell or organism exhibiting normal traits (e.g., a control or normal cell or organism). In yet another embodiment, a “preferred control” or “appropriate control” is a predefined value, level, feature, property, etc.

[0116] Design of Ava molecules The oligonucleotide conjugates described herein include oligonucleotides. Non-limiting examples of oligonucleotides include siRNA, antisense oligonucleotides (ASOs), and macro-RNAs.

[0117] In some embodiments, the siRNA molecule of the present invention is a double-stranded molecule comprising a sense strand and a complementary antisense strand, wherein the antisense strand is sufficiently complementary to a target sequence such as an mRNA sequence for mediating RNAi (e.g., an htt mRNA sequence, a cyclophyllin B mRNA sequence, etc.). Preferably, the siRNA molecule has a nucleotide length of about 10 to 50 or more, i.e., each strand contains 10 to 50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a nucleotide length of about 16 to 30 nucleotides per strand, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, and one of the strands is sufficiently complementary to the target region. Preferably, the chains are arranged such that at least one, two, or three bases are present at the ends of the unaligned chains (i.e., no complementary bases are produced in the opposing chains), so that when the chains are annealed, an overhang of one, two, or three residues occurs at one or both ends of the double helix. Preferably, the siRNA molecule has a nucleotide length of about 10 to 50 or more, i.e., each chain contains 10 to 50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a nucleotide length of about 16 to 30, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides per chain, with one chain substantially complementary to the target sequence and the other chain identical or substantially identical to the first chain.

[0118] In general, siRNA can be designed using any method known in the art, for example, by using the following protocols:

[0119] 1. siRNA may be specific to a target sequence. Preferably, the first strand is substantially complementary to the target sequence, and the other strand is substantially complementary to the first strand. In one embodiment, the target sequence is outside the coding region of the target gene. Exemplary target sequences are selected from the 5' untranslated region (5'-UTR) or intron region of the target gene. Cleavage of mRNA at these sites should eliminate translation of the corresponding mutant protein. Target sequences from other regions of the gene are also suitable for targeting. The sense strand is designed based on the target sequence. Furthermore, siRNA with a lower G / C content (35-55%) may be more active than siRNA with a G / C content higher than 55%. Therefore, in one embodiment, the present invention includes nucleic acid molecules having a G / C content of 35-55%.

[0120] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. Preferably, the RNA silencing agent of the present invention does not induce a PKR response (i.e., is sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that cannot produce a PRK response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0121] The siRNA molecule of the present invention has sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. Generally, it is preferable that siRNA containing a nucleotide sequence that is sufficiently identical to the target sequence portion of the target gene results in RISC-mediated cleavage of the target gene. Therefore, in a preferred embodiment, the sense strand of the siRNA is designed to have a sequence that is sufficiently identical to a portion of the target. For example, the sense strand may have 100% identity with respect to the target site. However, 100% identity is not required. It is preferable that there is greater than 80% identity between the sense strand and the target RNA sequence, for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 100% identity. This disclosure has the advantage that certain sequence modifications can be tolerated to enhance the efficiency and specificity of RNAi. In one embodiment, the sense strand has 4, 3, 2, 1, or 0 mismatched nucleotides having a target region that differs depending on the target region, such as a wild-type allele and a mutant allele, and the other strand is identical or substantially identical to the first strand. Furthermore, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective in mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions may be effective in inhibition.

[0122] Sequence identity can be determined by sequence comparison and alignment algorithms known in the art. To determine the percentage of identity between two nucleic acid sequences (or two amino acid sequences), the sequences are aligned for optimal comparison (for example, gaps can be introduced into the first or second sequence for optimal alignment). Then, the nucleotides (or amino acid residues) at the corresponding nucleotide (or amino acid) positions are compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage (%) homology = number of identical positions / total number of positions × 100), and optionally, a penalty is imposed on the score for the number and / or length of introduced gaps.

[0123] The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. In one embodiment, alignment is generated in specific parts of aligned sequences that have sufficient identity, but not in parts with a low degree of identity (i.e., local alignment). A preferred non-restrictive example of a local alignment algorithm used for sequence comparison is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264-68, as revised as found in Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-77. Such an algorithm is incorporated into the BLAST program (version 2.0), Altschul, et al. (1990) J.Mol.Biol.215:403-10.

[0124] In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the length of the aligned sequence (i.e., gap alignment). To obtain gap alignment for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps, and percentage identity is determined over the entire length of the aligned sequence (i.e., global alignment). A preferred non-restrictive example of a mathematical algorithm used for global sequence comparison is the Myers and Miller algorithm, CABIOS (1989). Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties can be used.

[0125] 3. The antisense or guide strand of an siRNA is routinely the same length as the sense strand and contains complementary nucleotides. In one embodiment, the guide and sense strands are perfectly complementary, i.e., the strands have blunt ends when aligned or annealed. In another embodiment, the siRNA strands may be paired to have 1 to 4, e.g., 2 nucleotide 3' overhangs. The overhangs may contain (or consist of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs may contain (or consist of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide materials. Thus, in another embodiment, the nucleic acid molecule may have 2 nucleotide 3' overhangs, such as TT. The overhang nucleotides may be either RNA or DNA. As described above, it is desirable to select a target region where the mutant:wild-type mismatch is a purine:purine mismatch.

[0126] 4. Using any method known in the art, compare potential targets with appropriate genome databases (human, mouse, rat, etc.) to exclude any target sequences that have significant homology to other coding sequences. One such sequence homology search method is known as BLAST and is available on the National Center for Biotechnology Information website.

[0127] 5. Select one or more sequences that meet the evaluation criteria.

[0128] Further general information regarding the design and use of siRNA can be found in “The siRNA User Guide,” available on the website of The Max-Plank-Institut fur Biophysikalishe Chemie.

[0129] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) that can hybridize with a target sequence (e.g., hybridize in 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C, for 12–16 hours, followed by washing). Additional preferred hybridization conditions include hybridization at 70°C in 1×SSC or 50°C in 1×SSC, 50% formamide, followed by washing at 70°C in 0.3×SSC, or hybridization at 70°C in 4×SSC or 50°C in 4×SSC, 50% formamide, followed by washing at 67°C in 1×SSC. The hybridization temperature of a hybrid expected to be less than 50 base pairs in length must be 5–10°C lower than the melting temperature (Tm) of the hybrid, which is determined according to the following equation: For hybrids with a length of less than 18 base pairs, Tm(°C) = 2 (number of A + T bases) + 4 (number of G + C bases). For hybrids with a length of 18 to 49 base pairs, Tm(°C) = 81.5 + 16.6 (log 10[Na+]) + 0.41(%G+C) - (600 / N), where N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] = 0.165 M per 1 × SSC). Examples of additional stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 9 and 11, and in Current Protocols in Molecular Biology, 1995, F.M.Ausubel et al., eds., John Wiley & Sons, Inc., Sections 2.10 and 6.3–6.4 (incorporated herein by reference).

[0130] Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity with the appropriate genome. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to ensure that the negative control lacks homology to any other genes in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

[0131] 6. To verify the effectiveness of siRNA in disrupting target mRNA (e.g., wild-type or mutant huntingtin mRNA), siRNA may be incubated with target cDNA (e.g., huntingtin cDNA) in a Drosophila-based in vitro mRNA expression system. 32 The newly synthesized target mRNA (e.g., huntingtin mRNA), radiolabeled with 3P, is detected by autoradiography on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include the omission of siRNA and the use of non-target cDNA. Alternatively, a control siRNA is selected that has the same nucleotide composition as the selected siRNA but does not exhibit significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. Homology searches can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0132] The siRNA may be designed to target any of the target sequences described above. The siRNA comprises an antisense strand that is sufficiently complementary to the target sequence in order to mediate the silencing of the target sequence. In certain embodiments, the RNA silencing agent is siRNA.

[0133] The siRNA-mRNA complementation site that provides optimal mRNA specificity and maximum mRNA cleavage is selected.

[0134] siRNA-like molecules The siRNA-like molecule of the present invention has a sequence that is "sufficiently complementary" to the target sequence of mRNA (e.g., htt mRNA) in order to induce gene silencing by either RNAi or translational repression (i.e., has a sequence-containing strand). The siRNA-like molecule is designed in the same manner as an siRNA molecule, but the degree of sequence identity between the sense strand and the target RNA approximates that is observed between miRNA and its target. Generally, when the degree of sequence identity between the miRNA sequence and the corresponding target gene sequence decreases, the tendency for post-transcriptional gene silencing to be mediated by translational repression rather than RNAi increases. Therefore, in alternative embodiments where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementary sites) dispersed within the target mRNA (e.g., within the 3'-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Because the translational repression mechanism is cooperative, in certain embodiments, multiple complementary sites (e.g., 2, 3, 4, 5, or 6) may be targeted.

