Modified miR-135, its conjugates and uses thereof

Abstract

Disclosed is a composition comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO: 37 and the complementary strand as set forth in SEQ ID NO: 40. Also disclosed is a conjugate comprising the synthetic miR-135 molecule and a composition comprising a cell targeting moiety.

Description

[Technical field]

[0001] Related Weekly Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 138,555, filed January 18, 2021, and U.S. Provisional Patent Application No. 63 / 272,329, filed October 27, 2021, the contents of which are incorporated by reference in their entireties.

[0002] SEQUENCE LISTING STATEMENT A 21,023 byte ASCII file entitled 89938SequenceListing.txt, dated January 17, 2022, which was submitted concurrently with this application, is incorporated by reference herein.

[0003] The present invention in some embodiments thereof relates to therapeutic miR-135 molecules and, more particularly, but not exclusively, to uses thereof. [Background technology]

[0004] Mood disorders, such as major depression and anxiety disorders, represent some of the most common and growing health problems worldwide, affecting approximately 10% of the population. Despite decades of research, the mechanisms of depression onset, transmissibility, and available treatments are only partially understood. Currently, only about one-third of patients respond to available treatments, so there is a great demand for a better understanding of the pathology.

[0005] Current dogma regarding the etiology of depression is a complex interplay between environmental factors and genetic predisposition, suggesting a mechanistic role for epigenetic processes.

[0006] Serotonin (5HT) is a monoamine neurotransmitter produced in the brain by the raphe nucleus (RN) and distributed throughout the brain to regulate various cognitive, emotional and physiological functions. The association between dysregulation of serotonin activity and depression is well established [Michelsen KA. et al., Brain Res Rev (2007) 55(2):329-42]. The genetic circuitry responsible for its production, secretion, reuptake and inactivation as well as the levels of 5HT are dysregulated in depression. In addition, recently offered antidepressants target the function of 5HT system-related proteins, resulting in increased levels of 5HT in the synapse [Krishnan V and Nestler EJ, Nature (2008) 455: 894-902]. Existing therapies require long-term administration before symptomatic relief is observed.

[0007] MicroRNAs (miRs) are a subset of endogenous small (~22 nucleotides) non-coding RNA molecules that post-transcriptionally repress gene expression. MiRs are transcribed as primary miR molecules, processed into precursor miRs with stem-loop structures in the cell nucleus, and transported to the cytoplasm for further processing into active mature miRs. Mature miRs are then incorporated into RNA-induced silencing complexes and function primarily by binding to the 3' untranslated region (3'UTR) of specific mRNA molecules. Binding occurs via a seed sequence, a 6-8 nucleotide sequence at the 5' end of the miR, where the complementary seed base pairs with a sequence on the target mRNA 3'UTR. miR binding leads to direct mRNA destabilization or translational repression, ultimately resulting in reduced protein levels of the target genes.

[0008] MiRs are abundant in the nervous system, and early studies focused on normal processes such as development, neuronal cells in cancer and neurodegenerative disorders, and orthopedics [Kosik KS. Nat Rev Neurosci (2006) 7:911-20]. Several miR screening studies have reported that miR levels in various adult rodent or human brain structures are affected by a range of behavioral and pharmacological manipulations [O'Connor RM et al., Mol Psychiatry (2012) 17: 359-376]. In addition, miRs have been suggested to play a role in psychiatric disorders such as schizophrenia, autism, as well as depression and anxiety in both human and mouse models [Miller BH and Wahlestedt C, Brain Res (2010) 1338: 89-99 and Issler O and Chen A, Nat Rev Neurosci. (2015) 16: 201-212]. Several studies have recently implicated miRs in the regulation of 5HT-related genes [Millan MJ. Curr Opin Pharmacol (2011) 11(1):11-22], highlighting the emerging role of miRs in regulating the 5HT axis and their possible association with depression-related disorders.

[0009] WO 2013 / 018060 discloses microRNAs (e.g., miR-135) and compositions comprising same for the treatment and diagnosis of conditions related to serotonin-releasing hormone, adrenaline-releasing hormone, noradrenaline-releasing hormone, glutamate-releasing hormone, and corticotropin-releasing hormone.

[0010] WO 2015 / 118537 discloses a method for treating bipolar disorder by administering to a subject a therapeutically effective amount of miR-135, a precursor thereof, or a nucleic acid molecule encoding miR-135 or a precursor thereof.

[0011] US Patent Application Publication No. 2017 / 0037404 discloses methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. The synthetic nucleic acid molecules may be modified.

[0012] WO 2011 / 131693 discloses conjugates comprising (i) a nucleic acid complementary to a target nucleic acid sequence, the expression of which prevents or reduces expression of the target nucleic acid (e.g., miRNA), and (ii) a selection agent capable of binding with high affinity to a neurotransmitter transporter (e.g., serotonin reuptake inhibitor). WO 2011 / 131693 discusses the use of these conjugates for delivery of nucleic acids to cells of interest, for treatment of disease, and for diagnostic purposes. Summary of the Invention

[0013] In one aspect of some embodiments of the invention, a composition is provided comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:37 and the complementary strand as set forth in SEQ ID NO:40.

