Dendritic conjugates for brain delivery of therapeutic oligonucleotides

Abstract

This disclosure provides compositions, systems, and methods for delivering therapeutic oligonucleotides to the brain. The oligonucleotides are conjugated to a dendron comprising a hydrophilic end group, a phosphate group, and / or a hydrophobic chain.

Description

[Technical Field]

[0001] This application claims the benefits of U.S. Provisional Patent Application No. 63 / 453,324, filed on March 20, 2023. The entire contents of the aforementioned patent application are incorporated herein by reference.

[0002] Description of federally funded research and development. This invention was made with government support under grant number CA261151, awarded by the National Institutes of Health. The U.S. Government reserves certain rights in this invention.

[0003] This disclosure also relates to dendritic conjugates for the delivery of therapeutic oligonucleotides to the brain. Specifically, compositions, systems, and methods for delivering oligonucleotide conjugates, comprising oligonucleotides and dendrons, to the brain are provided herein. [Background technology]

[0004] 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).

[0005] The adhesion of biomolecules and proteins to the surface of therapeutic agents can have several advantages (see Francia, V., Schiffelers, R.M., Cullis, P.R. & Witzigmann, D. “The Biomolecular Corona of Lipid Nanoparticles for Gene Therapy,” Bioconjugate Chemistry 31, 2046-2059 (2020), which is incorporated herein by reference in its entirety).

[0006] Strategic design of conjugates of oligonucleotides is necessary because the structure and binding affinity play central roles in cellular uptake and therapeutic efficacy.

[0007] Therefore, there is still a need for novel compositions, systems, and methods for effectively delivering therapeutic oligonucleotides. SUMMARY OF THE INVENTION

[0008] Compositions and methods for delivering oligonucleotide conjugates comprising an oligonucleotide and a dendron are provided herein. The oligonucleotide conjugates can efficiently knockdown genes in the brain. Several different oligonucleotide conjugates comprising different oligonucleotides and dendrons demonstrated brain delivery upon administration.

[0009] s In one aspect, the present disclosure provides a method for delivering an oligonucleotide conjugate to the brain of a subject, the method comprising administering the oligonucleotide conjugate to the subject, the oligonucleotide conjugate comprising i) an oligonucleotide comprising a 5’ end and a 3’ end and complementarity to a target nucleic acid, and ii) a dendron attached to the oligonucleotide and comprising a terminal group, a phosphate group, and / or a hydrophobic chain, the oligonucleotide conjugate being formulated for brain administration.

[0010] In certain embodiments, the terminal group may be a hydrophilic group containing hydroxides, amines, phosphate esters, sulfur, and / or sugars, an amine, an amide, an ether, an ester, a N or O-containing heterocycle, a thiol, a thioether, and / or saturated or unsaturated C 1-24 A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings.

[0011] In certain embodiments, the hydrophobic chain is saturated or unsaturated C 1-24 It is an alkyl group.

[0012] In certain embodiments, the dendron includes two branched chains.

[0013] In certain embodiments, the dendron includes four branched chains.

[0014] In certain embodiments, the dendron includes eight branched chains.

[0015] In certain embodiments, the dendron is attached to the 5' and / or 3' ends of the oligonucleotide.

[0016] In certain embodiments, the oligonucleotide includes an antisense oligonucleotide or siRNA.

[0017] In certain embodiments, the siRNA comprises a sense strand and an antisense strand.

[0018] In certain embodiments, the dendron is bonded to the 5' and / or 3' ends of the sense chain, or to the 5' and / or 3' ends of the antisense chain.

[0019] In certain embodiments, the dendron is attached to the 3' or 5' end of the sense chain.

[0020] In certain embodiments, the antisense strand comprises a length of approximately 15 to 25 nucleotides.

[0021] In certain embodiments, the sense strand comprises a length of approximately 15 to 25 nucleotides.

[0022] In certain embodiments, the antisense strand is 20 nucleotides long, 21 nucleotides long, or 22 nucleotides long.

[0023] In certain embodiments, the sense strand is 15 nucleotides long, 16 nucleotides long, 18 nucleotides long, or 20 nucleotides long.

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

[0025] In certain embodiments, the siRNA includes a double-stranded region of 15, 16, 18, or 20 base pairs.

[0026] In certain embodiments, the siRNA includes at least one blunt end.

[0027] In certain embodiments, the siRNA includes at least one single-stranded nucleotide overhang.

[0028] In certain embodiments, the siRNA contains naturally occurring nucleotides.

[0029] In certain embodiments, the siRNA comprises at least one modified nucleotide.

[0030] 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, phosphoramic acids, nucleotides containing unnatural bases, or mixtures thereof.

[0031] In certain embodiments, the siRNA includes at least one modified nucleotide bond.

[0032] In certain embodiments, the modified nucleotide bond includes a thiophosphate nucleotide bond.

[0033] In certain embodiments, the siRNA contains 4 to 16 thiophosphate nucleotide links.

[0034] In certain embodiments, the siRNA contains 8 to 13 thiophosphate nucleotide links.

[0035] In certain embodiments, the siRNA comprises nucleotides that are at least 80% chemically modified.

[0036] In certain embodiments, the siRNA is fully chemically modified.

[0037] In certain embodiments, the sense strand includes one or more nucleotide mismatches between the antisense strand and the sense strand.

[0038] In certain embodiments, the antisense chain comprises a 5'-phosphate, a 5'-alkylphosphonate, a 5'-alkylenephosphonate, or a 5'-alkenylphosphonate.

[0039] In certain embodiments, the antisense chain comprises a 5'-vinylphosphonate.

[0040] 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.

[0041] In certain embodiments, oligonucleotide conjugates are administered sequentially or simultaneously.

[0042] In a particular embodiment, the oligonucleotide conjugate has the structure of formula I, [ka] (In the formula,

[0043] A is a oligonucleotide,

[0044] Each instance of B independently comprises one or more hydrophobic chains, amines, amides, esters, N or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, where the hydrophobic chain is saturated or unsaturated C 1-24 Containing alkyl chains,

[0045] Each instance of C is independently a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an amine, amide, ether, ester, N or O-containing heterocycle, a thiol, thioether, and / or saturated or unsaturated C. 1-24 A hydrophobic group containing an alkyl chain, and / or one or more aromatic rings,

[0046] D is a branched unit that, each time it appears, independently contains one or more alkyl chains, amides, ethers, esters, and amines, where the branched unit contains 2 to 4 branches.

[0047] m is either 0 or 1 independently each time it appears.

[0048] In certain embodiments, the oligonucleotide conjugate has the structure of formula II. [ka] In certain embodiments, the oligonucleotide conjugate has the structure of formula III, [ka] (In the formula, n is an independent integer between 1 and 24 each time it appears.)

[0049] In certain embodiments, C is OH, and n is independently 1, 6, or 12 each time it appears.

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

[0051] In certain embodiments, the oligonucleotide conjugate has the structure of formula V. [ka] (In the formula, n is an independent integer between 1 and 24 each time it appears.)

[0052] In certain embodiments, C is OH, and n is independently 1, 6, or 12 each time it appears.

[0053] In certain embodiments, the oligonucleotide conjugate has the structure of formula VII. [ka]

[0054] In certain embodiments, the oligonucleotide conjugate formulation contains approximately 0.1 to 20 mg of oligonucleotide conjugate per kg of body weight.

[0055] In certain embodiments, oligonucleotide conjugates are administered to subjects by intrastriatal (IS) injection, intraventricular (ICV) injection, intratumoral (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, subarachnoid injection, or a combination thereof.

[0056] In certain embodiments, the oligonucleotide conjugate has low toxicity.

[0057] In certain embodiments, the oligonucleotide has one of the sequences of sequence numbers 8 and 10.

[0058] In another aspect, the present disclosure relates to a method for providing treatment to a patient who requires treatment for a brain disorder, disorder, or injury, and to the patient,

[0059] i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and

[0060] ii) Administering an oligonucleotide conjugate comprising a dendron, which is bound to an oligonucleotide and includes a terminal group, a phosphate group, and / or a hydrophobic chain,

[0061] This invention provides a method for formulating oligonucleotide conjugates for cerebral administration.

[0062] In certain embodiments, the brain disease, disorder, or injury is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, autism, concussion, dystonia, dementia, epilepsy, Huntington's disease, muscular dystrophy, neurological disorders, Parkinson's disease, sleep disorders, Tourette syndrome, or a combination thereof.

[0063] In certain embodiments, brain diseases, disorders, or injuries are associated with the HTT gene and / or the APP gene.

[0064] In certain embodiments, oligonucleotide conjugates inhibit the expression of the HTT gene and / or the APP gene.

[0065] In certain embodiments, the oligonucleotide has one of the sequences of sequence numbers 8 and 10.

[0066] In another aspect, the Disclosure relates to a method for administering a therapeutically effective dose of an oligonucleotide conjugate to the brain of a subject, the method comprising administering the oligonucleotide conjugate to the subject, the oligonucleotide conjugate is

[0067] i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and

[0068] ii) A dendron bonded to the oligonucleotide and comprising a terminal group, a phosphate group, and / or a hydrophobic chain,

[0069] This invention provides a method for formulating oligonucleotide conjugates for cerebral administration.

[0070] In another aspect, the disclosure provides an oligonucleotide conjugate for use in the treatment of a brain disease, disorder, or injury in a patient requiring treatment, wherein the oligonucleotide conjugate is administered to the patient and formulated for brain administration, and furthermore, the oligonucleotide conjugate is

[0071] i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and

[0072] ii) A dendron bonded to an oligonucleotide, comprising a terminal group, a phosphate group, and / or a hydrophobic chain.