[0135] The ability of an siRNA-like double-stranded molecule to mediate RNAi or translational repression can be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent in the complementary region. In one embodiment where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central part of the complementary region such that the double helix formed by the miRNA guide strand and the target mRNA contains a central "bulge" (Doench JG et al., Genes & Dev., 2003). In another embodiment, 2, 3, 4, 5, or 6 consecutive or non-identical nucleotides are introduced. The non-identical nucleotides may be selected to form fluctuating base pairs (e.g., G:U) or mismatch base pairs (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further preferred embodiment, the "bulge" is centered at nucleotide positions 12 and 13 from the 5' end of the miRNA molecule.

[0136] Modified RNA silencing agent In certain aspects of the present invention, the RNA silencing agent (or any part thereof) of the present invention may be modified as described above to further improve the activity of the agent. For example, the RNA silencing agent described above may be modified with any of the modifications described below. The modifications may act in part to further improve target recognition, to improve the stability of the agent (e.g., to prevent degradation), to promote cell uptake, to improve targeting efficiency, to improve the effectiveness of binding (e.g., to the target), to improve patient tolerance to the agent, and / or to reduce toxicity.

[0137] 1) Modifications to improve target identification In certain embodiments, the RNA silencing agent of the present invention may be substituted with an unstable nucleotide to improve single-nucleotide target recognition (see U.S. Patent Application No. 11 / 698,689 filed January 25, 2007, and U.S. Provisional Patent Application No. 60 / 762,225 filed January 25, 2006, both of which are incorporated herein by reference). Such modifications may be sufficient to neutralize the specificity of the RNA silencing agent to non-target mRNA (e.g., wild-type mRNA) without significantly affecting the specificity of the RNA silencing agent to target mRNA (e.g., gain-of-function mutant mRNA).

[0138] In preferred embodiments, the RNA silencing agent of the present invention is modified by introducing at least one universal nucleotide into its antisense strand. The universal nucleotide comprises a base moiety that can indiscriminately base-pair with any of the four conventional nucleotide bases (e.g., A, G, C, U). Universal nucleotides are preferred because they have a relatively small effect on the stability of the RNA double helix, or the double helix formed by the guide strand and target mRNA of the RNA silencing agent. Exemplary universal nucleotides include those having an inosine base moiety or inosine analog base moiety selected from the group consisting of deoxyinosine (e.g., 2'-deoxyinosine), 7-deaza-2'-deoxyinosine, 2'-aza-2'-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2'-O-methoxyethyl-inosine, and 2'-OMe-inosine. In certain preferred embodiments, the universal nucleotide is an inosine residue or a natural analog thereof.

[0139] In certain embodiments, the RNA silencing agent of the present invention is modified by introducing at least one destabilizing nucleotide within 5 nucleotides of a specificity-determining nucleotide (i.e., a nucleotide that recognizes a disease-associated polymorphism). For example, the destabilizing nucleotide may be introduced at a position within 5, 4, 3, 2, or 1 nucleotide (or more) of the specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position 3 nucleotides from the specificity-determining nucleotide (i.e., so that two stabilizing nucleotides are present between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g., siRNA and shRNA), the destabilizing nucleotide may be introduced into the strand or strand portion that does not contain the specificity-determining nucleotide. In preferred embodiments, the destabilizing nucleotide is introduced into the same strand or strand portion that contains the specificity-determining nucleotide.

[0140] 2) Modifications to enhance efficacy and specificity In certain embodiments, the RNA silencing agents of the present invention can be modified to easily enhance their efficacy and specificity in mediating RNAi in accordance with asymmetric design rules (see U.S. Patents 8,309,704, 7,750,144, 8,304,530, 8,329,892, and 8,309,705). Such modifications facilitate the entry of the antisense strand of siRNA (e.g., siRNA designed using the method of the present invention, or siRNA produced from shRNA) into RISC in a manner favorable to the sense strand, thereby allowing the antisense strand to preferentially induce cleavage or translational repression of the target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. Preferably, the asymmetry of the RNA silencing agent is improved by reducing the base pair strength between the antisense strand 5' end (AS5') and the sense strand 3' end (S3') of the RNA silencing agent, relative to the binding strength or base pair strength between the antisense strand 3' end (AS3') and the sense strand 5' end (S'5) of the RNA silencing agent.

[0141] In one embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that the number of G:C base pairs between the 5' end of the first or antisense strand and the 3' end of the sense strand portion is less than the number of G:C base pairs between the 3' end of the first or antisense strand and the 5' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that there is at least one mismatch base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. Preferably, the mismatch base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C, and U:U. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced such that there is at least one fluctuation base pair, for example, G:U, between the 5' end of the first strand or antisense strand and the 3' end of the sense strand portion. In another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced to include at least one base pair containing a rare nucleotide, such as inosine (I). Preferably, the base pair is selected from the group consisting of I:A, I:U, and I:C. In yet another embodiment, the asymmetry of the RNA silencing agent of the present invention may be enhanced to include at least one base pair containing a modified nucleotide. In a preferred embodiment, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

[0142] 3) RNA silencing agents with improved stability The RNA silencing agent of the present invention may be modified to improve its stability in serum or growth medium for cell culture. To improve stability, the 3'-residue may be stabilized against degradation, and in particular, it may be selected to consist of a purine nucleotide such as adenosine or guanosine nucleotide. Alternatively, substitution of pyrimidine nucleotides with modification analogs, for example, substitution of uridine with 2'-deoxythymidine, is acceptable and does not affect the efficiency of RNA interference.

[0143] In a preferred embodiment, the present invention features an RNA silencing agent comprising a first and a second strand, wherein the second and / or first strand is modified by substituting internal nucleotides with modified nucleotides to improve in vivo stability compared to the corresponding unmodified RNA silencing agent. As defined herein, “internal” nucleotides are those located at any position other than the 5' or 3' end of a nucleic acid molecule, polynucleotide, or oligonucleotide. Internal nucleotides may be located within a single-stranded molecule or within a double-stranded or double-stranded molecule. In one embodiment, the sense strand and / or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and / or antisense strand are modified by substitutions of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more of internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by substitutions of all internal nucleotides.

[0144] In preferred embodiments of the present invention, the RNA silencing agent may comprise at least one modified nucleotide analog. The nucleotide analog may be located at a position where target-specific silencing activity, such as RNAi-mediated activity or translational repression activity, is substantially unaffected, for example, in the 5' and / or 3' terminal regions of the siRNA molecule. In particular, the terminals can be stabilized by incorporating the modified nucleotide analog.

[0145] Exemplary nucleotide analogs include sugar- and / or skeletal-modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone). For example, the phosphodiester bond in native RNA may be modified to include at least one nitrogen or sulfur heteroatom. In exemplary skeletal-modified ribonucleotides, the phosphate ester group attached to an adjacent ribonucleotide is replaced by a modifying group, for example, a phosphothioate group. In exemplary sugar-modified ribonucleotides, the 2'OH- group is replaced by a group selected from H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON, where R is a C1-C6 alkyl, alkenyl, or alkynyl, and halo is F, Cl, Br, or I.

[0146] In certain embodiments, the modifications are 2'-fluoro, 2'-amino, and / or 2'-thio modifications. Particularly preferred modifications include 2'-fluorocytidine, 2'-fluorouridine, 2'-fluoroadenosine, 2'-fluoroguanosine, 2'-aminocytidine, 2'-aminouridine, 2'-aminoadenosine, 2'-aminoguanosine, 2,6-diaminopurine, 4-thiouridine, and / or 5-aminoallyluridine. In certain embodiments, the 2'-fluororibonucleotide is any uridine or cytidine. Additional exemplary modifications include 5-bromouridine, 5-iodouridine, 5-methylcytidine, ribothymidine, 2-aminopurine, 2'-aminobutyrylpyreneuridine, 5-fluorocytidine, and 5-fluorouridine. 2'-deoxynucleotides and 2'-Omenucleotides may also be used within the modified RNA silencing agent moiety of the present invention. Additional modification residues include deoxy debase, inosine, N3-methyluridine, N6,N6-dimethyladenosine, pseudouridine, purine ribonucleoside, and ribavirin. In certain preferred embodiments, the 2' portion is a methyl group, such that the linking portion is a 2'-O-methyl oligonucleotide.

[0147] In exemplary embodiments, the RNA silencing agent of the present invention comprises locked nucleic acid (LNA). The LNA contains sugar-modified nucleotides that are resistant to nuclease activity (highly stable) and have single-nucleotide recognition for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1):439-447, Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2'-O,4'-C-ethylene-bridged nucleic acids that may have modifications such as 2'-deoxy-2''-fluorouridine. Furthermore, the LNA enhances the specificity of oligonucleotides by constraining the sugar moiety to a 3'-end conformation, thereby pre-organizing the nucleotides for base pairing and raising the melting temperature of the oligonucleotides by up to 10°C per base.