[0014] In one aspect of some embodiments of the present invention, a composition is provided comprising a synthetic miR-135 molecule comprising a nucleic acid sequence of miR-135b as set forth in any one of SEQ ID NOs: 10, 16, or 41-46, and a complementary strand as set forth in any one of SEQ ID NOs: 13 or 47.

[0015] In one aspect of some embodiments of the invention, a composition comprising a nucleic acid construct of a synthetic miR-135 molecule of some embodiments of the invention is provided.

[0016] In one aspect of some embodiments of the present invention, (i) a composition comprising a synthetic miR-135 molecule of some embodiments of the invention; (ii) a cell-targeting moiety; and A conjugate comprising:

[0017] In one aspect of some embodiments of the invention, there is provided a pharmaceutical composition comprising a composition of some embodiments of the invention or a conjugate of some embodiments of the invention and a pharma- ceutically acceptable carrier.

[0018] In one aspect of some embodiments of the invention, there is provided a method for treating a central nervous system (CNS)-related condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition of some embodiments of the invention, or a conjugate of some embodiments of the invention, thereby treating the CNS-related condition.

[0019] In one aspect of some embodiments of the present invention, there is provided a method for treating a depression-related disorder in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, thereby treating the depression-related disorder.

[0020] In one aspect of some embodiments of the present invention, there is provided a method for treating a cancerous disease in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, thereby treating the cancerous disease.

[0021] In one aspect of some embodiments of the present invention, there is provided a method for promoting bone regeneration or muscle cell differentiation in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a composition of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, thereby promoting bone regeneration or muscle cell differentiation.

[0022] In one aspect of some embodiments of the invention, there is provided a therapeutically effective amount of a composition of some embodiments of the invention, or a conjugate of some embodiments of the invention, for treating a CNS-related condition in a subject in need of such treatment.

[0023] In one aspect of some embodiments of the present invention, there is provided a therapeutically effective amount of a composition of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for treating a depression-related disorder in a subject in need of such treatment.

[0024] In one aspect of some embodiments of the present invention, there is provided a therapeutically effective amount of a composition of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for treating a cancerous disease in a subject in need of such treatment.

[0025] In one aspect of some embodiments of the invention, there is provided a therapeutically effective amount of a composition of some embodiments of the invention, or a conjugate of some embodiments of the invention, for treating a bone-related disease or condition or a muscle-related disease or condition in a subject in need of such treatment.

[0026] According to some embodiments of the invention, the miR-135 molecule comprises 50 or fewer nucleic acids.

[0027] According to some embodiments of the invention, the nucleic acid sequence of miR-135b as shown in SEQ ID NO: 37 and the complementary strand as shown in SEQ ID NO: 40 are present in separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

[0028] According to some embodiments of the present invention, the nucleic acid sequence of miR-135b as shown in SEQ ID NO: 37 and the complementary strand as shown in SEQ ID NO: 40 form a hairpin loop structure.

[0029] According to some embodiments of the present invention, the nucleic acid sequence of miR-135b as shown in SEQ ID NO:37 and its complementary strand as shown in SEQ ID NO:40 are 100% complementary over the entire length of SEQ ID NO:37 and SEQ ID NO:40, respectively.

[0030] According to some embodiments of the invention, the nucleic acid sequence of miR-135b and / or the complementary strand comprises one or more modifications selected from the group consisting of a sugar modification, a nucleobase modification, and an internucleotide linkage modification.

[0031] According to some embodiments of the invention, the sugar modification is selected from the group consisting of 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), locked nucleic acid (LNA) and 2'-fluoroarabino oligonucleotide (FANA).

[0032] According to some embodiments of the invention, the sugar modification in miR-135b is present on at least one nucleotide at the 3' end of the nucleic acid sequence.

[0033] According to some embodiments of the invention, the sugar modification in miR-135b is present on at least one nucleotide at the 5' end of the nucleic acid sequence.

[0034] According to some embodiments of the invention, the sugar modification in the complementary strand comprises a modification of the last nucleotide at the 3' end of the nucleic acid sequence.

[0035] According to some embodiments of the invention, the sugar modification in the complementary strand comprises a modification of the first two nucleotides at the 5' end of the nucleic acid sequence.

[0036] According to some embodiments of the invention, the sugar modification is a 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), and / or 2'-fluoro (2'-F) modification.

[0037] According to some embodiments of the invention, the sugar modifications are 2'-O-methoxyethyl (2'-O-MOE) and 5' ribose methylation (2'-O-MOE-5'-Me).

[0038] According to some embodiments of the invention, the internucleotide linkage modification is selected from the group consisting of phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl phosphonates, alkyl phosphonates, chiral phosphonates, phosphinates, phosphoramidates, aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, boranophosphates, phosphodiesters, phosphonoacetic acids (PACE) and peptide nucleic acids (PNAs).