[0073] In another aspect, the present disclosure relates to a pharmaceutical composition for providing treatment to a patient who requires treatment for a brain disease, disorder, or injury,

[0074] i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and

[0075] ii) Oligonucleotide conjugates comprising a dendron bonded to an oligonucleotide and containing a terminal group, a phosphate group, and / or a hydrophobic chain,

[0076] A pharmaceutically acceptable carrier, and,

[0077] The pharmaceutical composition is formulated for administration to the brain.

[0078] A method for inhibiting genes in cells, the method is

[0079] i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and

[0080] ii) Introducing an oligonucleotide conjugate into cells, which includes a dendron bound to an oligonucleotide and containing a terminal group, a phosphate group, and / or a hydrophobic chain.

[0081] (b) The cells produced in step (a) are maintained for a sufficient time to obtain degradation of the gene mRNA transcripts, thereby inhibiting gene expression in the cells.

[0082] In certain embodiments, the gene is the HTT gene, and / or the target is the HTT mRNA.

[0083] In certain embodiments, the gene is the APP gene, and / or the target is the APP mRNA.

[0084] These and other aspects of the applicant's teachings are described herein.

[0085] This patent or application document includes at least one drawing drawn in color. Copies of this patent or patent application publication, including the color drawing(s), will be provided by the Patent Office upon request and payment of the necessary fees.

[0086] The aspects, features, benefits, and advantages of the embodiments described herein will become apparent with reference to the following description, examples, claims, and accompanying drawings. [Brief explanation of the drawing]

[0087] [Figure 1A-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 1A-2] Same as above. [Figure 1B-1] Same as above. [Figure 1B-2] Same as above. [Figure 1B-3] Same as above. [Figure 1B-4] Same as above. [Figure 2A-1]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 2A-2] Same as above. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 3] Fluorescence images of brain sections after D-siRNA injection are shown. D-siRNA was injected into one hemisphere, and a vehicle (control) was injected into the other hemisphere. After intrastriatal injection (2 nmol), D-siRNA was observed to distribute beyond the injection site. [Figure 4A] This study shows HTT mRNA expression in the frontal lobe, striatum, thalamus, hippocampus, medial cortex, posterior cortex, cerebellum, and brainstem regions of the brain after injection of NTC-D-siRNA, HTT-D-siRNA, and Di-HTT-siRNA. Injections were administered locally (intraventricular injection, ICV) at a dose of 5 nmol (2.5 mg / kg), and tissue analysis was performed at 1 month (N=4, dendrimers included injection errors) (Figure 4A) and 3 months (N=5) (Figure 4B). In addition, injections were administered locally (intraventricular injection, ICV) at a dose of 10 nmol (10 mg / kg), and tissue analysis was performed at 2 months (N=5) (Figure 4C). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A-1]Figure 5A shows the relative levels of HTT and APP RNA expression after injection of ASO and siRNA. Figure 5A shows the structures of unconjugated and conjugated siRNA and their relative hydrophobicity. Figure 5B shows HTT RNA expression after injection of ASO and siRNA. Figure 5C shows that lipid-conjugated siRNA exhibits consistent moderate silencing of two mRNA targets in a GBM xenograft model. In particular, the schematic diagram on the left shows bilateral intratumoral and intrastriatal injection of lipid-conjugated siRNA. The top graph shows silencing of human HTT and mouse Htt mRNA with Chol-siRNA or DCA-siRNA, and the middle graph shows silencing of human HTT and mouse Htt mRNA with EPA-siRNA or D-siRNA. The bottom graph shows the results of silencing of human APP and mouse App mRNA with EPA-siRNA and D-siRNA, measured using qPCR from xenografted and ungrafted striatum. [Figure 5A-2] Same as above. [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 6] This shows the relative levels of HTT and APP RNA expression in normal brain cells and GBM cells after siRNA injection. Lipid-conjugated siRNA is shown to be functionally delivered to GBM8 xenografts via ICV injection. The top schematic shows bilateral ICV injection of a total of 30 nmol of siRNA. The top graph shows silencing of human HTT and mouse Htt mRNA measured using qPCR from xenografted and non-grafted striatum. The middle schematic shows bilateral ICV injection of a total of 10 nmol of siRNA, and shows silencing of human APP and mouse App mRNA measured using qPCR from xenografted and non-grafted striatum (middle graph) and from the olfactory bulb and cerebellum (bottom graph). [Figure 7-1]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 7-2] Same as above. [Figure 8] 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 Quanigene bDNA assay. Data were normalized to housekeeping genes (Hprt) and expressed as a percentage of untreated control cells. n=3. Mean ± standard deviation (SD). [Modes for carrying out the invention]

[0088] For clarity, it will be understood that the following discussion will describe various aspects of the embodiments of the applicant's teachings. It should be noted that no particular embodiment is intended as an exhaustive description or as a limitation to the broader embodiments discussed herein. Any embodiment described in conjunction with a particular embodiment is not necessarily limited to that embodiment and may be implemented in conjunction with any other embodiment(s).

[0089] This disclosure relates to dendritic conjugates for delivering therapeutic oligonucleotides to the brain. Specifically, this disclosure provides compositions, systems, and methods for delivering dendron-bound therapeutic oligonucleotides. Oligonucleotide conjugates disclosed herein can be delivered to the brain upon administration.

[0090] The oligonucleotide conjugates described herein facilitate the convenient, efficient, and non-toxic delivery of oligonucleotides (e.g., siRNA, antisense oligonucleotides (ASOs), macroRNAs) and can promote potent silencing of therapeutic targets in brain cells in vivo.

[0091] Unless otherwise specified, the nomenclature used herein in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein, as well as nucleic acid chemistry and hybridization, is 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 specified, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as described herein, in accordance with the manufacturer's specifications or as commonly achieved in the art. The terminology used herein in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical / medical chemistry, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for the chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, delivery, and treatment of patients.

[0092] 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 noted. The use of the term "including," and other forms such as "include," "includes," and "included," is not limited to these.

[0093] To make this disclosure easier to understand, we first define certain terms.

[0094] definition In this specification, the use of the singular form includes the plural form unless otherwise specified. Where used herein, the singular forms "a," "an," and "the" include plural references unless the context explicitly indicates otherwise. Furthermore, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limited to this.

[0095] Whenever an aspect is described herein with the phrase "including," it is understood that other similar aspects are also provided, described in terms of "consisting of" and / or "essentially consisting of."

[0096] Where used herein, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the range, and, where appropriate, fractions thereof (e.g., one-tenth and one-hundredth of an integer), unless otherwise specified.

[0097] The terms “approximately” or “essentially include,” as determined by those skilled in the art, indicate a value or composition that is within an acceptable margin of error for that particular value or composition and is partially dependent on the method of measurement or determination of that value or composition, i.e., the limits of the measuring system. When a particular value or composition is provided in this application and claims, unless otherwise specified, the meaning of “approximately” or “essentially include” should be understood as being within an acceptable margin of error for that particular value or composition.

[0098] As used herein, the term “and / or” should be interpreted as meaning that each of the two specified features or components is specifically disclosed, whether or not the other is present. Accordingly, as used herein in phrases such as “A and / or B,” the term “and / or” is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Similarly, as used in phrases such as “A, B, and / or C,” the term “and / or” is intended to include each of the following embodiments, namely 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).

[0099] 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 cytosine (e.g., cytidine or a chemically modified derivative thereof).

[0100] The term "nucleoside" refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine, and thymidine. Further exemplary nucleosides include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and N2,N2-dimethylguanosine (also known as a “rare” nucleoside). The term "nucleotide" refers to a nucleoside having one or more phosphate groups attached to the sugar moiety within an ester linkage. Exemplary nucleotides include nucleoside monophosphates, diphosphates, and triphosphates. The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably herein and refer to polymers of nucleotides linked together by phosphate diester or thiophosphate ester links between the 5' and 3' carbon atoms.

[0101] 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 converted during protein synthesis when ribosomes bind to mRNA.

[0102] As used herein, the term “small interfering RNA” (“siRNA”) (also known in the art as “short interfering RNA”) refers to RNA (or RNA analogues) containing about 10 to 50 nucleotides (or nucleotide analogues) that can direct or mediate RNA interference. siRNA is a double helix formed by a sense strand and an antisense strand that are sufficiently complementary to each other to form the double helix described above. 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 refers to 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 longer siRNA retains its ability to mediate RNAi without further processing of the short siRNA, e.g., enzymatic processing.

[0103] The terms “nucleotide analog,” “modified nucleotide,” “modified nucleotide,” or “chemically modified nucleotide” refer to non-standard nucleotides, such as ribonucleotides or deoxyribonucleotides, which do not exist in nature. Exemplary nucleotide analogs are modified at any position to alter the specific chemical properties of a nucleotide, while retaining 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-propyneuridine, 5-propenyluridine; the 6-position, e.g., 6-(2-amino)propyluridine; and the 8-position of adenosine and / or guanosine, e.g., 8-bromoguanosine, 8-chloroguanosine, 8-fluoroguanosine. Furthermore, examples of nucleotide analogs include deazanucleotides, such as 7-deaza-adenosine, O and N modified (e.g., alkylated, e.g., N6-methyladenosine, or those 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.

[0104] Nucleotide analogs may also include modifications to the sugar moiety of the nucleotide. For example, the 2'OH group may be substituted with 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 analog includes a 2'-O-methyl modification. In certain embodiments, the nucleotide analog includes a 2'-fluoro modification.