[0148] In another exemplary embodiment, the RNA silencing agent of the present invention comprises peptide nucleic acid (PNA). The PNA comprises modified nucleotides in which the sugar-phosphate moiety of the nucleotide is replaced with a neutral 2-aminoethylglycine moiety that can form a polyamide skeleton, thereby providing high resistance to nuclease digestion and conferring improved binding specificity to molecules (Nielsen, et al., Science, (2001), 254:1497-1500).

[0149] Nucleic acid base-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleic acid base instead of naturally occurring nucleic acid bases, are also preferred. The bases can be modified to block the activity of adenosine deaminase. Examples of modified nucleic acid bases, but not limited to these, include uridine and / or cytidine modified at position 5, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6-methyladenosine, which are preferred. It should be noted that the above modifications may be combined.

[0150] In other embodiments, crosslinking can be used to alter the pharmacokinetics of RNA silencing agents, for example, to extend their half-life in the body. Therefore, the present invention comprises RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The present invention also comprises RNA silencing agents that are conjugated (e.g., at their 3' end) to another part (e.g., a non-nucleic acid part such as a peptide), an organic compound (e.g., a dye), etc., or unconjugated. Modifying siRNA derivatives in this manner can improve cellular uptake of the resulting siRNA derivative or enhance its cell targeting activity compared to the corresponding siRNA, is useful for tracing the siRNA derivative in cells, or improves the stability of the siRNA derivative compared to the corresponding siRNA.

[0151] Other exemplary modifications include (a) 2' modifications, e.g., on U of the sense or antisense chain, but particularly on the sense chain, providing a 2'OMe portion, or a 3' overhang, e.g., providing a 2'OMe portion at the 3' terminus (where the 3' terminus means the 3' atom or most of the 3' portion of the molecule, e.g., at most of the 3'P or 2' positions as indicated by the context); (b) modifications of the skeleton, e.g., by substitution of 0 at S in the phosphate skeleton, e.g., providing a phosphorothioate modification, e.g., on U or A or both, particularly on the antisense chain, e.g., by substitution of P at S; (c) substitution of U in the C5 aminolinker; (d) substitution of A at G (preferably the sequence change is located on the sense chain rather than the antisense chain); and (d) modifications at the 2', 6', 7', or 8' positions. Exemplary embodiments include embodiments in which one or more of these modifications are present on the sense but not on the antisense chain, or embodiments in which the antisense chain has few such modifications. Further exemplary modifications include the use of methylated P at the 3' end of the 3' overhang, for example; combinations of 2' modifications, for example; provision of a 2'OMe moiety and modification of the skeleton, for example; substitution of P with S, for example; provision of phosphorothioate modification; or the use of methylated P at the 3' end of the 3' overhang, for example; modification with a 3' alkyl group; or the use of debasalized pyrrolidone at the 3' end of the 3' overhang, for example; naproxen, ibuprofen, or modification with other moieties that inhibit degradation at the 3' end.

[0152] 4) Modifications that enhance cellular uptake In other embodiments, the RNA silencing agent may be modified with a chemical moiety to enhance cellular uptake by target cells (e.g., nerve cells). Accordingly, the present invention includes RNA silencing agents that are conjugated (e.g., at their 3' end) to another moiety (e.g., a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. Conjugation can be achieved using methods known in the art, for example, Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (described for nucleic acids packed into polyalkylcyanoacrylate (PACA) nanoparticles), Fattal et al., J. Control Release 53(1-3): 137-43 (1998) (described for nucleic acids bound to nanoparticles), Schwab et al., Ann. Oncol. 5 Suppl. 4: 55-8 (1994) (described for nucleic acids bound to inserts, hydrophobic groups, polycations, or PACA nanoparticles), and Godard et al., Eur. J. Biochem. 232(2): 404-10 (1995) (described for nucleic acids bound to nanoparticles).

[0153] In certain embodiments, the RNA silencing agent of the present invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand containing a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of the siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3' end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, or a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic components include cholic acid, adamantane acetate, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

[0154] 5) Tethering ligand Other entities can be tethered to the RNA silencing agent of the present invention. For example, ligands are tethered to the RNA silencing agent to improve stability, hybridization thermodynamics with target nucleic acids, targeting to specific tissues or cell types, or cell permeability, for example, by endocytosis-dependent or independent mechanisms. Ligands and associated modifications can also enhance sequence specificity, thereby reducing off-site targeting. Tethered ligands may include one or more modified bases or sugars that can function as intercalators. These are preferably located within internal regions, such as within the bulge of the RNA silencing agent / target double helix. Intercalators may be aromatic, e.g., polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems having two, three, or four fused rings. Universal bases described herein may be included in ligands. In one embodiment, ligands may include cleavage groups that contribute to the inhibition of target genes by cleaving target nucleic acids. The cleavage group may be, for example, bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., O-phenanthroline), polyamine, tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. Examples of metal ion chelating groups include Lu(III) or EU(III) macrocyclic complexes, Zn(II) 2,9-dimethylphenanthroline derivatives, Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the bulge region by free metal ions such as Lu(III). In some embodiments, a peptide ligand can be tethered to an RNA silencing agent to promote, for example, the cleavage of target RNA at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage.The tethering ligand can be an aminoglycoside ligand, which can impart improved hybridization properties or improved sequence specificity to the RNA silencing agent. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of acridine analogs can enhance sequence specificity. For example, neomycin B has high affinity for RNA compared to DNA, but low sequence specificity. The acridine analog neo-5-acridine has high affinity for the HIV Rev-response element (RRE). In some embodiments, guanidine analogs (guanidinoglycosides) of aminoglycoside ligands are tethered to the RNA silencing agent. Within guanidinoglycosides, the amine group on the amino acid is replaced with a guanidine group. The attachment of a guanidine analog can increase the cellular permeability of RNA silencing agents. The tethering ligand may be a polyarginine peptide, peptoid, or peptide mimetic that can increase the cellular uptake of oligonucleotide agents.

[0155] Exemplary ligands are preferably covalently bound to a ligand-conjugate carrier, either directly or indirectly via intervening tethering. In exemplary embodiments, the ligand is attached to the carrier via intervening tethering. In exemplary embodiments, the ligand alters the distribution, targeting, or lifetime of the RNA silencing agent into which it is incorporated. In exemplary embodiments, the ligand provides, for example, higher affinity to selected targets, such as molecules, cells or cell types, compartments, such as cell or organ compartments, tissues, organs, or regions of the body, compared to species in which such ligands are absent.

[0156] Exemplary ligands can improve transport, hybridization, and specificity properties, and may also improve the nuclease resistance of polymer molecules and / or natural or modified ribonucleotides, including the resulting natural or modified RNA silencing agents or any combination of monomers described herein. Ligands generally include, for example, therapeutic modifiers to increase uptake, such as diagnostic compounds or reporter groups to monitor distribution, crosslinkers, nuclease resistance-constituting moieties, and natural or aberrant nucleic acid bases. Common examples include lipophilic substances, lipids, steroids (e.g., ubaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, friederin, epifriederanol-deranolic acid-derived lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binders, integrin target molecules, polycations, peptides, polyamines, and peptide mimetic compounds. Ligands can be naturally occurring substances (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin), carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid), amino acids, or lipids. Ligands may also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-coglycolated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphatidine.Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudo-peptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helix peptides.

[0157] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to specific cell types such as the kidney, gland (e.g., thyroid), brain, eye, and / or male testicular cells. The targeting group can also be thyroid stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine, polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrene), lys-tyr-lys tripeptide, aminoglycoside, guanidium aminoglycodies, artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol (and its thio analogs), cholic acid, chenodeoxycholic acid, lithocholic acid, adamantaneacetic acid, 1-pyrenebutanoic acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, such as C 10 C 11 C 12 C 13 C 14 C 15 C16 , C 17 , C 18 , C 19 , or C 20 fatty acids) and their ethers, for example, C 10 , C 11 , C 12 , C 13 , C 14 , C 15 , C 16 , C 17 , C 18 , C 19 , or C 20 Alkyl compounds (e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octadecyl)glycerol), geranyloxyhexyl groups, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine), and peptide conjugates (e.g., Antennapedia, Tat peptide). Examples include alkylating agents, phosphates, amino acids, mercaptos, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, substituted alkyl groups, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of the tetraazamacrocycle), dinitrophenyl, HRP, or AP.