[0039] According to some embodiments of the invention, the internucleotide linkage modification in miR-135b is present at the last nucleotide at the 5' end of the nucleic acid sequence.

[0040] According to some embodiments of the invention, the internucleotide linkage modification comprises a phosphate.

[0041] According to some embodiments of the invention, the internucleotide linkage modification comprises phosphorothioate.

[0042] According to some embodiments of the invention, the phosphorothioate modification is present between the last two nucleotides at the 3' end of the nucleic acid sequence of miR-135b or the complementary strand.

[0043] According to some embodiments of the invention, the phosphorothioate modification is present between the last two nucleotides at the 5' end of the nucleic acid sequence of miR-135b or the complementary strand.

[0044] According to some embodiments of the invention, the nucleic acid sequence of miR-135b including the modification is set forth in any one of SEQ ID NOs: 10, 16 or 41-46.

[0045] According to some embodiments of the invention, the nucleic acid sequence of the complementary strand containing the modification is as set forth in any one of SEQ ID NOs: 13 or 47.

[0046] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO:10, and the complementary strand is shown in SEQ ID NO:13.

[0047] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in SEQ ID NO:41, and the complementary strand is set forth in SEQ ID NO:13.

[0048] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in SEQ ID NO:42, and the complementary strand is set forth in SEQ ID NO:13.

[0049] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in SEQ ID NO:10, and the complementary strand is set forth in SEQ ID NO:47.

[0050] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO:16, and the complementary strand is shown in SEQ ID NO:13.

[0051] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in SEQ ID NO:41, and the complementary strand is set forth in SEQ ID NO:47.

[0052] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in SEQ ID NO:42, and the complementary strand is set forth in SEQ ID NO:47.

[0053] According to some embodiments of the invention, the nucleic acid sequence of miR-135b is set forth in any one of SEQ ID NOs: 10, 16, or 41-46, and the complementary strand is set forth in any one of SEQ ID NOs: 13 or 47, each present in a separate nucleic acid sequence molecule forming a double-stranded synthetic miR-135 molecule.

[0054] According to some embodiments of the present invention, the nucleic acid sequence of miR-135b is set forth in any one of SEQ ID NOs: 10, 16, or 41 to 46, and the complementary strand is set forth in any one of SEQ ID NOs: 13 or 47, which form a hairpin structure.

[0055] According to some embodiments of the present invention, the nucleic acid sequence of miR-135b is set forth in any one of SEQ ID NOs: 10, 16, or 41 to 46, and the complementary strand is set forth in any one of SEQ ID NOs: 13 or 47, and the complementarity is 100%.

[0056] According to some embodiments of the invention, the cell targeting moiety is conjugated to the 5' end of the nucleic acid sequence of the complementary strand.

[0057] According to some embodiments of the invention, the cell targeting moiety is conjugated to the 5' end of the nucleic acid sequence of miR-135b.

[0058] According to some embodiments of the invention, the cell targeting moiety is conjugated to the 3' end of the nucleic acid sequence of the complementary strand.

[0059] According to some embodiments of the invention, the cell targeting moiety is conjugated to the 3' end of the nucleic acid sequence of miR-135b.

[0060] According to some embodiments of the invention, the synthetic miR-135 molecule and the cell targeting moiety are linked by a linking group.

[0061] According to some embodiments of the invention, the linking group comprises a compound selected from the group consisting of phosphodiesters, phosphoramidites, phosphorothioates, carbamates, methylphosphonates, guanidinium, sulfamic acids, sulfamides, formacetals, thioformacetals, sulfones, amides, and mixtures thereof.

[0062] According to some embodiments of the invention, the linking group comprises a C10 linker.

[0063] According to some embodiments of the invention, the cell targeting moiety specifically binds to a molecule expressed or displayed on brain cells.

[0064] According to some embodiments of the invention, the cell targeting moiety specifically binds to a neurotransmitter transporter.

[0065] According to some embodiments of the invention, the cell targeting moiety is selected from the group consisting of serotonin reuptake inhibitors (SRIs), selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic-specific serotonergic antidepressants (NASSAs), noradrenaline reuptake inhibitors (NRIs), dopamine reuptake inhibitors (DRIs), endocannabinoid reuptake inhibitors (eCBRIs), adenosine reuptake inhibitors (AdoRIs), excitatory amino acid reuptake inhibitors (EAARIs), glutamate reuptake inhibitors (GluRIs), GABA reuptake inhibitors (GRIs), glycine reuptake inhibitors (GlyRIs) and norepinephrine-dopamine reuptake inhibitors (NDRIs).

[0066] According to some embodiments of the invention, the selective serotonin reuptake inhibitor (SSRI) is selected from the group consisting of sertraline, sertraline-structural analogs, fluoxetine, fluvoxamine, paroxetine, indalpine, zimelidine, citalopram, dapoxetine, escitalopram, and mixtures thereof.