[0105] The phosphate group of a nucleotide can also be modified, for example, by substituting one or more oxygen atoms of the phosphate group with sulfur (e.g., thiophosphate esters), or by making other substitutions so that the nucleotide can perform its intended function. For example, see 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, including their analogs, for example, in vivo or in vitro.

[0106] The term "RNA analog" refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) that has at least one modified nucleotide compared to the corresponding unmodified or unmodified RNA, but retains the same or similar properties or functions as the corresponding unmodified or unmodified RNA. As described above, oligonucleotides may be linked by a linkage that results in a reduced hydrolysis rate of the RNA analog compared to RNA molecules with phosphate diester linkages. For example, the nucleotides of the analog may include methylenediol, ethylenediol, oxymethylthio, oxyethylthio, oxycarbonyloxy, amide phosphate, amide phosphate, and / or thiophosphate linkages. Some RNA analogs include sugars and / or skeletal-modified ribonucleotides and / or deoxyribonucleotides. Such modifications 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 native RNA that has the ability to mediate RNA interference.

[0107] As used herein, the term “RNA interference” (“RNAi”) refers to the selective intracellular degradation of RNA. RNAi occurs naturally within cells and removes foreign RNA (e.g., viral RNA). Natural RNAi proceeds via fragments cleaved from free dsRNA, directing the degradation mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by humans, for example, to silencing the expression of a target gene.

[0108] RNAi agents, such as RNA silencing agents, have a chain that is "sufficiently complementary to the target mRNA sequence in order to direct target-specific RNA interference (RNAi)." This means that the chain has a sequence sufficient to cause the destruction of the target mRNA by the RNAi mechanism or process.

[0109] As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) means an RNA molecule that, if produced by recombinant technology, is substantially free of other cellular material or culture medium, and an RNA molecule that, if chemically synthesized, is substantially free of chemical precursors or other chemicals.

[0110] 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), querring, 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.

[0111] The term "in vitro" has a recognized meaning in the art, including, for example, purified reagents or extracts, such as cell extracts. The term "in vivo" also has a recognized meaning in the art, including, for example, living cells, such as immortalized cells, primary cells, cell lines, and / or cells within an organism.

[0112] As used herein, “target” means 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 the expression of. 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 a subject.

[0113] As used herein, the term “target gene” refers to a gene whose expression is substantially inhibited or “silenced.” This silencing can be achieved, for example, by cleaving the mRNA of the target gene or by silencing the RNA by translational repression of the target gene. The term “non-target gene” refers to 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 and non-target genes may differ by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In another embodiment, the target and non-target genes may share less than 100% sequence identity. In another embodiment, the non-target gene may be a homolog (e.g., an ortholog or paralog) of the target gene.

[0114] 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 a post-transcriptional silencing mechanism. Examples of RNA silencing agents include small (<50 b.p.) non-coding RNA molecules, such as RNA double-stranded molecules 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 molecules, 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.

[0115] As used herein, the term “rare nucleotide” refers to naturally occurring nucleotides that occur infrequently, such as naturally occurring deoxyribonucleotides or ribonucleotides that do not occur infrequently, such as guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides, but not limited to, include inosine, 1-methylinosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine, and 2,2N,N-dimethylguanosine.

[0116] The term "engineered," as in engineered RNA precursors or engineered nucleic acid molecules, indicates that the 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. Once created or selected, the sequence is 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.

[0117] 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 the genome of a virus, mammal, or plant) and can direct or mediate RNA silencing. “miRNA disorder” refers to a disease or disorder characterized by abnormal expression or activity of miRNA.

[0118] As used herein, the term “dual-functional oligonucleotide” refers to an RNA silencing agent having the formula TL-μ, where T is the mRNA targeting portion, L is the ligation portion, and μ is the miRNA recruitment portion. As used herein, the terms “mRNA targeting portion,” “targeting portion,” “mRNA targeting portion,” or “targeting portion” refer to a domain, portion, or region of a dual-functional oligonucleotide that is sufficiently large and sufficiently complementary to a portion or region of mRNA selected or targeted for silencing (i.e., the portion has a sequence sufficient to capture the target mRNA).

[0119] As used herein, the terms “linking moiety” or “linking portion” refer to a domain, part, or region of an RNA silencing agent that covalently joins or links mRNA.

[0120] As used herein, the term “antisense strand” of an RNA silencing agent, such as siRNA, refers to a strand substantially complementary to approximately 10–50 nucleotides, e.g., approximately 15–30, 16–25, 18–23, or 19–22 nucleotides, of the mRNA of the targeted gene for silencing. The antisense strand, or first strand, has a sequence that is sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., sufficient complementarity to cause the destruction of the desired target mRNA by an RNAi mechanism or process (RNAi interference), or sufficient complementarity to induce translational repression of the desired target mRNA.

[0121] The terms “sense strand” or “second strand” of an RNA silencing agent, such as siRNA, refer to a strand complementary to the antisense strand or first strand. The antisense strand and sense strand can 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 intermediate contains a miRNA strand that is sufficiently complementary to approximately 10–50 nucleotides of the mRNA of the gene targeted for silencing, and a miRNA* strand that is sufficiently complementary to form a double-stranded structure with the miRNA strand.

[0122] As used herein, the term “guide strand” refers to a strand of RNA silencing agent, such as a double-stranded siRNA or antisense strand of an siRNA sequence, that enters the RISC complex and directs the cleavage of the target mRNA.

[0123] As used herein, the term “asymmetry,” as in the asymmetry of the double-stranded region of an RNA silencing agent (e.g., the stem of shRNA), refers to an unevenness in binding strength or base pair strength between the ends of the RNA silencing agent (e.g., between a terminal nucleotide on the first strand or stem portion and a terminal nucleotide on the opposing second strand or stem portion). This results in the 5' end of one strand of the double helix being more frequently in a transient unpaired state, e.g., a single-stranded state, than the 5' end of the complementary strand. This structural difference determines that one strand of the double helix is ​​preferentially incorporated into the RISC complex. The strand whose 5' end is less firmly paired with the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

[0124] As used herein, the terms “bond strength” or “base pair strength” primarily refer to the strength of interactions between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double-strand (e.g., an siRNA double-strand), and between these nucleotides (or nucleotide analogs), such as through H bonds and van der Waals interactions.

[0125] As used herein, “5' end,” such as at the 5' end of an antisense strand, refers to, for example, the 5' terminal nucleotides between 1 and approximately 5 nucleotides at the 5' end of the antisense strand. As used herein, “3' end,” such as at the 3' end of a sense strand, refers to a region complementary to the 5' terminal nucleotides of the complementary antisense strand, for example, a region of 1 to approximately 5 nucleotides.

[0126] 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 such that the base pair has a lower binding strength than a conventional base pair (i.e., a Watson-Crick base pair). In certain embodiments, the destabilized nucleotide can form an inconsistent 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.

[0127] As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide double helix (e.g., the strands of an RNA silencing agent and the double helix formed by a target mRNA sequence), primarily through H bonds between said nucleotides (or nucleotide analogs), van der Waals interactions, and the like. As used herein, the terms “bond strength” or “base pair strength” refer to the strength of a base pair.

[0128] As used herein, the term “incompatible base pair” refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs that are not, for example, normal complementary G:C, A:T, or A:U base pairs. As used herein, the term “ambiguous base pair” (also known as non-distinguishable base pair) refers to a base pair formed by universal nucleotides.

[0129] 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 between bases on a complementary polynucleotide when forming base pairs. Universal nucleotides are primarily hydrophobic molecules and can efficiently pack into antiparallel double-stranded nucleic acids (e.g., double-stranded DNA or RNA) through stacking interactions. The base portion of a universal nucleotide typically contains a nitrogen-containing aromatic heterocyclic moiety.

[0130] As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has sequences (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, respectively.

[0131] 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.

[0132] 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.

[0133] Various methodologies of this disclosure include a step of comparing values, levels, features, properties, etc., with a “preferred control,” which is interchangeably referred to herein as “appropriate control.” A “preferred control” or “appropriate control” is any control or standard well known to those skilled in the art and useful for comparison purposes. 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 characteristics or properties, genotype, phenotype, etc., can 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 cells or organisms, e.g., a control or, e.g., normal cells or organisms exhibiting normal traits. In yet another embodiment, a “preferred control” or “appropriate control” is a predefined value, level, feature, property, etc.

[0134] Design of Ava molecules The oligonucleotide conjugates described herein include oligonucleotides. Non-limiting examples of oligonucleotides include siRNA, antisense oligonucleotides (ASOs), and macroRNAs.

[0135] In some embodiments, the siRNA molecule of the present invention is a double helix comprising a sense strand and a complementary antisense strand, wherein the antisense strand is sufficiently complementary to a target sequence such as an RNAi-mediated mRNA sequence (e.g., an htt mRNA sequence, a cyclophyllin B mRNA sequence, etc.). Preferably, the siRNA molecule has a length of about 10 to 50 or more nucleotides, i.e., each strand contains 10 to 50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecule has a length of about 16 to 30 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, and one of the strands is sufficiently complementary to the target region. Preferably, the chains are aligned such that when the chains are annealed, there are at least 1, 2, or 3 base residues at the ends of the unaligned chains (i.e., there are no bases complementary to the opposing chains) so that when the chains are annealed, there are 1, 2, or 3 residue protrusions at one or both ends of the double helix. Preferably, the siRNA molecules have a length of about 10 to 50 or more nucleotides, i.e., each chain contains 10 to 50 nucleotides (or nucleotide analogs). More preferably, the siRNA molecules have a length of about 16 to 30 nucleotides, for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each chain, with one chain substantially complementary to the target sequence and the other chain identical or substantially identical to the first chain.