[0158] Ligands can be proteins, such as glycoproteins, or peptides, such as molecules that have a specific affinity for a colligand, or antibodies, such as antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or osteocytes. Examples of ligands include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, polyvalent mannose, or polyvalent fucose. Ligands can be, for example, lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0159] A ligand can be a substance, such as a drug, that can increase the uptake of RNA silencing agents into cells by disrupting the cytoskeleton of a cell, for example, by disrupting the microtubules, microfilaments, and / or intermediate filaments of a cell. Drugs may include, for example, taxone, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latranculine A, phalloidin, swinholide A, indanosine, or myoservin. Ligands can also increase the uptake of RNA silencing agents into cells by, for example, activating an inflammatory response. Exemplary ligands having such effects include tumor necrosis factor alpha (TNFα), interleukin-1 beta, or gamma interferon. In one embodiment, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA-binding ligands allow for the distribution of conjugates to target tissues, such as non-renal target tissues of the body. For example, the target tissue may be the liver, including the parenchymal cells of the liver. Other molecules that can bind to HSA can also be used as ligands. For example, naproxen or aspirin can be used. Lipid or lipid-based ligands can be used to (a) improve the conjugate's resistance to degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) modulate binding to serum proteins, e.g., HSA. Lipid-based ligands can be used to modulate, e.g., control the binding of the conjugate to target tissue. For example, a lipid or lipid-based ligand that binds more strongly to HSA is less likely to target the kidney and therefore less likely to be removed from the body. A lipid or lipid-based ligand that does not bind less strongly to HSA can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid-based ligand binds to HSA.Lipid-based ligands can bind to HSA with sufficient affinity such that the conjugate is preferably distributed to non-renal tissue. However, the affinity is preferably not so strong that it cannot reverse the HSA-ligand binding. In another preferred embodiment, the lipid-based ligand binds weakly to HSA or does not bind at all such that the conjugate is preferably distributed to the kidney. Other parts that target kidney cells can be used instead of or in addition to the lipid-based ligand.

[0160] In another embodiment, the ligand is a portion taken up by target cells, e.g., proliferating cells, e.g., a vitamin. These are particularly useful in treating undesirable cell proliferation, e.g., disorders characterized by malignant or non-malignant forms, e.g., cancer cells. Exemplary vitamins include vitamins A, E, and K. Other exemplary vitamins include vitamin B (e.g., folic acid, B12, riboflavin, biotin, pyridoxal) or other vitamins or nutrients taken up by cancer cells. HSA and low-density lipoprotein (LDL) are also included.

[0161] In another embodiment, the ligand is preferably a cell permeabilizer, such as a helical cell permeabilizer. Preferably, the agent is amphiphilic. Exemplary agents are peptides such as tat or antennopedia. If the agent is a peptide, it may be modified, including the use of peptidyl mimes, invertomers, non-peptide or pseudo-peptide bonds, and D-amino acids. The helical agent is preferably an alpha-helical agent and preferably has an oleophilic phase and an oleophobic phase.

[0162] The ligand may be a peptide or a peptide mimetic. A peptide mimetic (also referred to herein as an oligopeptide mimetic) is a molecule that can fold into a defined three-dimensional structure similar to that of a natural peptide. The attachment of peptides and peptide mimetics to oligonucleotide agents can affect the pharmacokinetic distribution of RNA silencing agents by improving cellular recognition and absorption, among other things. The peptide or peptide mimetic moiety may be about 5 to 50 amino acids long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (for example, mainly consisting of Tyr, Trp, or Phe). The peptide moiety may be a dendrimeric peptide, a restrictive peptide, or a cross-linked peptide. The peptide moiety may be an L-peptide or a D-peptide. Alternatively, the peptide moiety may contain a hydrophobic membrane translocation sequence (MTS). Peptides or peptide mimetic compounds can be encoded by random DNA sequences, such as peptides identified from phage presentation libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptide mimetic compound tethered to an RNA silencing agent via an incorporated monomer unit is a cell-targeted peptide, such as an arginine-glycine-aspartate (RGD)-peptide or RGD mimic. The peptide moiety may range in length from approximately 5 to approximately 40 amino acids. The peptide moiety may have structural modifications, such as to improve stability or induce conformational properties. Any of the structural modifications described below may be utilized.

[0163] Oligonucleotide conjugates The oligonucleotide conjugates described herein comprise an oligonucleotide and a dendron. The dendron may have a potent, reversible, and non-covalent albumin bond, which can be advantageously used to minimize degradation, reduce macrophage uptake and degradation, prevent nonspecific uptake by cells, and / or provide improved delivery of the oligonucleotide conjugate to the lungs. The dendron may also have a nanomolar affinity for albumin, which advantageously ensures that the albumin-oligonucleotide conjugate complex remains stable throughout in vivo distribution, cell interactions, and / or throughout the treatment.

[0164] In some embodiments, the oligonucleotide conjugate has the structure of formula I, [ka] During the ceremony, A is a oligonucleotide, B, for each occurrence, independently comprises one or more hydrophobic chains, amines, amides, esters, N- or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, wherein the hydrophobic chains are saturated or unsaturated C 1-24 Containing alkyl chains, For each appearance, C is independently a hydroxide, amine, phosphate, sulfur, and / or a hydrophilic group containing sugar, amine, amide, ether, ester, N- or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C. 1-24 A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings, D is a branched unit that, for each occurrence, independently contains one or more alkyl chains, amides, ethers, esters, and amines, and the branched unit contains 2 to 4 branches. m is either 0 or 1 for each occurrence, independently.

[0165] In some embodiments, the oligonucleotide conjugate has the structure of formula II, [ka] During the ceremony, A is a oligonucleotide, B, for each occurrence, independently comprises one or more hydrophobic chains, amines, amides, esters, N- or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, wherein the hydrophobic chains are saturated or unsaturated C 1-24 Containing alkyl chains, For each appearance, C is independently a hydroxide, amine, phosphate, sulfur, and / or a hydrophilic group containing sugar, amine, amide, ether, ester, N- or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C. 1-24 A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings, D is a branched unit that, for each occurrence, independently contains one or more alkyl chains, amides, ethers, esters, and amines, and the branched unit contains 2 to 4 branches.

[0166] In some embodiments, the oligonucleotide conjugate has the structure of formula III, [ka] In the formula, n is an integer from 1 to 24, independently for each occurrence. In some embodiments, C is OH, and n is 1, 6, or 12, independently for each occurrence. In certain embodiments, the oligonucleotide conjugate has the structure of formula IV. [ka]

[0167] In some embodiments, the oligonucleotide conjugate has the structure of formula V, [ka] During the ceremony, A is a oligonucleotide, B, for each occurrence, independently comprises one or more hydrophobic chains, amines, amides, esters, N- or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, wherein the hydrophobic chains are saturated or unsaturated C 1-24 Containing alkyl chains, For each appearance, C is independently a hydroxide, amine, phosphate, sulfur, and / or a hydrophilic group containing sugar, amine, amide, ether, ester, N- or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C. 1-24 A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings, D is a branched unit comprising, independently for each occurrence, one or more alkyl chains, amides, ethers, esters, and amines, and the branched unit comprises 2 to 4 branches. In some embodiments, the oligonucleotide conjugate has the structure of formula VI, [ka] In the formula, n is an integer from 1 to 24, independently for each occurrence. In some embodiments, C is, independently for each occurrence, a hydroxide, amine, phosphate, sulfur, and / or hydrophilic group containing sugar, amine, amide, ether, ester, N- or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C 1-24 It comprises a hydrophobic group including an alkyl chain and / or one or more aromatic rings. In some embodiments, C is OH. In some embodiments, n is independently 1, 6, or 12 for each occurrence. In certain embodiments, the oligonucleotide conjugate has the structure of formula VII. [ka]

[0168] Branched oligonucleotide conjugate In certain embodiments, the oligonucleotide conjugate is a branched oligonucleotide conjugate.

[0169] The branched oligonucleotide conjugates described herein comprise two or more glucosamines or their derivatives that are conjugated together. Different branched oligonucleotide conjugates described herein (e.g., branched oligonucleotide conjugates with two, three, or four glucosamines or their derivatives) can enhance oligonucleotide delivery, including specific delivery to kidney, glandular (e.g., thyroid), brain, eye, and male testicular cells.

[0170] In certain embodiments, the branched oligonucleotide conjugate comprises i) an oligonucleotide having a 5' end and a 3' end, as well as complementarity to the target nucleic acid, and ii) a branched functional moiety bonded to the oligonucleotide and containing two or more glucosamines or derivatives thereof.

[0171] In certain embodiments, the branched functional moiety is a triple amine functional moiety such as a phosphatidylcholine (PC) esterified triple amine (PC-triple amine).

[0172] In certain embodiments, two or more glucosamines or derivatives thereof in a branched oligonucleotide conjugate are linked to one or more branched functional moieties, independently selected from linkers, spacers, and branching points.

[0173] In certain embodiments, the linker includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, or any combination thereof.

[0174] In certain embodiments, the branching point includes a polyvalent organic species or a derivative thereof.

[0175] In another embodiment, the branching point is an amino acid derivative. In yet another embodiment, the branching point is selected from the following formula. [ka]

[0176] A polyvalent organic species is a carbon atom with a moiety containing three or more valencies (i.e., attachment sites to moieties such as S, L, or N as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene), tricarboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid), tetracarboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid), tertiary amines (e.g., tripropargylamine, triethanolamine), triamines (e.g., diethylenetriamine), tetramines, and species containing combinations of hydroxyl, thiol, amino and / or carboxyl moieties (e.g., amino acids such as lysine, serine, and cysteine).