[0067] According to some embodiments of the invention, when the cell targeting moiety is sertraline, the conjugate has the structure: [ka]

[0068] According to some embodiments of the invention, when the cell targeting moiety is sertraline, the conjugate has the structure: [ka]

[0069] According to some embodiments of the invention, when the cell targeting moiety is sertraline, the conjugate has the structure: [ka]

[0070] According to some embodiments of the invention, when the cell targeting moiety is sertraline, the conjugate has the structure: [ka]

[0071] According to some embodiments of the invention, the cell targeting moiety specifically binds to a tumor-associated antigen.

[0072] According to some embodiments of the invention, the cell targeting moiety specifically binds to a molecule expressed or displayed on bone cells, muscle cells or gastrointestinal cells.

[0073] According to some embodiments of the invention, the compositions or conjugates of some embodiments of the invention further comprise at least one cell membrane penetrating moiety or a moiety for transport across the blood-brain barrier (BBB).

[0074] According to some embodiments of the invention, the compositions or conjugates of some embodiments of the invention further comprise a linker between the cell membrane penetrating moiety or the moiety for transport across the BBB and the synthetic miR-135 molecule.

[0075] According to some embodiments of the invention, a cell membrane penetrating moiety or a moiety for transport across the BBB is linked to miR-135b and / or its complementary strand and / or a cell targeting moiety.

[0076] According to some embodiments of the invention, administration is by a mode of administration selected from the group consisting of intranasal, intracerebroventricular, intrathecal, oral, local injection, and intravenous administration.

[0077] According to some embodiments of the invention, the composition is for intranasal, intracerebroventricular, intrathecal, oral, local injection, or intravenous administration.

[0078] According to some embodiments of the invention, the CNS-related condition is a psychiatric disorder.

[0079] According to some embodiments of the invention, the CNS-related condition is selected from the group consisting of depression, anxiety, autism spectrum disorder, schizophrenia, bipolar disorder, stress, fatigue, cognitive dysfunction, panic attacks, obsessive-compulsive behavior, addiction, social phobia, sleep disorders, eating disorders, memory disorders, cognitive disorders, growth disorders and reproductive disorders.

[0080] According to some embodiments of the invention, the depression-related disorder is selected from the group consisting of major depression, obsessive-compulsive disorder (OCD), pervasive developmental disorder (PDD), post-traumatic stress disorder (PTSD), anxiety disorder, bipolar disorder, eating disorder and chronic pain.

[0081] According to some embodiments of the invention, the cancerous disease is selected from the group consisting of ovarian cancer, colorectal cancer and prostate cancer.

[0082] According to some embodiments of the invention, the subject has a bone-related disease or condition selected from the group consisting of osteoporosis, fractures or bone defects, primary or secondary hyperparathyroidism, osteoarthritis, periodontal disease or defects, osteolytic diseases, post-plastic surgery sequelae, post-orthopedic implant sequelae, and post-dental implant sequelae.

[0083] According to some embodiments of the invention, the subject has a muscle-related disease or condition selected from the group consisting of a muscular degenerative disease, a neuromuscular disease, a spinal muscular atrophy, an inflammatory myopathy, and a metabolic myopathy.

[0084] According to some embodiments of the invention, the subject is a human subject.

[0085] Unless otherwise defined, all technical and / or scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described in this application can be used in carrying out or testing embodiments of the present invention, exemplary methods and / or materials are described below. In the case of conflicts such as definitions, this patent specification shall prevail. Moreover, the materials, methods and examples are merely illustrative and are not necessarily intended to be limiting.

[0086] Some embodiments of the present invention are herein described, by way of example only, with reference to the accompanying drawings, in which: specific reference will now be made to the details of the drawings, stressing that the features shown therein are exemplary and are intended for illustrative discussion of embodiments of the present invention, in which the description taken together with the drawings will make apparent to those skilled in the art how embodiments of the present invention may be practiced. [Brief description of the drawings]