[0136] In general, siRNA can be designed using any method known in the art, for example, the following protocols.

[0137] 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 result in the loss of translation of the corresponding mutant protein. Target sequences from other regions of the htt gene are also suitable for targeting. The sense strand is designed based on the target sequence. Furthermore, siRNAs with lower G / C content (35-55%) may be more active than those with higher G / C content (55%). Therefore, in one embodiment, the present invention includes nucleic acid molecules having a G / C content of 35-55%.

[0138] 2. The sense strand of the siRNA is designed based on the sequence of the selected target site. The RNA silencing agents of the present invention do not induce a PKR response (i.e., are sufficiently short in length). However, longer RNA silencing agents may be useful, for example, in cell types that cannot produce a PKR response, or in situations where the PKR response is downregulated or suppressed by alternative means.

[0139] The siRNA molecule of the present invention has sufficient complementarity with the target sequence so that the siRNA can mediate RNAi. Generally, to effectively cleave the target gene by RISC, siRNA containing a nucleotide sequence sufficiently identical to the target sequence portion of the target gene is preferred. Therefore, in preferred embodiments, the sense strand of the siRNA is designed to have a sequence sufficiently identical to the target region. For example, the sense strand may be 100% identical to the target site. However, 100% identity is not required. The identity between the sense strand and the target RNA sequence is preferably greater than 80%, 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%. The present invention has the advantage of allowing for specific sequence modifications to enhance the efficiency and specificity of RNAi. In one embodiment, the sense strand has 4, 3, 2, 1, or 0 mismatched creotides with a target region, such as a target region, which differs by at least one base pair between the wild-type allele and the mutant allele. For example, the target region contains a gain-of-function mutation, 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 substitutions or insertions of nucleotide analogs may be effective in inhibition.

[0140] 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 arrangement). 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 corresponds to 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 given to the score for the number and / or length of introduced gaps.

[0141] 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, revised 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.

[0142] In another embodiment, the arrangement is optimized by introducing appropriate gaps, and percentage identity is determined over the length of the aligned sequences (i.e., the gapped arrangement). To obtain a gapped arrangement 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 arrangement is optimized by introducing appropriate gaps, and percentage identity is determined over the entire length of the aligned sequences (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.

[0143] 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; that is, the strands are blunt-ended when aligned or annealed. In another embodiment, the siRNA strands may be paired to have 1-4, e.g., 2-nucleotide 3' overhangs. The overhangs may contain (or be composed of) nucleotides corresponding to the target gene sequence (or its complement). Alternatively, the overhangs may contain (or be composed of) deoxyribonucleotides, e.g., dT, or nucleotide analogs, or other suitable non-nucleotide substances. Thus, in another embodiment, the nucleic acid molecule may have a 2-nucleotide 3' overhang 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.

[0144] 4. By using any method known in the art, potential targets can be compared to appropriate genome databases (human, mouse, rat, etc.) to eliminate the need to consider any target sequences that have significant homology with other coding sequences. One such sequence homology search method is known as BLAST and is available on the National Center for Biotechnology Information website.

[0145] 5. Select one or more sequences that meet the evaluation criteria.

[0146] 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.

[0147] Alternatively, siRNA can be functionally defined as a nucleotide sequence (or oligonucleotide sequence) that can hybridize with a target sequence (e.g., hybridize with 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM MEDTA at 50°C or 70°C for 12–16 hours, followed by washing). Additional preferred hybridization conditions include hybridization with 1×SSC at 70°C or 1×SSC at 50°C with 50% formamide, followed by washing with 0.3×SSC at 70°C, or hybridization with 4×SSC at 70°C or 4×SSC at 50°C with 50% formamide, followed by washing with 1×SSC at 67°C. The hybridization temperature for hybrids expected to be less than 50 base pairs in length should be 5–10°C lower than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following formula: For hybrids with a length of less than 18 base pairs, Tm(°C) = 2(A + T base count) + 4(G + C base count). For hybrids of 18–49 base pairs in length, Tm(°C) = 81.5 + 16.6(log10[Na+]) + 0.41(%G+C) - (600 / N) (wherein N is the number of bases in the hybrid and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1xSSC = 0.165M)). Examples of additional stringency conditions for polynucleotide hybridization can be found in Sambrook, J., E.F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F.M. Usubel et al., eds., John Wiley & Sons, Inc., sections These are described in sections 2.10 and 6.3–6.4, which are incorporated herein by reference.

[0148] Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but lack 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 confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

[0149] 6. To verify the effectiveness of siRNA in disrupting mRNA (e.g., wild-type or mutant huntingtin mRNA), siRNA may be co-administered with target cDNA (e.g., huntingtin cDNA) in a Drosophila-based in vitro mRNA expression system. 32 A 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 have 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 confirm that the negative control lacks homology to any other gene in the appropriate genome. Furthermore, negative control siRNA can be designed by introducing one or more base mismatches into the sequence.

[0150] siRNA can be designed to target any of the target sequences described above. siRNA comprises an antisense strand that is sufficiently complementary to the target sequence to mediate the silencing of the target sequence. In certain embodiments, the RNA silencing agent is siRNA.

[0151] The siRNA-mRNA complementation site is selected to provide optimal mRNA specificity and maximum mRNA cleavage.

[0152] siRNA-like molecules The siRNA-like molecules of the present invention have a sequence that is "sufficiently complementary" to the target sequence of mRNA (e.g., htt mRNA) in order to direct gene silencing by RNAi or translational repression (i.e., a strand having the sequence). The siRNA-like molecules are designed in the same manner as siRNA molecules, 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 another embodiment 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.

[0153] 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, two, three, four, five, or six consecutive or discontinuous non-identical nucleotides are introduced. The non-identical nucleotides may be selected to form fluctuating base pairs (e.g., G:U) or mismatched 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 on the nucleotides at positions 12 and 13 from the 5' end of the miRNA molecule.

[0154] Modified RNA silencing agent In certain embodiments of the present invention, the RNA silencing agent (or any part thereof) of this application 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 by 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.

[0155] 1) Modifications to improve target identification In certain embodiments, the RNA silencing agents of the present invention may be substituted with destabilized nucleotides to improve single-nucleotide target recognition (see U.S. Patent Application No. 11 / 698,689 and U.S. Provisional 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 any apparent effect on the specificity of the RNA silencing agent to target mRNA (e.g., gain-of-function mutant mRNA).

[0156] In a preferred embodiment, 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 impact on the stability of the RNA double helix, or the double helix formed by the guide strand of the RNA silencing agent and the target mRNA. 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 a particularly preferred embodiment, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

[0157] 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 from the specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position 3 nucleotides away 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 a 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.

[0158] 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. This allows the antisense strand to preferentially induce cleavage or translational repression of the target mRNA, thereby increasing or improving the efficiency of target cleavage and silencing. Preferably, the asymmetry of the RNA silencing agent is enhanced 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.

[0159] 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 mismatched base pair between the 5' end of the first or antisense strand and the 3' end of the sense strand portion. Preferably, the mismatched 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 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 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 such that there is at least one base pair containing a rare nucleotide, for example, inosine(I). Preferably, the base pairs are 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 so that at least one base pair containing a modified nucleotide is present. In a particular 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.

[0160] 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 may be selected to consist of a purine nucleotide, such as adenosine or guanosine nucleotide. Alternatively, substitution of pyrimidine nucleotides with modification analogs, such as substitution of uridine with 2'-deoxythymidine, is acceptable and does not affect the efficiency of RNA interference.

[0161] In a preferred embodiment, the present invention features an RNA silencing agent comprising a first strand and a second strand, wherein the second strand and / or the first strand are modified by substitution of internal nucleotides with modified nucleotides to enhance 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 are modified by substitution of at least one internal nucleotide. In another embodiment, the sense strand and / or antisense strand are modified by 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%, 95%, or more internal nucleotides. In yet another embodiment, the sense strand and / or antisense strand are modified by substitutions of all internal nucleotides.

[0162] In a preferred embodiment of the present invention, the RNA silencing agent may include 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. Specifically, the terminals may be stabilized by incorporating the modified nucleotide analog.

[0163] Exemplary nucleotide analogs include sugar- and / or skeletal-modified ribonucleotides (i.e., modifications to the phosphate-sugar backbone). For example, phosphate diester links in native RNA can be modified to include at least one nitrogen or sulfur heteroatom. In exemplary skeletal-modified ribonucleotides, phosphate ester groups bound to adjacent ribonucleotides are replaced by modifying groups, such as thiophosphate ester groups. 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 group, and halo is F, Cl, Br, or I.

[0164] 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'-OMe nucleotides can 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 a particularly preferred embodiment, the 2' portion is a methyl group, such that the linking portion is a 2'-O-methyl oligonucleotide.

[0165] 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 modifiable 2'-O, 4'-C-ethylene-bridged nucleic acids, such as 2'-deoxy-2"-fluorouridine. Furthermore, the LNA increases the specificity of oligonucleotides by constraining the sugar moiety to the 3'-end conformation, thereby pre-organizing the nucleotides for base pairing and raising the melting temperature of the oligonucleotides by approximately 10°C per nucleotide.

[0166] 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 capable of forming 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).

[0167] Furthermore, ribonucleotides with modified nucleic acid bases, 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 may 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.