[0177] In certain embodiments, the spacer includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, or combinations thereof.

[0178] Pharmaceutical composition and administration method In one embodiment, a pharmaceutical composition is provided herein comprising a therapeutically effective amount of one or more oligonucleotide conjugates described herein and a pharmaceutically acceptable carrier.

[0179] The pharmaceutical compositions of the present invention are formulated to be compatible with their intended route of administration (e.g., intradermal).

[0180] In this specification, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc., that are suitable for drug administration. The use of media and agents relating to pharmaceutically active substances is well known to those skilled in the art. Any conventional media or agent is intended to be used in a composition unless it is incompatible with the active compound. Complementary active compounds may also be incorporated into the composition.

[0181] Compositions, methods, and systems, such as pharmaceutical compositions containing oligonucleotide conjugates, provided herein may include any pharmaceutically acceptable salts, esters, or salts of such esters. The disclosure herein also relates to pharmaceutically acceptable salts, prodrugs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents of oligonucleotide conjugates. Preferred pharmaceutically acceptable salts include, but are not limited to, sodium salts and potassium salts. In some embodiments, the disclosure provides salts of oligonucleotide conjugates. In some embodiments, the salt is a sodium salt. In some embodiments, the salt is a potassium salt.

[0182] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions and sterile powders for the immediate preparation of sterile solutions or dispersions for injection. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and fluid enough to be easily injected. The composition must be stable under manufacturing and storage conditions and protected from contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be achieved, for example, by lecithin. The required particle size can be maintained by the use of coating agents such as those mentioned above, or by the use of surfactants in the case of dispersions. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars, mannitol, sorbitol, and polyalcohols such as sodium chloride, in the composition. Sustained absorption of the injectable composition can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0183] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound, along with one or a combination of the components listed above as needed, into a suitable solvent, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze-drying, from which a powder is obtained from a pre-sterilized filtered solution containing the active ingredient and any desired additional components.

[0184] Data obtained from cell culture assays and animal studies can be used in formulating a range of doses for use in humans. Doses of such compounds are preferably within a circulating concentration range that includes an ED50 with little to no toxicity. Doses may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the present invention, the therapeutically effective dose can first be estimated from a cell culture assay. The dose can be formulated in animal models to achieve a circulating plasma concentration range that includes an EC50 (i.e., the concentration of the test compound that achieves half of the maximum response) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high-performance liquid chromatography.

[0185] kit In certain other embodiments, the present invention provides a kit comprising a suitable container for containing a pharmaceutical formulation of an RNA silencing agent, e.g., a double-stranded RNA silencing agent, or an sRNA agent (e.g., a precursor, e.g., a larger RNA silencing agent that can be processed to become an sRNA agent, or an RNA silencing agent, e.g., a double-stranded RNA silencing agent, or an sRNA agent, or DNA encoding such a precursor). In certain embodiments, the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for RNA silencing agent preparation and a container for at least another carrier compound. The kit may be packaged in multiple different configurations, such as one or more containers in one box. For example, different components can be combined according to instructions provided with the kit. These components can be combined, for example, to prepare and administer a pharmaceutical composition according to the methods described herein. The kit may also include a delivery device.

[0186] Treatment method In one embodiment, a method is provided herein for selectively delivering one or more oligonucleotide conjugates described herein to the skin of a patient, the method comprising administering the compound to the patient.

[0187] In certain embodiments, methods for providing treatment to a patient requiring treatment for a skin disease, disorder, or injury are provided herein, comprising administering a compound of formula (I) to the patient. Non-limiting examples of such diseases or disorders include acne, pemphigus, alopecia areata, psoriasis, atopic dermatitis, Raynaud's phenomenon, epidermolysis bullosa, rosacea, hidradenitis suppurativa, scleroderma, ichthyosis, vitiligo, and congenital onychoplakia. In some embodiments, the skin disease, disorder, or injury is related to the JAK1 gene.

[0188] In certain embodiments, the Specified Invention provides a method for providing cancer treatment to a patient requiring such treatment, comprising, for example, administering a compound of formula (I) to the patient via intradermal injection. Non-limiting examples of cancer include melanoma, brain cancer, breast cancer, cervical cancer, colorectal cancer, eye cancer, fallopian tube cancer, testicular cancer, leukemia, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, pulmonary cancer, rectal cancer, kidney cancer, skin cancer, stomach cancer, and thyroid cancer.

[0189] As used herein, “treatment” or “to treat” is defined as the application or administration of a therapeutic agent (e.g., an RNA agent or vector or a transgene encoding such an RNA agent or vector) to a patient, or the application or administration of a therapeutic agent to tissue or cell lines isolated from a patient having a disease or disorder, symptoms of a disease or disorder, or a predisposition to a disease or disorder, for the purpose of treating, curing, reducing, mitigating, modifying, repairing, restoring, improving, or influencing such disease or disorder, symptoms of a disease or disorder, or predisposition to a disease or disorder.

[0190] In one embodiment, the present invention provides a method for preventing the above-mentioned disease or disorder in a subject by administering a therapeutic agent (e.g., an RNAi agent or vector or a transgene encoding the same) to the subject. Subjects at risk of the disease can be identified, for example, by one or a combination of the diagnostic assays or prognostic assays described herein. The administration of the prophylactic agent can be carried out before the appearance of symptoms characteristic of the disease or disorder, in order to prevent the disease or disorder or, alternatively, to slow its progression.

[0191] Another aspect of the present invention relates to a method for therapeutically treating a subject, i.e., a method for altering the onset of symptoms of a disease or disorder. In exemplary embodiments, the modulatory method of the present invention involves contacting cells expressing a gain-of-function mutant with a therapeutic agent (e.g., an RNAi agent, or a vector or transgene encoding it) that is specific to one or more target sequences in a gene, so as to achieve sequence-specific interference with the gene. These methods can be carried out in vitro (e.g., by culturing cells with the drug) or, alternatively, in vivo (e.g., by administering the drug to the subject).

[0192] In one embodiment, the subject is administered an initial dose and one or more maintenance doses of the RNA silencing agent. The maintenance dose or multiple maintenance doses are generally lower than the initial dose, for example, less than half of the initial dose. The maintenance regimen may include treating the subject with doses ranging from 0.01 to 1.4 mg / kg body weight per day, for example, 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day, or multiple doses. The maintenance dose is preferably administered once or less every 5, 10, or 30 days. Furthermore, the treatment regimen may last for a duration that varies depending on the nature of the specific disease, its severity, and the patient's overall condition. In a preferred embodiment, the dose may be delivered once or less per day, for example, once or less every 24, 36, 48 hours, or more, for example, once or less every 5 or 8 days. After treatment, the patient may be monitored for changes in the patient's condition and relief of symptoms of the disease state. The dosage of the compound may be increased if the patient does not respond significantly to the current dosage level, or the dosage may be decreased if relief of symptoms of the disease state is observed, the disease state is eliminated, or undesirable side effects are observed. [Examples]

[0193] While several experimental examples are proposed, these embodiments are intended to be non-limiting.

[0194] Example 1. Alkyl molecule synthesis Oligonucleotides were synthesized according to standard protocols on a MerMade 6 / 12 synthesizer (Bioautomation) and an AKTA Oligopilot100 (GE Healthcare Life Sciences). In summary, conjugated sense strands were synthesized on a 5–20 μmol scale on custom-synthesized lipid-functionalized controlled-pore glass (CPG) supports for DCA conjugates. Dendritic sense strands were synthesized on CPGs functionalized with UnyLinker (ChemGenes), and the dendritic portion was constructed at the 5' end using commercially available amidites (Cy3, C6, C12, and symmetrically branched from ChemGenes and Glen Research). All sense strands had a 2dT spacer between the strand and the conjugate. Antisense strands were synthesized on CPGs functionalized with UnyLinker. First, the antisense chain was deprotected with a solution of bromotrimethylsilane / pyridine (3:2, v / v) in dichloromethane for (E)-vinylphosphonate deprotection, then cleaved and deprotected with 28% aqueous ammonium hydroxide at 60°C for 20 hours. All chains were cleaved and deprotected with 28% aqueous ammonium hydroxide at 60°C for 20 hours, then dried under vacuum at 60°C and resuspended in Millipore H2O. Oligonucleotides were purified using an Agilent Prostar System (Agilent Technologies), with the lipid conjugate sense chain on a C18 column and the antisense chain on an ion exchange column. The purified oligonucleotides were desalted by size exclusion chromatography and characterized by liquid chromatography-mass spectrometry (LC / MS) analysis on an Agilent 6530 precise mass quadrupole time-of-flight (Q-TOF) LC / MS (Agilent Technologies). Figure 1 shows examples of fully chemically stabilized oligonucleotides (siRNAs) and dendritic (D)siRNAs synthesized using this method.