[0087] [Figure 1] Figure 1 shows the synthesis of miR-135 single-stranded oligonucleotides. The synthesis was carried out using a typical experimental procedure for solid-phase synthesis on a CPG (Controlled Pore Glass) support. A typical oligonucleotide synthesis proceeded through a series of cycles (as detailed in the "General Materials and Experimental Procedures" section below) consisting of four steps (deprotection, coupling, capping, and oxidation) that were repeated until the 5'-most nucleotide was attached. [Diagram 2]Figure 2 illustrates the synthesis of sertraline-conjugated miR-135. The addition of sertraline and linker to the 5'-end was performed as follows: 1. Carboxy C10 linker addition: After the last cycle (DMT-OFF) to facilitate the attachment of derivatized sertraline (sertraline attached to C6-NH2) to the 5'-end of the oligonucleotide, a carboxy C10 linker amidite block (in its N-hydroxysuccinimide ester form) was attached to the sequence in the final coupling step. 2. Sertraline addition: Derivatized sertraline was conjugated to the 5'-carboxy C10 modified oligonucleotide via an amide bond. This condensation was performed under organic conditions (diisopropylethylamine and dimethylformamide (DIPEA / DMF), 24 hours at room temperature, as detailed in the Examples below). [Diagram 3] 3A-B depict the results of luciferase reporter assays showing that duplex 11 significantly targets the 3'UTR of Slc6a4 (FIG. 3A) and the 3'UTR of HTR1a (FIG. 3B). [Figure 4-1] Figure 4A-E shows the effect of miR-135 mimics on serotonergic function in vivo. Of note, systemic 8-OH-DPAT administration (1 mg / kg body weight (BW), ip) did not induce hypothermia in naked (i.e., unconjugated) miR-135-treated (100 μg) mice at 24 hours (Figure 4A), 48 hours (Figure 4B), 72 hours (Figure 4C), and 96 hours (Figure 4D) after treatment. Two-way ANOVA showed a significant effect (***P<0.001) versus the control group (n=5-10). No differences in basal body temperature were observed between groups (Figure 4E). [Figure 4-2] Same as above [Diagram 5]5A-D illustrate the effect of sertraline-conjugated miR-135 mimic duplex 11 (miCure-135-1, shown in SEQ ID NOs: 10 and 13) on serotonergic function following direct dorsal raphe nucleus (DRN) administration. Of note, systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not result in hypothermia in sertraline-conjugated miR-135-treated (100 μg) mice at 24 hours (FIG. 5A), 48 hours (FIG. 5B), 96 hours (FIG. 5C) and 7 days (FIG. 5D) after treatment. Two-way ANOVA showed a significant effect (***P<0.001) versus control (n=10). [Figure 6-1] Figure 6A-H illustrates that acute treatment with low doses (30 μg) of sertraline-conjugated miR-135 (miCure-135-1) results in silencing of 5HT1a and SERT, resulting in an antidepressant-like response. Of note, systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not produce hypothermia in sertraline-conjugated miR-135-treated (30 μg) mice on days 1 (Figure 6A), 2 (Figure 6B), 4 (Figure 6C), and 7 (Figure 6D) of treatment. Two-way ANOVA showed a significant effect (***P<0.001) versus the control group (n=10). No differences in basal body temperature were observed between groups (n=10-40) (Figure 6E). Increased extracellular serotonin was evident in the medial prefrontal cortex (mPFC) of miCure-135-1-treated mice during the tail suspension test. Significant effect (*P<0.05) vs. controls (Figure 6F). A single intracerebral miCure-135-1 administration (30 μg) resulted in reduced immobility in the tail suspension test (n=8-9) (Figure 6G). Autoradiography of [3H]-citalopram binding showed reduced SERT density in the dorsal raphe in treated mice compared to controls (n=5-7), *p<0.05 (Figure 6H). [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 7]Figure 7A-C illustrates that acute intranasal administration of miCure-135-1 (166 μg) results in silencing of 5HT1a, resulting in an antidepressant-like response. Of note, systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not produce hypothermia on day 5 following intranasal delivery in sertraline-conjugated miR-135-treated (166 μg) mice. Two-way ANOVA showed a significant effect (*P<0.05) versus the control group (n=5) (Figure 7A). A single intranasal miCure-135-1 administration (166 μg) showed reduced immobility in the tail suspension test 4 days after treatment (n=8-9). *P<0.05 versus control (Figure 7B). Mice that received a single intranasal dose of miCure-135-1 (166 μg) explored the light compartment for a longer period of time than controls in the dark / light locomotion test (n = 11–12). *P < 0.05 vs. control (Figure 7C). [Figure 8] Figure 8 shows additional miR-135 modifications that were performed. Modifications are as follows: capital letters (e.g., A,U,C,G): RNA; lower case letters (e.g., a,u,c,g): 2'-O-Me modification; Um: (2'-O-MOE-5'-Me) uracil modification; Am: (2'-O-MOE) adenine modification; lower case 's': phosphorothioate, no indication means normal phosphodiester bond; P: phosphate; underline: 2'-fluoro, i.e., 2'-F. [Figure 9A] Figures 9A-9B show the results of luciferase reporter assays showing that miCure-135-1, miCure-135-2, miCure-135-3, miCure-135-9, miCure-135-10 and miCure-135-11 significantly target the Slc6a4 3'UTR (Figure 9B) and HTR1a 3'UTR (Figure 9A). Numbers above the bars are the number of statistically significant differences found (among five experiments). [Figure 9B] Same as above [Figure 10A]Figures 10A-E represent immune responses of human peripheral blood mononuclear cells (PBMCs) following in vitro treatment with three different miR-135 conjugated duplexes. Of note, miCure-135-10 induced high secretion of TNF-alpha cytokine, especially at a concentration of 300 nM, whereas miCure-135-1 only mildly activated this cytokine and miCure-135-2 induced almost no secretion (Figure 10A). The same secretion pattern was shown for the IFN-alpha-2a cytokine (Figure 10B), where miCure-135-10 induced high levels of secretion, miCure-135-1 induced moderate levels of secretion, and miCure-135-2 induced no secretion. None of the duplexes tested induced secretion of IL-10 (Figure 10C), IL-1 beta (Figure 10D), or IL-6 (Figure 10E). In Figures 10A and 10C to 10E, dir. incubation means direct incubation. [Figure 10B] Same as above [Figure 10C] Same as above [Figure 10D] Same as above [Figure 10E] Same as above [Figure 11A]Figures 11A-11F show immune responses of human peripheral blood mononuclear cells (PBMCs) following in vitro treatment with an additional set of three miR-135 conjugate duplexes. Notably, miR-135-11 and miCure-135-9 induced high secretion of IFN-alpha-2a, whereas miCure-135-3 induced little secretion (Figure 11A). Low levels of TNF-alpha secretion were induced by miCure-135-11 and miCure-135-9, whereas miCure-135-3 induced no secretion (Figure 11B). At a concentration of 300 nM, miCure-135-9 showed low levels of IFN-gamma