[0168] 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, where the two strands are crosslinked. The present invention also comprises RNA silencing agents that are conjugated (e.g., at their 3' end) or unconjugated to another portion (e.g., a non-nucleic acid portion such as a peptide) or an organic compound (e.g., a dye). Such modified siRNA derivatives may improve the uptake of the resulting siRNA derivative into cells or enhance its cell-targeting activity compared to the corresponding siRNA, be useful for tracking the siRNA derivative within cells, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

[0169] Other exemplary modifications include: (a) 2' modifications, e.g., in the sense or antisense chain, but particularly providing a 2'OMe portion on U in the sense chain, or, for example, providing a 2'OMe portion in a 3' protrusion at the 3' end (wherein the 3' end means the 3' atom of the molecule, or the most 3' portion, e.g., the most 3' P or 2' position, as indicated by the context); (b) skeletal modifications, e.g., in the phosphate skeleton, by substitution of O with S, e.g., providing thiophosphate ester modifications to U or A or both in the antisense chain, e.g., by substitution of P with S; (c) substitution of U with a C5 aminolinker; (d) substitution of A with G (the sequence change may, in certain embodiments, be located in the sense chain rather than the antisense chain); and (e) modifications at the 2', 6', 7', or 8' positions. Exemplary embodiments are embodiments in which one or more of these modifications are present on the sense but not on the antisense chain, or in embodiments in which the antisense chain has fewer such modifications. Further exemplary modifications include, for example, the use of methylated P in the 3' protrusion at the 3' end; 2' modifications, such as providing a 2'OMe moiety, and modifications of the skeleton, such as substitution of P with S, such as providing a thiophosphate ester modification; or, for example, the use of methylated P in the 3' protrusion at the 3' end; modifications with a 3' alkyl group; for example, modifications with debasic pyrrolidone in the 3' protrusion at the 3' end; modifications with naproxen, ibuprofen, or other moieties that inhibit degradation at the 3' end.

[0170] 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) or an organic compound (e.g., a dye), etc. The binding is carried out by methods known in the art, e.g., Lambert et al., Drug Deliv. Rev.:47(1),99-112(2001) (described on nucleic acids loaded onto polyalkylcyanoacrylate (PACA) nanoparticles), Fattal et al., J. Control Release 53(1-3):137-43(1998) (described on nucleic acids bound to nanoparticles), Schwab et al., Ann. Oncol.5 Suppl.4:55-8 (1994) (described on nucleic acids linked to inserts, hydrophobic groups, polycations, or PACA nanoparticles), and Godard et al., Eur. J. Biochem.232(2):404-10(1995) (described on nucleic acids linked to nanoparticles).

[0171] 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.

[0172] 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. Tethering factor 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, the ligand may include cleavage groups that contribute to the inhibition of target genes by cleaving the target nucleic acid. 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 cleavage of target RNA at the bulge region, for example. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (Cycram) can be conjugated to a peptide (e.g., via 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-binding 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 lower 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.

[0173] Exemplary ligands are preferably covalently coupled to ligand-conjugate carriers, either directly or indirectly via intervening tethering factors. In exemplary embodiments, the ligand is attached to the carrier via intervening tethering factors. 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.

[0174] 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-helical peptides.

[0175] The ligand can also include a targeting group, such as a cell or tissue targeting agent, for example, a lectin, glycoprotein, lipid, or protein, such as an antibody, that binds to a specific cell type such as the kidney, gland (e.g., thyroid), brain, eye, and / or male testicular cells. The targeting group can also be a 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 peptidomimetic. Other examples of ligands include dyes, intercalating agents (e.g., acridine and substituted acridines), 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, guanidinium aminoglycoside guanidinium aminoglycoside, artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol (and its thio analogs), cholic acid, chenodeoxycholic acid, lithocholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, glycerol (e.g., its esters (e.g., mono, bis, or tris fatty acid esters, e.g., C 10 、C 11 、C 12 、C 13 、C 14 、C 15 、C 16 、C17 , C 18 , C 19 or C 20 fatty acids) and 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 group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, 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., ante Napedia peptides, Tat peptides), alkylating agents, phosphates, amino acids, mercaptos, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyls, substituted alkyls, 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, Eu tetraazama macrocycles) 3+ Examples include complexes, dinitrophenyl, HRP, or AP.

[0176] Ligands can be proteins such as glycoproteins, peptides such as molecules that have a specific affinity for the coligand, 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. Examples of ligands include lipopolysaccharides, p38 MAP kinase activators, or NF-κB activators.

[0177] 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 the conjugate 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 resistance to degradation of the conjugate, (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 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 very strongly to HSA can be used to target the conjugate to the kidney. In a preferred embodiment, the lipid-based ligand binds to HSA. The lipid-based ligand can bind to HSA with sufficient affinity so that the conjugate is distributed to non-renal tissue.However, it is desirable that the affinity is not so strong as to prevent the reversal of HSA-ligand binding. In another preferred embodiment, the lipid-based ligand binds weakly to HSA or does not bind at all, so that the conjugate is preferably distributed in the kidney. Other parts that target kidney cells can also be used instead of, or in addition to, the lipid-based ligand.

[0178] 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.

[0179] 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.

[0180] The ligand may be a peptide or a peptide mimetic. Peptide mimes (also referred to herein as oligopeptide mimes) are molecules capable of folding into a defined three-dimensional structure similar to that of natural peptides. Attachment of peptides and peptide mimes to oligonucleotide agents can influence 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 acid lengths, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid lengths. The peptide or peptide mimetic may be, for example, a cell-permeable peptide, a cationic peptide, an amphiphilic peptide, or a hydrophobic peptide (e.g., 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 the 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 can undergo structural modifications, for example, to improve stability or induce conformational properties. Any of the structural modifications described below may be used.

[0181] Oligonucleotide conjugates The oligonucleotide conjugates described herein comprise oligonucleotides and dendrons. Dendrons may possess potent, reversible, and non-covalent albumin binding, which can be advantageously used to minimize degradation, reduce macrophage uptake and degradation, prevent nonspecific uptake by cells, and / or provide enhanced delivery of oligonucleotide conjugates to the brain. Dendrons may also possess nanomolar affinity for albumin, which advantageously ensures that the albumin-oligonucleotide conjugate complex remains stable throughout in vivo distribution, cell interactions, and / or treatment.

[0182] In some embodiments, the oligonucleotide conjugate has the structure of formula I, [ka] (In the formula, A is a oligonucleotide, Each instance of B independently comprises one or more hydrophobic chains, amines, amides, esters, N or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, where the hydrophobic chain is saturated or unsaturated C 1-24 Containing alkyl chains, Each instance of C is independently a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an amine, amide, ether, ester, N or O-containing heterocycle, a 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, each time it appears, independently contains one or more alkyl chains, amides, ethers, esters, and amines, where the branched unit contains 2 to 4 branches. m is either 0 or 1 independently each time it appears.

[0183] In some embodiments, the oligonucleotide conjugate has the structure of formula II, [ka] (In the formula, A is a oligonucleotide, Each instance of B independently comprises one or more hydrophobic chains, amines, amides, esters, N or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, where the hydrophobic chain is saturated or unsaturated C 1-24 Containing alkyl chains, Each instance of C is independently a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an amine, amide, ether, ester, N or O-containing heterocycle, a 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, each instance, independently comprises one or more alkyl chains, amides, ethers, esters, and amines (wherein the branched unit contains 2 to 4 branches). In some embodiments, the oligonucleotide conjugate has the structure of formula III, [ka] (wherein n is an integer from 1 to 24, independently of each occurrence.) In some embodiments, C is OH, and n is 1, 6, or 12, independently of each occurrence. In some embodiments, the oligonucleotide conjugate has the structure of formula IV. [ka]

[0184] In some embodiments, the oligonucleotide conjugate has the structure of formula V, [ka] (In the formula, A is a oligonucleotide, Each instance of B independently comprises one or more hydrophobic chains, amines, amides, esters, N or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, where the hydrophobic chain is saturated or unsaturated C 1-24 Containing alkyl chains, Each instance of C is independently a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an amine, amide, ether, ester, N or O-containing heterocycle, a 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, each instance, independently comprises one or more alkyl chains, amides, ethers, esters, and amines, and the branched unit contains 2 to 4 branches. In some embodiments, oligonucleotide conjugates have the structure of formula VI. [ka] (wherein the formula, n is an integer from 1 to 24, independently of each occurrence.) In some embodiments, C is, independently of each occurrence, a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an amine, amide, ether, ester, N or O-containing heterocycle, thiol, thioether, and / or saturated or unsaturated C 1-24 The molecule 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, each time it appears. In some embodiments, the oligonucleotide conjugate has the structure of formula VII. [ka]

[0185] Branched oligonucleotide conjugate In certain embodiments, the oligonucleotide conjugate is a branched oligonucleotide conjugate.

[0186] 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.

[0187] 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.

[0188] In certain embodiments, the branched functional moiety is a triple amine functional moiety such as a phosphatidylcholine (PC) esterified triple amine (PC-triple amine).

[0189] 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.

[0190] In certain embodiments, the linker includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphates, phosphonates, phosphoramidates, esters, amides, triazoles, or any combination thereof.

[0191] In certain embodiments, the branching point includes a polyvalent organic species or a derivative thereof.

[0192] In another embodiment, the branch point is an amino acid derivative. In yet another embodiment, the branch point is selected from the following formula: [ka]

[0193] Polyvalent organic species are those containing carbon and a valency of three or more (i.e., bonding sites with sites 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).

[0194] In certain embodiments, the spacer includes ethylene glycol chains, alkyl chains, peptides, RNA, DNA, phosphate esters, phosphonic acid esters, amide phosphate esters, esters, amides, triazoles, or combinations thereof.

[0195] 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.