[0195] The sequences of the compounds and their modifications are shown in Table 1 below (#: PS skeleton, m: 2'-o-methyl, f: 2'-fluoro, C12: hexaethylene spacer, C6: triethylene spacer, SB: symmetric branching, and V: (E)-vinylphosphonate). [Table 1-1] [Table 1-2] [Table 1-3]

[0196] Example 2. Injection of lipid-conjugated siRNA into mice Animal studies of siRNA conjugates were conducted at the RNA Therapeutic Research Institute in accordance with the animal husbandry ethics approval and guidelines of the University of Massachusetts Medical School Institutional Animal Care and Use Committee (IACUC, protocol number 202000010). In all experiments, 7-8 week old female FVB / NJ mice (The Jackson Laboratory) were used and, unless otherwise specified, were administered by sc or IV injection at a concentration of 20 mg / kg either untargeted control siRNA (NTC) or lipid-conjugated siRNA (n=5-6 per group).

[0197] For distribution studies, three mice were injected per sample. For efficacy studies, 5 to 8 mice were studied per gene per sample. For toxicity studies, three mice were injected per sample.

[0198] Example 3. In vivo mRNA silencing experiment One week after injection, mice were euthanized and perfused with PBS. Tissue was collected and stored overnight in RNAlater (Sigma) at 4°C. mRNA was quantified using QuantiGene 2.0 Assay (Affymetrix). 1.5 mm punches (three per tissue) were placed in a QIAGEN Collection Microtube holding 3 mm tungsten beads and lysed in 300 μl of Homogenizing Buffer (Affymetrix) containing 0.2 mg / ml protease K (Invitrogen) using QIAGEN TissueLyser II. The samples were then centrifuged at 1000 × g for 10 minutes and incubated at 55°C–60°C for 1 hour. Lysates and diluted probe sets (mouse Htt, mouse Ppib, or mouse Hprt) were added to bDNA capture plates, the signals were amplified, and detection was performed. The light emission was detected using a Tecan M1000 (Tecan, Morrisville, NC, USA).

[0199] Example 4. Peptide nucleic acid (PNA) hybridization assay The tissue concentration of the antisense strand was determined using a peptide nucleic acid (PNA) hybridization assay. Tissue punches were placed in a QIAGEN Collection Microtube containing 3 mm tungsten beads and lysed in 300 μl of MasterPure tissue lysate (EpiCentre) containing 0.2 mg / ml protease K (Invitrogen) using QIAGEN TissueLyser II. The lysates were then centrifuged at 1000 × g for 10 minutes and incubated at 55°C–60°C for 1 hour. Sodium dodecyl sulfate (SDS) was precipitated from the lysates by adding 20 μl of 3 M potassium chloride and pelletizing centrifugation at 5000 × g for 15 minutes. The conjugated siRNA in the removed supernatant was hybridized to a Cy3-labeled PNA probe that was completely complementary to the antisense strand (PNABio, Thousand Oaks, CA, USA). The samples were analyzed by HPLC (Agilent, Santa Clara, CA, USA) on a DNAPac PA100 anion exchange column (Thermo Fisher Scientific) using a sodium perchlorate gradient as follows: Buffer A: 50% water; 50% acetonitrile; 25 mM Tris-HCl, pH 8.5; 1 mM ethylenediaminetetraacetic acid. Buffer B: 800 mM sodium perchlorate in Buffer A. Gradient conditions: 10% Buffer B within 4 minutes, 50% Buffer B for 1 minute, and 50% to 100% Buffer B within 5 minutes. Cy3 fluorescence was monitored and the peaks were integrated. The final concentration was confirmed using a calibration curve generated by spiked tissue lysates from untreated animals with a known amount of lipid-conjugated siRNA. The spiked samples for calibration and the experimental samples were processed and analyzed under the same laboratory conditions.

[0200] Example 5. Fluorescence microscopy examination Organs were collected from euthanized mice, washed with PBS, molded in OCT embedding medium, and frozen. Frozen sections were cut to a thickness of 5 μm using Leica Cryostat CM1950, and the slides were air-dried overnight. The slides were then hydrated twice in PBS buffer for 5 minutes at room temperature, followed by mounting coverslips onto the slides using ProLong® Gold Antifade Mountant with DAPI staining for the nuclei.

[0201] Example 6. Lipoprotein size exclusion chromatography For lipoprotein profiling, we followed the same protocol previously described by Osborn (see Osborn, MF et al. “Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways,” Nucleic Acids Research 47, 1070-1081 (2018) (the entire work is incorporated herein by reference)). Briefly, mice were intravenously injected with 10 mg / kg of Cy3-labeled oligonucleotide. Fifteen minutes later, whole mouse blood (approximately 500 μl) was collected in a sterile EDTA-coated tube after incising the cheek with a lancet. The sample was rotated at 10,000 RPM for 10 minutes at 4°C. 50 μl of plasma was injected directly into a Superose 6 Increase 10 / 300 size exclusion column (GE Healthcare). Oligonucleotide migration was monitored at 570 nm, and lipoprotein content was monitored by absorbance at 280 nm. For subcutaneous injections, samples were collected one hour after the injection.

[0202] Example 7. Reverse-phase HPLC analysis of D-siRNA vs. DCA-siRNA LC data of oligonucleotides were performed on an Agilent 6530 high-precision mass Q-TOF under the following conditions: Buffer A: 100 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 9 mM triethylamine (TEA) in LC-MS grade water; Buffer B: 100 mM HFIP and 9 mM TEA in LC-MS grade methanol; Column: Agilent AdvanceBio Oligonucleotide C18; 5-100% B for 11 minutes; Temperature: 60°C; Flow rate: 0.5 ml / min. LC peaks were monitored at 260 nm.

[0203] Example 8. Cell culture and in vitro dose response HeLa cells were maintained in DMEM (Cellgro, 10-013-CV) supplemented with 10% fetal bovine serum (FBS) (Gibco, 26140) and 100 U / mL pen / strep (Invitrogen, 15140), and grown at 37°C and 5% CO2. Seven-point dose-response curves were generated by treating HeLa cells with various concentrations of siRNA formulated with RNAiMax (Invitrogen, 13778-150) at 37°C and 5% CO2 for 72 hours. Transfection was performed without antibiotics in 50:50 DMEM / OptiMEM (Gibco, 31985-070) and 3% FBS. Cells were lysed by incubation at 55°C for 30 minutes using a diluted QuantiGene (QG) lysis mixture (Invitrogen, QP0524) containing protease K (Invitrogen, 25530-049). Gene silencing was evaluated by QG branched DNA (bDNA) assay using the following probe sets: mouse Htt (SB-14150) and mouse Hprt (SB-15463), according to the manufacturer's instructions (see brief description below). Htt data were normalized to housekeeping Hprt and expressed as a percentage of the untreated control. n=3.

[0204] Example 9. In vivo dose response Eight-week-old female mice were subcutaneously injected with a panel of Cy3-labeled siRNA variants. After 24 hours, the femurs of the mice were dissected, and bone marrow cells were isolated from the dissected femurs by washing them with PBS on a 70 μm cell strainer placed in a 50 mL conical tube using a 23 G needle. The bone marrow and spleen were then crushed using a 5 mL plunger, followed by rinsing the strainer with DMEM medium. The cell solution collected in a 50 mL tube was centrifuged at 350 g for 5 minutes at room temperature and washed once with PBS. Blood cells were lysed using ACK lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA in DW) and subsequently washed with DMEM (500 × g, 10 min, 4°C). Next, for flow cytometry analysis, the cell pellet was suspended in flow cytometry buffer (0.5% BSA, 2 mM EDTA in DMEM).

[0205] For leukocyte staining, VioGreen conjugate CD45 antibody (clone REA737, Miltenyi Biotec), APC conjugate GR-1 antibody (clone REA810, Miltenyi Biotec), FITC conjugate CD11b antibody (clone REA592, Miltenyi Biotec), PE-Vio770 conjugate CD11c antibody (clone REA754, Miltenyi Biotec), and PE-Vio615 F40 / 80 (clone REA126, Miltenyi Biotec) were used. For lymphocyte staining, PE-vio770 conjugate CD19 (clone REA749) and APC conjugate CD3 antibody (clone REA641) were used. Cells were stained at 4°C for 30 minutes. Next, the cells were washed twice with 700 μl of flow cytometry buffer and resuspended in flow cytometry buffer containing 1 μM SYTOX® Blue (Thermo Fisher Scientific). The stained cells were analyzed using MACSQuant® VYB Flow Cytometer (Miltenyi Biotec), and the data were analyzed using FlowJo software (v10.6, BD Biosciences, Ashland, OR, USA).

[0206] Example 10. Selective protein binding profile of D-siRNA to albumin As previously developed by Osborn, we evaluated the lipoprotein binding profile via size exclusion chromatography (see Osborn, MF et al. “Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways,” Nucleic Acids Research 47, 1070-1081 (2018) (the entirety of which is incorporated herein)).