secretion, whereas miCure-135-3 and miCure-135-9 showed no secretion (Figure 11C). None of the three tested duplexes resulted in significant production of the cytokines IL-10 (FIG. 11D), IL-1b (FIG. 11E), or IL-6 (FIG. 11F). [Figure 11B] Same as above [Figure 11C] Same as above [Figure 11D] Same as above [Figure 11E] Same as above [Figure 11F] Same as above [Figure 12-1]Figure 12A-G shows the effects of sertraline-conjugated miR-135 mimics (miCure-135-2, miCure-135-3 and miCure-135-9) on serotonergic function and their antidepressant-like effects. Systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not produce hypothermia in miCure-135-2-treated (30 μg) mice on days 2 (Figure 12A), 4 (Figure 12B) and 7 (Figure 12C) of treatment. Two-way ANOVA showed significant effects (*P<0.05, ***P<0.001) versus the control group (n=4-5). Systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not produce hypothermia in miCure-135-9-treated (30 μg) mice on the second day of treatment (Figure 12D). Two-way ANOVA showed a significant effect (P<0.05) versus the control group (n=4). There were no differences in basal body temperature between groups (Figure 12E) (n=4–19). Infusion of a local selective serotonin reuptake inhibitor (citalopram, 10 μM) by reverse-dialysis resulted in an increase in extracellular 5-hydroxytryptamine (5-HT) in the PFC of sertraline-conjugate control (100 μg)-treated mice compared to miCure-135-3 (100 μg)-treated mice (Figure 12F). Two-way ANOVA showed a significant effect (*P<0.05) versus the control group (n=6). A single intracerebral administration of miCure-135-3 (100 μg) resulted in a decrease in immobility in the tail suspension test (n=6), *p<0.05 (FIG. 12G). [Figure 12-2] Same as above [Figure 12-3] Same as above [Figure 13-1]Figures 13A-F show that intranasal and intracerebroventricular (ICV) administration of sertraline-conjugated miR-135 mimics maintains effects on serotonergic function in vivo. Of note, systemic 8-OH-DPAT administration (1 mg / kg BW, ip) did not result in hypothermia 48 hours after treatment in mice treated with miCure-135-3 (50 μg) (Figure 13A), (100 μg) (Figure 13B) and (200 μg) (Figure 13C). Similar effects were also illustrated 24 hours after intranasal administration of miCure-135-2 (33 μg / day) for 7 days (Figure 13D) and 96 hours after ICV administration of miCure-135-3 (200 μg / day) for 7 days (Figure 13E). Two-way ANOVA showed a significant effect (*P<0.05) versus the control group (n=6). Mice treated with intranasal administration of miCure-135-3 (200 μg / day) for 7 days showed a smaller increase in extracellular 5-hydroxytryptamine (5-HT) in the PFC following infusion of a local selective serotonin reuptake inhibitor (citalopram) by retrodialysis compared to sertraline conjugate control (200 μg / day)-treated mice (FIG. 13F). Two-way ANOVA showed a significant effect (*P<0.05) versus the control group (n=6). [Figure 13-2] Same as above [Figure 14-1]Figure 14A-G illustrates that acute intranasal administration of miCure-135-3 (2500 μg) affects serotonergic function and produces an antidepressant-like response. Immunoblot images and quantification show that protein levels of SERT (Figure 14A-B) and HTR1AR (Figure 14C-D) in the dorsal raphe are reduced in treated mice compared to controls (n=6-7), *p<0.05. Extracellular serotonin was reduced in the mPFC of control but not treated mice after 8-OH-DPAT administration (1 mg kg-1, ip) (n=5-6). There was a significant effect of group (P<0.05 vs. control (Figure 14E). Infusion of a local selective serotonin reuptake inhibitor (citalopram 10 μM) by retrodialysis resulted in an increase in extracellular 5-hydroxytryptamine (5-HT) in the PFC of sertraline conjugate control (2500 μg)-treated mice compared to miCure-135-3 (2500 μg)-treated mice (Figure 14F). Two-way ANOVA showed a P value = 0.05 vs. control group (n = 6). A single intranasal administration of miCure-135-3 (2500 μg) resulted in a decrease in immobility in the tail suspension test (as described in detail in the "General Materials and Experimental Procedures" section below) (n = 8-14), *p<0.05 (Figure 14G). [Figure 14-2] Same as above [Figure 15] Figure 15 illustrates that acute intranasal administration of miCure-135-3 (2500 μg) produces an antidepressant-like response in mice with induced depression-like behavior. Mice underwent a 28-day protocol to induce depression-like behavior (see General Materials and Experimental Procedures section below) and then received a single intranasal administration of control or miCure-135-3. Results show that a single intranasal miCure-135-3 administration (2500 μg) 3 days after treatment resulted in reduced immobility in the tail suspension test (n=8-10), *P<0.05 vs. control. [Figure 16-1]Figure 16 A-D illustrates the localization of miCure-135-3 in the dorsal raphe nucleus following intranasal administration. Selective accumulation of alexa488-labeled miCure-135-3 (green) in tryptophan hydroxylase 2-positive (TPH2-positive) neurons following intranasal administration (1000 μg). Confocal images show colocalization of alexa488-labeled miCure-135-3 (yellow) in dorsal raphe nucleus (DR) 5-HT neurons (TPH2-positive, red). Cell nuclei were stained with DAPI (4,6-diamidino-2-phenylindole, blue). Each row represents a different mouse (n=3, Figure 16A). High-magnification micrographs of the boxes shown in 'exm1' in Figure 16A are provided to show colocalization of alexa488-labeled miCure-135-3 in dorsal raphe nucleus (DR) 5-HT neurons (Figure 16B-C). Scale bars: left=100 μm, right=25 μm. In contrast, alexa488-labeled miCure-135-3 was absent from cells in brain regions adjacent to the administration site (olfactory bulb) or in the ventricles (hippocampus and striatum) (Figure 16D). [Figure 16-2] Same as above [Figure 16-3] Same as above [Figure 17-1] Figure 17A-D illustrates immunohistological measurements of cell viability in the raphe nucleus after acute administration of miCure-135-3 (100 μg). Conjugate control, miCure-135-3 or a positive control previously reported to have no effect on cell viability were rapidly delivered to the dorsal raphe nucleus. Adjacent 30 μm thick sections through the midbrain raphe nucleus were stained with the neuronal NeuN marker (Figure 17A), the microglial Iba1 marker (Figure 17B), or the serotonergic TPH marker (Figure 17C). Representative images depicting the midbrain raphe nucleus stained with NeuN, Iba1 and TPH under different treatments are shown in Figure 17D. Of note, no differences were found for any of the markers between all experimental groups. A representative image is shown in Figure 17C, with scale bars at 100 μm. [Figure 17-2] Same as above [Figure 17-3] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] The present invention in some embodiments thereof relates to therapeutic miR-135 molecules and, more particularly, but not exclusively, to uses thereof.