[0196] The pharmaceutical compositions of the present invention are formulated to suit their intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous (IV), intradermal, subcutaneous (SC or SQ), intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents, e.g., water for injection, saline, fixative oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, e.g., benzyl alcohol or methylparaben; antioxidants, e.g., ascorbic acid or sodium bisulfite; chelating agents, e.g., ethylenediaminetetraacetic acid; buffers, e.g., acetates, citrates, or phosphates; and agents for adjusting tonicity, e.g., sodium chloride or dextrose. pH can be adjusted with an acid or base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in glass or plastic ampoules, disposable syringes, or multi-dose vials.

[0197] 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 retarders, etc., that are suitable for drug administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agent is incompatible with the active compound, its use in the composition is intended. Supplementary active ingredients may also be incorporated into the composition.

[0198] Compositions, systems, and methods, 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.

[0199] 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 a coating agent, in the case of a dispersion, by maintaining the required particle size, and by the use of a surfactant. 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, polyalcohols, such as mannitol, sorbitol, and 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.

[0200] 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.

[0201] Data obtained from cell culture assays and animal studies can be used when formulating dose ranges for use in humans. Doses of such compounds are preferably within the range of circulating concentrations including an ED50 that is little to no toxicity. Doses may vary within this range depending on the form of administration 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 cell culture assays. Doses can be formulated in animal models to achieve a circulating plasma concentration range including the 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.

[0202] kit In certain other embodiments, the present invention provides a kit comprising a suitable container for 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 the preparation of the RNA silencing agent and one 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 the instructions provided in 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.

[0203] Treatment method In one embodiment, a method for selectively delivering one or more oligonucleotide conjugates described herein to the brain of a patient is provided herein, the method comprising administering the compound to the patient.

[0204] In certain embodiments, methods for providing treatment to patients requiring treatment for a disease, disorder, or injury of the brain are provided herein, comprising administering a compound of formula (I) to the patient. Non-limiting examples of such diseases or disorders include amyotrophic lateral sclerosis (ALS), Alzheimer's disease, autism, concussion, dystonia, dementia, epilepsy, Huntington's disease, muscular dystrophy, neurological disorders, Parkinson's disease, sleep disorders, and Tourette syndrome.

[0205] 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 it) to a patient, or the application or administration of a therapeutic agent to tissue or cell lines isolated from a patient, the patient having the disease or disorder, the symptoms of the disease or disorder or a predisposition to the disease or disorder, and the treatment being performed to treat, cure, reduce, alleviate, modify, repair, restore, improve or influence the disease or disorder, the symptoms of the disease or disorder or a predisposition to the disease or disorder.

[0206] 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. Administration of the prophylactic agent can be performed before the appearance of symptoms characteristic of the disease or disorder, thereby preventing the disease or disorder or slowing its progression.

[0207] 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 vector or a transgene encoding the same) that is specific to one or more target sequences in a gene, thereby achieving 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).

[0208] The dose of RNA silencing agents delivered directly to the brain may range from approximately 0.00001 mg to approximately 3 mg per brain, or preferably approximately 0.0001 mg to 0.001 mg, approximately 0.03 mg to 3.0 mg, approximately 0.1 mg to 3.0 mg, or approximately 0.3 mg to 3.0 mg per brain. In one embodiment, the unit dose is administered less frequently than once a day, for example, less frequently than every 2, 4, 8, or 30 days. In another embodiment, the unit dose is not administered at a constant frequency (e.g., regularly). For example, the unit dose may be administered only once. In one embodiment, the effective dose is administered in combination with other conventional therapies.

[0209] 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. They may be reduced if undesirable side effects are observed. 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 particular 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 hours, 36 hours, 48 ​​hours, or more, for example, once or less every 5 or 8 days. After treatment, the patient may be monitored for changes in their 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]

[0210] Several experimental examples are planned, but these embodiments are intended to be non-limiting.

[0211] Example 1. Synthesis of oligonucleotides Oligonucleotides were synthesized according to standard protocols using a MerMade 6 / 12 synthesizer (Bioautomation) and an AKTA Oligopilot 100 (GE Healthcare Life Sciences). Briefly, conjugated sense strands were synthesized in 5-20 μmol quantities on lipid-functionalized controlled-pore glass (CPG) supports customized 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; 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. These were first deprotected with (E)-vinylphosphonic acid in a dichloromethane solution of bromotrimethylsilane / pyridine (3:2, v / v), and then cleaved and deprotected with 28% aqueous ammonium hydroxide at 60°C for 20 hours. All chains were cleaved and protected with 28% aqueous ammonium hydroxide at 60°C for 20 hours, then dried under vacuum at 60°C and resuspended in Millipore H2O. The oligonucleotides were purified using an Agilent Prostar System (Agilent Technologies), with the lipid conjugate sense chains on a C18 column and the antisense chains on an ion exchange column. The purified oligonucleotides were desalted by size exclusion chromatography and analyzed by liquid chromatography-mass spectrometry (LC / MS) using an Agilent 6530 precise mass quadrupole time-of-flight (Q-TOF) LC / MS (Agilent Technologies). Figure 1 shows examples of fully chemically stabilized oligonucleotides (siRNA) and dendritic (D)siRNA synthesized by this method.

[0212] The compound sequences 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: symmetrically branched, V: (E)-vinylphosphonate ester). DIO = branched porous glass [Table 1-1] [Table 1-2]

[0213] 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).

[0214] 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.

[0215] 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 by a Tecan M1000 (Tecan, Morrisville, NC, USA). The light emission was detected by a Tecan M1000 (Tecan, Morrisville, NC, USA).

[0216] 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 dododecyl sulfate (SDS) was precipitated from the lysate by adding 20 μl of 3M 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) with 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%~100% Buffer B within 5 minutes. Cy3 fluorescence was monitored and peaks were integrated. Final concentrations were confirmed using calibration curves generated by spiked tissue lysates from untreated animals with known amounts of lipid-conjugated siRNA. Calibrated spiked samples and experimental samples were processed and analyzed under the same laboratory conditions.

[0217] 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.

[0218] Example 6. Lipoprotein size exclusion chromatography For lipoprotein profiling, we followed the same protocol previously described by Osborn (the entire protocol is incorporated herein by reference; 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)). Briefly, mice were intravenously injected with 10 mg / kg of Cy3-labeled oligonucleotide. After 15 minutes, the cheek was incised with a lancet, and whole mouse blood (approximately 500 μl) was collected in a sterile EDTA-clad tube. The sample was rotated at 4°C and 10,000 RPM for 10 minutes. 50 μl of plasma was directly injected 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. In the case of subcutaneous injection, the sample was collected one hour after the injection.

[0219] Example 7. Reverse-phase HPLC analysis of D-siRNA and DCA-siRNA The LC data of the oligonucleotide was performed using an Agilent 6530 Precision Mass Q-TOF under the following conditions: Buffer A: an LC-MS grade aqueous solution of 100 mM 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and 9 mM triethylamine (TEA); Buffer B: an LC-MS grade methanol solution of 100 mM HFIP and 9 mM triethylamine (TEA); 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.

[0220] 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. A 7 - point dose - response curve was 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. Transfections were performed in 50:50 DMEM / OptiMEM (Gibco, 31985 - 070) and 3% FBS without antibiotics. Cells were lysed by incubating at 55 °C for 30 minutes with a diluted QuantiGene (QG) lysis mixture (Invitrogen, QP0524) containing Proteinase 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 the following brief description). Htt data was normalized to the housekeeping Hprt and presented as a percentage of the untreated control. n = 3.

[0221] 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 the dissected femurs were used with a 23G needle to wash the bone marrow cells of the femurs by flushing them onto a 70-μm cell strainer placed in a 50-mL conical tube with PBS. Next, the bone marrow and spleen were fragmented using a 5-mL plunger, and then the strainer was rinsed 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. The blood cells were lysed using ACK lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA in DW), and then washed with DMEM (500×g, 10 minutes, 4 °C). Next, for flow cytometry analysis, the cell pellet was suspended in flow cytometry buffer (0.5% BSA, 2 mM EDTA in DMEM).

[0222] 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).

[0223] Example 10. Selective protein binding profile of D-siRNA to albumin We evaluated the lipoprotein binding profile via size exclusion chromatography, as previously developed 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 study of which is incorporated herein). The binding profile of the conjugates is partially hydrophobic; more hydrophobic conjugates (docosanoic acid, DCA, etc.) bind to low-density and high-density lipoproteins (LDL and HDL) in plasma, and to albumin to a lesser extent. The dendritic portion is larger and contains more aliphatic material (69 carbon atoms) compared to DCA (28 carbon atoms). In addition to the terminal hydroxyl groups, multiple phosphate groups that demarcate the structure of the conjugate enhance solubility and prevent aggregation. As a result, 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).

[0224] After hydrophobicity comparison by HPLC, the serum protein binding profiles of each conjugate after injection into mice were examined as described above. Briefly, Cy3-labeled D-siRNA or DCA-siRNA was administered to animals subcutaneously (sc) or intravenously (iv) at a dose of 10 mg / kg, and plasma was collected 15 minutes (for iv) and 60 minutes (for sc) post-injection. These time points were chosen to maximize the circulating concentration of the compound in the blood.

[0225] Subsequently, plasma was fractionated by size exclusion chromatography, and the elution time of Cy-3-siRNA was monitored (570 nm). Sucrose-6 elution profiles for several major plasma proteins (HDL, VLDL, LDL, albumin / globulin) were pre-established. The retention time of D-siRNA after injection into mice was 67 minutes, overlapping with the retention time of albumin, whereas DCA-siRNA had a multiprotein binding profile primarily associated with LDL and HDL (Figure 2C). The protein binding tendency was consistent across both administration routes. This data confirms selective and high-affinity binding to albumin, which is assumed to determine in vivo behavior as well as in vitro behavior. This data also suggests that D-siRNA binds strongly to albumin and circulates in the blood as a protein-RNA complex until it is explanted to various organs and tissues, regardless of the mode of injection (subcutaneous or intravenous).