[0207] The binding profile of a conjugate partially depends on its hydrophobicity; more hydrophobic conjugates (docosanoic acid, DCA, etc.) bind to low-density and high-density lipoproteins (LDL and HDL) in plasma, and to a lesser extent, to albumin. The dendritic portion is larger and contains more aliphatic material (69 carbons) compared to DCA (28 carbons). In addition to terminal hydroxyl groups, multiple phosphates puncturing the conjugate structure can result in increased solubility and prevention of aggregation. Therefore, D-siRNA is less hydrophobic than DCA-siRNA, which is evident from its lower retention when analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) (Figures 2A and 2B).

[0208] Following hydrophobicity comparisons by HPLC, the serum protein binding profiles of each conjugate after injection in mice were examined as described above. Briefly, animals were injected subcutaneously (sc) or intravenously (iv) with Cy3-labeled D-siRNA or DCA-siRNA at a dose of 10 mg / kg, and plasma was collected 15 minutes (for iv) and 60 minutes (for sc) after injection. The time points were selected to maximize the circulating levels of the compound in the blood.

[0209] Next, plasma was fractionated by size exclusion chromatography, and Cy-3-siRNA elution time was monitored (570 nm). Sucrose-6 elution profiles for several major plasma proteins (HDL, VLDL, LDL, albumin / globulin) were pre-established. After injection into mice, D-siRNA had a retention time of 67 minutes compared to DCA-siRNA, which had a multiprotein binding profile mainly associated with LDL and HDL, overlapping with the retention time for albumin (Error! Reference not found. B). Protein binding tendencies were consistent across both administration routes. This data confirms selective and high-affinity binding to albumin, which is assumed to determine the in vivo behavior, as determined in vitro. The data also suggest that D-siRNA binds firmly to albumin and can circulate in the blood as a protein-RNA complex, leaching into various organs and tissues, regardless of the mode of injection (subcutaneous or intravenous).

[0210] Example 11. Skin delivery of D-siRNA We evaluated the delivery of D-siRNA to the skin (Figures 3, 4, and 5).

[0211] Figure 3 shows the intradermal injection on the left. As previously described, 50 µl of a 200 µM Cy3-labeled siRNA conjugate was injected into the prepared and washed human skin. Photographs were taken to visually track the spread of siRNA in the human skin. The skin was then left in culture medium for 24 hours, and another photograph was taken to track the spread of siRNA in the human skin. The images clearly show better spread using D-siRNA on the left compared to more localized siRNA using the more hydrophobic DCA-siRNA. On the right side of the photograph, intradermal delivery of siRNA was performed as previously described, but here incubated for 72 hours. The treated skin was then separated into dermis and epidermis, homogenized, and mRNA was collected. Subsequently, the quantification of target mRNA (JAK1 mRNA in this case) was measured using a Quantagene singleplex assay. The data (JAK1 mRNA) were expressed as a percentage of the untargeted control, and HTT-targeted siRNA was also used as a control for each conjugate, as it does not silence JAK1 mRNA.

[0212] Intradermal injection of Cy3-labeled, unconjugated siRNA, as well as DCA, cholesterol, diol, and dendrimer-conjugated siRNA, was also evaluated (Figure 4). Of all the conjugates tested, dendrimer-conjugated siRNA was observed to spread the most and reach all edges of the skin sample. Chemically stabilized siRNA was also observed to accumulate in multiple cell types after intradermal injection into human skin (Figure 5).

[0213] Example 12. Regulation of CXCL mRNA expression by D-siRNA We evaluated the regulation of CXCL mRNA expression by D-siRNA (Figure 6).

[0214] The regulation of CXCL mRNA expression in human skin cells after administration of PBS, NTC siRNA, Unc-JAK1 siRNA, DCA-JACK1 siRNA, and D-JACK1 was evaluated. Human skin samples were injected with siRNA and stimulated with IFN gamma to examine CXCL-9, CXCL-10, and CXCL-11 mRNA expression (Figure 6A). siRNAs targeting JAK1 were observed to significantly silence downstream chemokine signaling after IFNg stimulation (Figure 6B).

[0215] Example 13. Safety Profile of D-siRNA Toxicity studies for D-siRNA were conducted by injecting mice with escalating doses of 100 mg / kg, and monitoring blood chemistry and complete blood count (CBC) over 24 hours (Figure 7). The data showed no significant changes to any parameters measured when mice were injected with D-siRNA. However, a clear decrease in platelet levels was observed with DCA-siRNA. This is an undesirable outcome, as it is associated with an increased risk of stroke.

[0216] We evaluated the reduction in innate immune activation and inflammatory effects of D-siRNA. Hydrophobic conjugates such as cholesterol and DCA stimulate the innate immune system when they are taken up by monocytes and leukocytes (e.g., white blood cells). DCA and cholesterol-conjugated siRNAs have similar accumulation in the bone marrow and uptake by immune cells, with DCA showing slightly reduced uptake compared to cholesterol.

[0217] Flow cytometry was used to evaluate the uptake of Cy3-labeled D-siRNA in bone marrow and spleen compared to cholesterol-siRNA. 24 hours after subcutaneous injection, mouse bone marrow and spleen were harvested and stained for lymphocyte and leukocyte subtypes to assess the broad profile of immune cell uptake. The gating strategy involved first selecting singlet cells (FSC-H vs. FSC-A), followed by selecting viable immune cells (Sytox Blue). -The cells consisted of (CD11b- / CD45+). Lymphocytes (CD11b-) and leukocytes (CD11b+) were then separated based on CD11b expression. Leukocytes were further analyzed into subpopulations of neutrophils (CD11c-, GR-1+), eosinophils (CD11c-, GR-1-), and CD11c+ monocytes. Dendritic cells (F40 / 80-) and macrophages (F40 / 80+) were gated from CD11c+ cells only in the spleen (lower count in bone marrow). Lymphocytes were separated into T- cells (CD19-, CD3+) and B- cells (CD19+, CD3-). The fractions and intensities of Cy3-labeled siRNA in each cell population were then measured using FlowJo software (Figures 8A and 8B).

[0218] D-siRNA exhibits a clear and significant reduction in engagement with all types of gated immune cells in both the spleen and bone marrow compared to cholesterol-siRNA. Specifically, D-siRNA has significantly reduced uptake in leukocytes throughout the spleen and bone marrow, as well as in subpopulations of macrophages in the spleen (these are involved in cytokine responses in the innate immune system). This supports the idea that amphipathic dendritic conjugates are an effective and safer alternative to other more hydrophobic conjugates (e.g., DCA and cholesterol).

[0219] Example 14. Activity comparison between 5'-conjugate D-siRNA and 3'-conjugate D-siRNA The effect of dendritic conjugates on the targeting of D-siRNA activity was evaluated (Figure 9). 3'-conjugated D-siRNAs were synthesized using reverse amidites, and the sense strand was constructed. Htt-targeted D-siRNAs, along with DCA-siRNAs and unconjugated siRNAs, were transfected into HeLa cells for 3 days in a dose-dependent manner (n=3). mRNA silencing efficacy was then quantified using QuantiGene® (Affymetrix), normalized to the housekeeping gene, Hprt (hypoxanthine-guanine phosphoribosyltransferase), and presented as untreated control percentages. No significant differences were observed, confirming that D-siRNAs functioned equally well when positioned at the 5' or 3' end of the sense strand. ****

[0220] Embedding by reference The content of all cited references (including literature, patents, patent applications, patent publications, and websites) that may be cited throughout this application is expressly incorporated in their entirety by reference for any purpose, as is the case with references cited within them. This disclosure uses immunological, molecular biological, and cell biological techniques that are well known as prior art unless otherwise specified.

[0221] This disclosure also incorporates, by reference, a whole range of techniques well known in the fields of molecular biology and drug delivery. These techniques include, but are not limited to, those described in the following publications:

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[0265] Equal portions This disclosure can be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described above should be considered illustrative in all respects and not limiting to this disclosure. Accordingly, the scope of this disclosure is indicated not by the above specification but by the appended claims, and all modifications that fall within the meaning and equivalents of the claims are intended to be incorporated herein.

Claims

1. A method for delivering an oligonucleotide conjugate to the skin of a target, wherein the method comprises administering the oligonucleotide conjugate to the target, and the oligonucleotide conjugate is i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) A dendron bonded to the oligonucleotide and comprising a terminal group, a phosphate group, and / or a hydrophobic chain, The method wherein the oligonucleotide conjugate is formulated for skin administration.

2. The terminal group may be a hydrophilic group containing hydroxides, amines, phosphates, sulfur, and / or sugars, an amine, an amide, an ether, an ester, an N- or O-containing heterocycle, a thiol, a thioether, and / or saturated or unsaturated C １－２４ The method according to claim 1, wherein the hydrophobic group includes an alkyl chain and / or one or more aromatic rings.

3. The hydrophobic chain is saturated or unsaturated C １－２４ The method according to claim 1 or 2, wherein the alkyl group is used.