[0089] The principles and operation of the present invention may be better understood with reference to the drawings and the accompanying description.

[0090] Before describing at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated in the examples. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0091] MicroRNAs (miRs) are a subset of small RNA molecules that post-transcriptionally regulate gene expression and are abundant in a variety of tissues, including the brain.

[0092] In carrying out the present invention, the inventors have synthesized novel synthetic miR-135 molecules for therapeutic use, and have further demonstrated that the miR-135 molecules can be targeted to all target cells by coupling them to other molecules capable of specifically binding to the target cells.

[0093] Specifically, the present inventors designed and synthesized miR-135 mimic molecules, including two strands, i.e., a guide strand and a passenger strand, based on endogenous miR-135 with several modifications aimed at improving stability and cell permeability (see Tables 1 and 6 below and FIG. 8). The miR-135 mimic molecules (named duplexes 1-17 in Table 1 and duplexes 1-16 in Table 6) were then screened in vitro against known miR-135 targets (e.g., Htr1a and Slc6a4). One of the synthesized mimic miR-135 molecules (named duplex 11 in Table 1 and duplex 1 in Table 6, shown in SEQ ID NOs: 10 and 13) showed more significant binding to miR-135 targets (e.g., Htr1a and Slc6a4) in human cell line HEK293T than the other molecules synthesized and tested (see FIG. 3A-B and Example 1 below). The miR-135 mimetic molecules (i.e., duplex 11 in Table 1 and duplex 1 in Table 6) contained a guide sequence of miR-135b (shown in SEQ ID NO: 37) and a complementary sequence (shown in SEQ ID NO: 40) that differed from the native sequence. Furthermore, the synthetic miR-135 molecules (i.e., duplex 11 in Table 1 and duplex 1 in Table 6) were shown to have in vivo serotonergic function in animal models (see Figures 4A-E and Example 2 below).