[0226] Example 11. Brain delivery of ASO and siRNA The delivery of D-siRNA ASO (antisense oligonucleotide) and siRNA to the brain was evaluated (Figures 3-6).

[0227] Fluorescence image of the brain after injection

[0228] Wild-type (FVB) female mice (approximately 8-10 weeks old) were injected with a total of 2 nmol (1.0 mg / kg) of D-siRNA (unilateral) via intrastriatal injection. Brains were collected 48 hours after injection. After fixation in 10% neutral buffered formalin for 24 hours, the brains were embedded in paraffin and coronal sections (4 micrometers) were collected. The coronal sections were stained with additional DAPI (nuclear stain) and imaged at 10x magnification using a Leica DMi8 microscope (Figure 3).

[0229] HTT mRNA expression in the brain after siRNA injection

[0230] Wild-type (B6) mice (female, 6-8 weeks old) were administered 5 nmol (total, 2.5 mg / kg) siRNA (bilateral injection, 2.5 nmol in each ventricle) and 10 nmol (total, 5 mg / kg) siRNA (bilateral injection, 5 nmol in each ventricle) by intracerebroventricular injection. Brains were collected 1-3 months after injection. Subsequently, mRNA levels of Htt and Hprt (hypoxanthine-guanine phosphoribosyltransferase, housekeeping gene) were measured using QuantiGene® (Affymetrix), normalized by Hprt (hypoxanthine-guanine phosphoribosyltransferase), and presented as a percentage relative to non-targeted control (NTC) (mean ± standard deviation (SD)). D-siRNA was found to spread more easily and be more active at lower doses and over longer periods. Low-dose (5 nmol) D-siRNA was found to be as effective as Dio-siRNA three months after injection, while high-dose (10 nmol) D-siRNA was found to be as effective as Dio-siRNA two months after injection (Figure 4).

[0231] HTT mRNA expression after injection of ASO and siRNA

[0232] Athymic (Nu / J) mice (female, 6 weeks old) carrying patient-derived GBM8 tumor xenografts were administered either by bilateral intraventricular injection (ICV) of 30 nmol (total, 15 mg / kg) of siRNA (15 nmol in each ventricle) or bilateral intratumoric (IT) / intracranial (IS) injection (2 nmol each in the tumor-bearing striatum and the contralateral striatum). Brains were collected one week after injection. mRNA levels of Htt and Hprt (normalization housekeeping genes) were measured using Taqman qPCR (Biorad) and expressed as percentages relative to NTC_D-siRNA (IT delivery) or vehicle (ICV delivery), depending on the route of administration (Figure 5B).

[0233] HTT and APP RNA expression after injection of unconjugated and conjugated siRNA

[0234] A panel of lipid-conjugated siRNAs was evaluated and revealed that they induce gene silencing in brain tumors while exhibiting a different toxicity profile in the central nervous system (CNS). Ligand conjugation, that is, attaching lipids, sugars, peptides, aptamers, or other target molecules to oligonucleotides, is one strategy used to improve delivery efficiency. Lipids are a particular type of binding molecule that can enhance the activity of oligonucleotides by extending tissue residence time, promoting cellular uptake, and improving the efficiency of endosomal extrusion. siRNA modified with cholesterol ligands has been shown to exhibit gene silencing in GBM xenografts. To improve functional delivery to GBM cells, a broader panel of lipid conjugates was evaluated. Four lipid-conjugated siRNAs were tested (Figure 5A). Three of the four binding molecules are biologically relevant lipids: cholesterol (Chol-siRNA), docosanoic acid (DCA-siRNA, a 22-carbon saturated fatty acid), and eicosapentaenoic acid (EPA-siRNA, a 20-carbon unsaturated fatty acid). The fourth conjugate is a synthetic dendritic structure (D-siRNA), which has been shown to exhibit high binding affinity to albumin. Each conjugate is covalently attached to an asymmetric, chemically modified monovalent siRNA scaffold, either via the 3' end of the sense strand (Chol, DCA, EPA) or the 5' end of the sense strand (dendrimer).

[0235] The chemical composition of lipid conjugates can affect the hydrophobicity of oligonucleotides, which may influence their subsequent accumulation in tissues and their functionality. To assess hydrophobicity, the retention times of selected siRNAs were analyzed using liquid chromatography (LC) (Figure 5A). Unconjugated siRNAs showed the shortest retention times and were found to be the least hydrophobic siRNAs in this panel. EPA-siRNA, D-siRNA, and DCA-siRNAs showed longer retention times and higher hydrophobicity compared to unconjugated siRNAs.

[0236] In an in vivo assay, mice were injected with 2 nmol of lipid-conjugated siRNA into their tumors (Figure 5C). Chol-siRNA HTT / Htt and DCA-siRNA HTT / Htt Mice injected with [substance name] exhibited phenotypic features of acute oligonucleotide-induced neurotoxicity. These phenotypes included seizures, hyperactivity, and ataxia that began immediately after recovery from anesthesia and were more severe and persistent than the reversible motor phenotypes seen after injection of some oligonucleotides. The condition of these mice did not improve, and they were humanely euthanized before reaching the planned experimental time point. Consequently, mRNA levels from these mice were measured two days after injection, rather than one week later.

[0237] Chol-siRNA HTT / Htt and DCA-siRNA HTT / Htt The compounds were found to reduce human HTT mRNA (i.e., within tumor xenografts) by 35% and 30%, respectively (Figure 5C, top graph). Both compounds showed a higher silencing effect in normal brain cells, reducing mouse Htt mRNA within xenografts by approximately 55%. The levels of mouse Htt mRNA showed a similar reduction in both striatums with and without xenografts. The degree of HTT / Htt mRNA reduction observed after 2 days does not reflect the maximum silencing achievable in GBM or normal brain cells using these siRNA compounds, as longer treatment periods are typically required to achieve the maximum silencing effect in vivo. Chol-siRNA and DCA-siRNA improved HTT silencing in GBM cells compared to ASOHTT / Htt and unbound monovalent and bivalent siRNAHTT / Htt, but their neurotoxicity significantly limited their therapeutic index. Consequently, Chol-siRNA and DCA-siRNA were not tested against APP / App. Chol-siRNA and DCA-siRNA are used in ASO HTT / Httand unconjugated monovalent and divalent siRNAs HTT / Htt Compared to other methods, Chol-siRNA or DCA-siRNA improved HTT silencing in GBM cells, but their neurotoxicity significantly limited the therapeutic index. Consequently, Chol-siRNA or DCA-siRNA were not tested against APP / App.

[0238] In contrast, EPA-siRNA is a less hydrophobic conjugate. HTT / Htt or D-siRNA HTT / Htt Mice injected intratumorically with EPA-siRNA showed no major signs of oligonucleotide-induced neurotoxicity. mRNA levels from these mice were measured one week later. HTT / Htt It reduced human HTT mRNA by 56%, while D-siRNA HTT / Htt This efficiently reduced human HTT mRNA by 47% (Figure 5C, middle graph). Thus, these two moderately hydrophobic conjugates exhibited the most potent silencing effect on HTT transcripts (i.e., within tumor xenografts) observed to date. Mouse Htt mRNA levels showed a similar reduction in both striatum with and without xenografts. Silencing with EPA-siRNA and D-siRNA was more potent in normal brain cells, reducing mouse Htt mRNA levels by 80% and 75%, respectively. Mouse Htt mRNA levels showed a similar reduction in both striatum with and without xenografts.

[0239] To verify whether this effective silencing could be applied to other targets, EPA-siRNA and D-siRNA were tested against APP / App. After one week, the levels of APP in human tumor cells were reduced by EPA-siRNA. APP / App and DsiRNA APPBoth decreased by more than 50% in both the / App (lower graph in Figure 5C). The levels of mouse App mRNA showed similar decreases in both the striatum with xenograft and the striatum without xenograft. These results demonstrate that EPA and the amphiphilic dendrimer conjugate exhibit gene silencing effects in vivo equivalent to or greater than those of unconjugated ASO and siRNA in GBM xenograft cells.

[0240] Functional delivery of lipid-conjugated siRNA with amphiphilic and moderate hydrophobicity to xenografts by using different delivery routes in the central nervous system (CNS).

[0241] Intratumoral delivery is a surgically feasible method for administering drugs to GBM tumors. However, when surgery is possible, most tumor masses are usually resected, reducing the clinical utility of these injections. Invasive GBM cells, i.e., tumor cells that have moved away from the center of the tumor and into adjacent normal brain tissue, are difficult to excise and ultimately cause tumor recurrence. Therefore, a delivery method that can widely distribute oligonucleotides throughout the brain tissue and reach treatment-resistant and migratory GBM cells is necessary to achieve clinical success in GBM treatment. Invasive GBM cells, i.e., tumor cells that have moved away from the center of the tumor and into adjacent normal brain tissue, are difficult to excise and ultimately cause tumor recurrence. Therefore, a delivery method that can widely distribute oligonucleotides throughout the brain tissue and reach treatment-resistant and migratory GBM cells is necessary to achieve clinical success in GBM treatment.

[0242] ICV injection in mice and intrathecal injection in patients can deliver oligonucleotides to the brain and spinal cord. Unconjugated, partially PS-modified ASOs and siRNAs are hydrophilic and help promote distribution within the CSF. Lipid-conjugated EPA-siRNAs and D-siRNAs are more hydrophobic and may affect distribution via the CSF. To verify this, siRNAs targeting selected huntingtin or amyloid precursor proteins were administered to GBM8 xenografts via bilateral bolus ICV injections, and gene expression was measured one week later.