4. The method according to any one of claims 1 to 3, wherein the dendron includes two branches.

5. The method according to any one of claims 1 to 3, wherein the dendron includes four branches.

6. The method according to any one of claims 1 to 3, wherein the dendron includes eight branches.

7. The method according to any one of claims 1 to 6, wherein the dendron is bonded to the 5' end and / or the 3' end of the oligonucleotide.

8. The method according to any one of claims 1 to 7, wherein the oligonucleotide corresponds to an antisense oligonucleotide or siRNA.

9. The method according to claim 8, wherein the siRNA includes a sense strand and an antisense strand.

10. The method according to claim 9, wherein the dendron is bonded to the 5' end and / or the 3' end of the sense chain, or to the 5' end and / or the 3' end of the antisense chain.

11. The method according to claim 9, wherein the dendron is bonded to the 3' or 5' end of the sense chain.

12. The method according to any one of claims 9 to 11, wherein the antisense strand comprises a length of about 15 to about 25 nucleotides.

13. The method according to any one of claims 9 to 12, wherein the sense strand comprises a length of about 15 to about 25 nucleotides.

14. The method according to any one of claims 9 to 13, wherein the antisense chain is 20 nucleotides long, 21 nucleotides long, or 22 nucleotides long.

15. The method according to any one of claims 9 to 14, wherein the sense strand is 15 nucleotides long, 16 nucleotides long, 18 nucleotides long, or 20 nucleotides long.

16. The method according to any one of claims 9 to 15, wherein the siRNA includes a double-stranded region of about 15 to about 20 base pairs.

17. The method according to any one of claims 9 to 16, wherein the siRNA includes a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

18. The method according to any one of claims 9 to 17, wherein the siRNA includes at least one blunt end.

19. The method according to any one of claims 9 to 17, wherein the siRNA comprises at least one single-stranded nucleotide overhang.

20. The method according to any one of claims 9 to 19, wherein the siRNA comprises naturally occurring nucleotides.

21. The method according to any one of claims 9 to 20, wherein the siRNA comprises at least one modified nucleotide.

22. The method according to claim 21, wherein the modified nucleotide comprises a 2'-O-methyl modified nucleotide, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, a debasalized nucleotide, a 2'-amino modified nucleotide, a 2'-alkyl modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base containing a nucleotide, or a mixture thereof.

23. The method according to any one of claims 8 to 22, wherein the siRNA includes at least one modified nucleotide bond.

24. The method according to claim 23, wherein the modified nucleotide linkage includes a phosphorothioate nucleotide linkage.

25. The method according to any one of claims 8 to 24, wherein the siRNA comprises 4 to 16 phosphorothioate nucleotide interbonds.

26. The method according to any one of claims 8 to 25, wherein the siRNA comprises 8 to 13 phosphorothioate nucleotide interbonds.

27. The method according to any one of claims 8 to 26, wherein the siRNA comprises at least 80% chemically modified nucleotides.

28. The method according to any one of claims 8 to 27, wherein the siRNA is completely chemically modified.

29. The method according to any one of claims 9 to 28, wherein the sense strand includes one or more nucleotide mismatches between the antisense strand and the sense strand.

30. The method according to any one of claims 9 to 29, wherein the antisense chain comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

31. The method according to claim 30, wherein the antisense chain comprises a 5' vinylphosphonate.

32. The method according to any one of claims 9 to 31, wherein the nucleotides located at positions 1 and 2 from the 3' end of the sense strand, and the nucleotides located at positions 1 and 2 from the 5' end of the antisense strand, are linked to adjacent ribonucleotides via phosphorothioate bonds.

33. The method according to any one of claims 1 to 32, wherein the oligonucleotide conjugate is administered sequentially or simultaneously.

34. The oligonucleotide conjugate has the structure of formula I, 【Chemistry 1】 During the ceremony, A is an oligonucleotide, B independently comprises, for each occurrence, one or more hydrophobic chains, amines, amides, esters, N- or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, wherein the hydrophobic chains are saturated or unsaturated C １－２４ Containing alkyl chains, For each appearance, C independently contains hydroxides, amines, phosphates, sulfur, and / or sugars, hydrophilic groups, amines, amides, ethers, esters, N- or O-containing heterocycles, thiols, thioethers, and / or saturated or unsaturated C. １－２４ A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings, D is a branched unit comprising, independently for each occurrence, one or more alkyl chains, amides, ethers, esters, and amines, wherein the branched unit comprises 2 to 4 branches. The method according to any one of claims 1 to 33, wherein m is independently 0 or 1 for each occurrence.

35. The method according to claim 34, wherein the oligonucleotide conjugate has the structure of formula II. 【Chemistry 2】

36. The oligonucleotide conjugate has the structure of formula III, 【Transformation 3】 The method according to claim 35, wherein n is an integer from 1 to 24, independently for each occurrence.

37. The method according to claim 36, wherein C is OH and n is independently 1, 6, or 12 for each occurrence.

38. The method according to any one of claims 34 to 37, wherein the oligonucleotide conjugate has the structure of formula IV. 【Chemistry 4】

39. The method according to claim 34, wherein the oligonucleotide conjugate has the structure of formula V. 【Transformation 5】

40. The oligonucleotide conjugate has the structure of formula VI, 【Transformation 6】 The method according to claim 39, wherein n is an integer from 1 to 24, independently for each occurrence.

41. The method according to claim 40, wherein C is OH and n is independently 1, 6, or 12 for each occurrence.

42. The method according to claim 41, wherein the oligonucleotide conjugate has the structure of formula VII. 【Transformation 7】

43. The method according to any one of the prior claims, wherein the oligonucleotide conjugate formulation comprises an oligonucleotide conjugate in an amount of about 0.1 mg / kg body weight to about 100 mg / kg body weight.

44. The method according to any one of the prior claims, wherein the oligonucleotide conjugate is administered intradermally.

45. The method according to any one of the prior claims, wherein the oligonucleotide conjugate has low toxicity.

46. The method according to any one of the prior claims, wherein the oligonucleotide has one of the sequences of sequence numbers 25 and 28.

47. A method for providing treatment to a patient who requires treatment for a skin disease, disorder, or injury, the patient i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) Administering an oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and having a hydrophilic terminal group, a phosphate group, and / or a hydrophobic chain, The method wherein the oligonucleotide conjugate is formulated for skin administration.

48. The method according to claim 47, wherein the disease, disorder, or injury of the skin is selected from the group consisting of acne, pemphigus, alopecia areata, psoriasis, atopic dermatitis, Raynaud's phenomenon, epidermolysis bullosa, rosacea, hidradenitis suppurativa, scleroderma, ichthyosis, vitiligo, congenital onychoplakia, and combinations thereof.

49. The method according to claim 47, wherein the skin disease, disorder, or injury is related to the JAK1 gene.

50. The method according to claim 49, wherein the oligonucleotide conjugate inhibits the expression of the JAK1 gene.

51. The method according to claim 49 or 50, wherein the oligonucleotide has one of the sequences of sequence numbers 25 and 28.

52. A method for administering a therapeutically effective amount of oligonucleotide conjugate to the skin of a target, wherein the method comprises administering the oligonucleotide conjugate to the target, and the oligonucleotide conjugate is i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) A dendron bonded to the oligonucleotide and comprising a hydrophilic terminal group, a phosphate group, and / or a hydrophobic chain, The method wherein the oligonucleotide conjugate is formulated for skin administration.

53. A method for inhibiting the expression of the JAK1 gene in cells, wherein the method is (a) Inside the cell, i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) Introducing an oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and containing a hydrophilic terminal group, a phosphate group, and / or a hydrophobic chain, (b) The method comprising maintaining the cells produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the JAK1 gene, thereby inhibiting the expression of the JAK1 gene in the cells.

54. Oligonucleotide conjugates for use in the treatment of a patient requiring treatment of a skin disease, disorder, or injury, wherein the oligonucleotide conjugate is administered to the patient and formulated for skin administration, and the oligonucleotide conjugate is i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) An oligonucleotide conjugate for use comprising a dendron bonded to the oligonucleotide and having a hydrophilic terminal group, a phosphate group, and / or a hydrophobic chain.

55. A pharmaceutical composition for providing treatment to a patient who requires treatment for a skin disease, disorder, or injury, i) Oligonucleotides including the 5' and 3' ends, and complementarity to the target nucleic acid, ii) An oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and having a hydrophilic end group, a phosphate group, and / or a hydrophobic chain, A pharmaceutically acceptable carrier, The pharmaceutical composition is formulated for skin administration.

56. It is a topical preparation, (a) Oligonucleotides including 5' and 3' ends, as well as complementarity to the target nucleic acid, ii) An oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and having a hydrophilic end group, a phosphate group, and / or a hydrophobic chain, (b) Optionally, a pharmaceutically acceptable carrier, (c) The topical formulation comprising, optionally, a pharmaceutically acceptable salt.

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