[0243] In the huntingtin ICV study, EPA-siRNA was used. HTT / Htt or D-siRNA HTT / Htt The total dose of 30 nmol was injected (Figure 6, upper schematic). EPA-siRNA HTT / Htt It was found that it reduced human HTT mRNA by 45% and the corresponding mouse HTT mRNA by 68% (Figure 6, top graph). D-siRNA HTT / Htt It was found that it reduced human HTT mRNA levels by 36% and mouse Htt mRNA levels by 65% ​​(Figure 6, top graph). The degree of reduction in human HTT and mouse Htt mRNA after IICV delivery was approximately 10% lower than the degree of reduction observed after intratumoral delivery (Figure 5C, middle graph). This difference is likely due to the function of low local drug concentrations, coupled with the fact that the xenograft is located in the striatum, a deep brain region, making it difficult for oligonucleotides to reach it via CSF even in a healthy brain.

[0244] In the ICV study of amyloid precursor protein, siRNA was injected at a total dose of 10 nmol (Figure 6, middle schematic). A lower dose than that used in the huntingtin ICV assay was used to ensure that saturation was not reached. Asymmetric, unconjugated monovalent and bivalent siRNAs were also used. These were predicted to be widely distributed within brain tissue and could be used as a baseline to compare the distribution efficiency of lipid-conjugated siRNAs. After one week, the tested siRNAs were found to reduce human APP mRNA levels by more than 35% and mouse APP mRNA levels by more than 50% (Figure 6, middle graph). To ensure the efficiency of ICV administration, mouse mRNA levels were evaluated in the contralateral striatum without a xenograft. Mouse Htt and App mRNA levels showed a similar degree of decrease in both the striatum with and without a xenograft (Figure 6, top and middle graphs).

[0245] To assess the extent to which EPA-siRNA and D-siRNA are distributed in the brain, mouse App expression was measured in the olfactory bulb and posterior cerebellar lobe (these are two of the most distant regions along the anterior-posterior axis of the mouse brain). APP / App It was found that monovalent and bivalent siRNA reduced mouse App mRNA levels by 40% in the olfactory bulb and 35% in the cerebellum. APP / App A similar decline was observed (Figure 6, schematic diagram and graph in the lower panel). Amphiphilic D-siRNA APP / Ap p also exhibited functional silencing, reducing mouse App mRNA levels in the cerebellum by 45%. In particular, in the olfactory bulb, D-siRNA was confirmed to reduce mouse App mRNA levels by 60%, which was slightly superior to unbound, asymmetric di-siRNA (Figure 6, lower graph).

[0246] Overall, these results demonstrate that moderately hydrophobic EPA-siRNA or amphiphilic D-siRNA can be functionally delivered to GBM xenografts via delivery pathways that enable drug distribution to distant tumor cells causing recurrent GBM. Furthermore, the functional distribution of EPA-siRNA or D-siRNA is comparable to that of unconjugated and asymmetric siRNA, suggesting that conjugation of these specific lipids does not inhibit efficient distribution within brain tissue.

[0247] Example 12. 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.

[0248] Example 13. 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 8). 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.

[0249] Built-in by reference The content of all references that may be cited throughout this application (including references to documents, patents, patent applications, patent publications, and websites) is expressly incorporated by reference in their entirety for any purpose, just as the references described herein are expressly incorporated. Unless otherwise specified, this disclosure uses immunological, molecular biological, and cell biological techniques that are well known as prior art. 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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[0352] Gonzalez-Barriga, A., Nillessen, B., Kranzen, J., van Kessel, I.D.G., Croes, H.J.E., Aguilera, B., de Visser, P.C., Datson, N.A., Mulders, S.A.M., van Deutekom, J.C.T. et al. (2017) Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro. Nucleic Acid Ther, 27, 144-158.

[0353] Wakimoto, H., Mohapatra, G., Kanai, R., Curry, W.T., Jr., Yip, S., Nitta, M., Patel, A.P., Barnard, Z.R., Stemmer-Rachamimov, A.O., Louis, D.N. et al. (2012) Maintenance of primary tumor phenotype and genotype in glioblastoma stem cells. Neuro Oncol, 14, 132-144.

[0354] Lee, J., Kotliarova, S., Kotliarov, Y., Li, A., Su, Q., Donin, NM, Pastorino, S., Purow, BW, Christopher, N., Zhang, W. et al. (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell, 9, 391-403.

[0355] Zhang, Q., Yang, L., Liu, YH, Wilkinson, JE and Krainer, AR (2023) Antisense oligonucleotide therapy for H3.3K27M diffuse midline glioma. Sci Transl Med, 15, eadd8280.

[0356] Equal parts This disclosure may 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 foregoing description but by the appended claims, and all modifications that fall within the meaning of the claims and the scope of equivalents are intended to be incorporated herein.

Claims

1. A method for delivering an oligonucleotide conjugate to the brain of a target, wherein the method comprises administering the oligonucleotide conjugate to the target, and the oligonucleotide conjugate is i) An oligonucleotide having a 5' end and a 3' end and being complementary 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 administration to the brain.

2. The terminal group may be a hydrophilic group containing hydroxides, amines, phosphate esters, 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 3' end of the sense chain, or to the 5' end and / or 3' end of the antisense chain.

11. The method according to claim 9, wherein the dendron is bonded to the 3' 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 strand 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 15 to 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 includes at least one single-stranded nucleotide overhang.

20. The method according to any one of claims 9 to 19, wherein the siRNA comprises a natural nucleotide.

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, an amide phosphate ester, a nucleotide containing a non-natural base, 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 bond includes a thiophosphate nucleotide bond.

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

26. The process according to any one of claims 8 to 25, wherein the siRNA comprises 8 to 13 thiophosphate nucleotide bonds.

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

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

29. The process 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 process 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 process according to claim 30, wherein the antisense chain comprises a 5'-vinylphosphonate.

32. The process according to any one of claims 9 to 31, wherein 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 thiophosphate ester bonds.

33. The process 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】 (In the formula, A is a oligonucleotide, Each instance of B independently comprises one or more hydrophobic chains, amines, amides, esters, N or O-containing heterocycles, thioethers, disulfides, and / or aromatic rings, where the hydrophobic chain is saturated or unsaturated C １－２４ Containing alkyl chains, Each instance of C independently includes a hydroxide, amine, phosphate ester, sulfur, and / or a hydrophilic group containing sugar, an 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, each time it appears, independently contains one or more alkyl chains, amides, ethers, esters, and amines, where the branched unit contains 2 to 4 branches. The method according to any one of claims 1 to 33, where m is independently 0 or 1 each time it appears.

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 of each other).

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

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 VI. 【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 of each other).

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

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 preparation comprises approximately 0.1 to 20 mg of oligonucleotide conjugate per kg of body weight.

44. The method according to any one of the prior claims, wherein the oligonucleotide conjugate is administered to the subject by intrastriatal (IS) injection, intraventricular (ICV) injection, intratumor (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, subarachnoid injection, or a combination thereof.

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 8 and 10.

47. A method for providing treatment to a patient who requires treatment for a brain disease, disorder, or injury, the patient i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and ii) Administering an oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and having terminal groups, phosphate groups, and / or hydrophobic chains, The method wherein the oligonucleotide conjugate is formulated for administration to the brain.

48. The method according to claim 47, wherein the brain disease, disorder, or injury is selected from the group consisting of amyotrophic lateral sclerosis (ALS), Alzheimer's disease, autism, concussion, dystonia, dementia, epilepsy, Huntington's disease, muscular dystrophy, neurological disorders, Parkinson's disease, sleep disorders, Tourette syndrome, or a combination thereof.

49. The method according to claim 47, wherein the brain disease, disorder, or injury is associated with the HTT gene and / or the APP gene.

50. The method according to claim 49, wherein the oligonucleotide conjugate inhibits the expression of the HTT gene and / or the APP gene.

51. The method according to claim 49 or 50, wherein the oligonucleotide has one of the sequences of sequence numbers 8 and 10.

52. A method for administering a therapeutically effective amount of oligonucleotide conjugate to the brain of a target, wherein the method comprises administering the oligonucleotide conjugate to the target, and the oligonucleotide conjugate is i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and 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 administration to the brain.

53. An oligonucleotide conjugate for use in the treatment of brain diseases, disorders, or injuries in patients requiring treatment, wherein the oligonucleotide conjugate is administered to the patient and formulated for brain administration, and further, the oligonucleotide conjugate is i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and ii) The oligonucleotide conjugate comprising a dendron bonded to the oligonucleotide and having a terminal group, a phosphate group, and / or a hydrophobic chain.

54. A pharmaceutical composition for providing treatment to a patient who requires treatment for a brain disease, disorder, or injury, i) Oligonucleotides comprising a 5' end and a 3' end, which are complementary to the target nucleic acid, and ii) Dendrons bonded to oligonucleotides, including terminal groups, phosphate groups, and / or hydrophobic chains, A pharmaceutically acceptable carrier, comprising an oligonucleotide conjugate, The pharmaceutical composition is formulated for administration to the brain.

55. A method for inhibiting genes in cells, wherein the method is (a) i) Oligonucleotides comprising a 5' end and a 3' end and complementary to the target nucleic acid, ii) Introducing an oligonucleotide conjugate into a cell, comprising a dendron bonded to the oligonucleotide and containing a 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 gene, thereby inhibiting the expression of the gene in the cells.

56. The method according to claim 55, wherein the gene is an HTT gene or an APP gene.

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