[0094] The present inventors further designed and synthesized sertraline-conjugated miR-135 molecules for non-invasive delivery to the brain. In vivo studies of sertraline-conjugated miR-135 mimic duplex 11 (named miCure-135-1 in this application) revealed that a single administration into the dorsal raphe nucleus (DRN) was sufficient to eliminate hypothermia induced by the selective HTR1a agonist 8-OH-DPAT in naive mice by day 7 (see Figures 5A-D and 6A-E, Examples 3-4 described below). Administration of sertraline-conjugated miR-135 molecules into the DRN was further shown to induce not only an antidepressant effect in treated mice, but also a better coping mechanism (Figures 6F-G and Example 4 described below). Administration of sertraline-conjugated miR-135 molecules into the DRN was also shown to silence the SERT gene (Figure 6H and Example 4 described below). In addition, non-invasive intranasal administration of sertraline-conjugated miR-135 silenced 5HT1a as shown by lower hypothermic responses in treated mice (FIG. 7A and Example 5 below), and resulted in antidepressant / anxiety-like responses (FIGS. 7B-C and Example 5 below).

[0095] Through further experiments, the inventors have demonstrated that additional sertraline-conjugated miR-135 mimics containing various chemical modifications, namely, miCure-135-1, miCure-135-2, miCure-135-3, miCure-135-9, miCure-135-10 and miCure-135-11 (see Table 6 below), are potent and significantly alter both 5HTR1A and SLC6a4 levels (using 3'UTR luciferase constructs, see Figures 9A-9B and Example 6 below). The effect of conjugated miR-135 mimics on immune activation was tested. As evident from the results (see Figures 10A-E and 11A-F, and Example 7 below), different conjugated miR-135 mimics induced various immune responses, with miCure-135-1, miCure-135-2, and miCure-135-3 inducing the lowest cytokine secretion from PBMCs. In addition, acute administration of sertraline-conjugated miR-135 mimics miCure-135-2, miCure-135-3, and miCure-135-9 into the dorsal raphe nucleus (DRN) affected serotonergic function and induced antidepressant-like responses (see Figures 12A-G, and Example 8 below). Furthermore, intranasal and intracerebroventricular administration of sertraline-conjugated miR-135 mimics successfully reduced serotonergic autoreceptors (5HT1a) and serotonin transporters (SERT) in the dorsal raphe nucleus (Figures 13A-F and Example 9 below).

[0096] Further experiments revealed that acute intranasal administration of miCure-135-3, a miR-135 mimic, reduces the protein levels of SERT and 5-HT1A autoreceptor (HTR1AR), and shows antidepressant effects (see below in Examples 10 and 11). Intranasal administration of miCure-135-3 leads to accumulation in dorsal raphe nucleus, especially in midbrain 5-HT neurons (see below in Example 12). In addition, intranasal administration of miCure-135-3, a miR-135 mimic, was found to be safe in vivo administration (see below in Example 13).

[0097] Thus, the synthetic miR-135 molecules of the invention, as well as conjugated forms thereof, may be used in therapy, such as for the treatment of CNS-related conditions, including psychiatric disorders.

[0098] Thus, in one embodiment of the invention, there is provided a composition comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b as shown in SEQ ID NO:37 and the complementary strand as shown in SEQ ID NO:40.

[0099] In one embodiment of the invention, a synthetic miR-135 molecule is provided comprising the nucleic acid sequence of miR-135b as shown in SEQ ID NO:37 and the complementary strand as shown in SEQ ID NO:40.

[0100] As used herein, "synthetic" refers to a non-naturally occurring molecule.

[0101] In one embodiment, the non-naturally occurring miR-135 comprises the sequence of natural mature miR-135b and comprises synthetic backbone and / or side chains. Typically, the synthetic miR-135 molecule functions as a natural miRNA (e.g., miR-135b) within a cell or under physiological conditions.

[0102] The term "miR-135b" as used herein refers to a microRNA molecule involved in post-transcriptional gene regulation, including the 5' (i.e., miR-135b, also referred to as miR-135b-5p) or 3' (i.e., miR-135b*, also referred to as miR-135b-3p) of miR-135b. Exemplary mature miR-135b includes, but is not limited to, the miR-135b set forth in Accession No. MIMAT0000758 (SEQ ID NO: 37). Exemplary mature miR-135b* includes, but is not limited to, the miR-135b* set forth in Accession No. MIMAT0004698 (SEQ ID NO: 38).

[0103] MicroRNAs are typically processed from pre-miRs (pre-microRNA precursors). Pre-miRs are a set of precursor miRNA molecules transcribed by RNA polymerase III that are efficiently processed into functional miRNAs, for example, when transfected into cultured cells. Pre-miRs can be used to specify the function of their targets by downregulating their expression in "(miRNA) gain-of-function" experiments. Pre-miR designs exist for all known miRNAs listed in the miRNA registry and can be easily designed for any study. MicroRNAs may be administered to cells as is or linked to a nucleic acid construct, as described in more detail below.

[0104] The microRNAs of the present teachings (e.g., miR-135) bind, attach, regulate, process, intervene, increase, stabilize, and / or destabilize any direct or indirect target of the microRNA (e.g., a miR-135 target). Such targets include, but are not limited to, DNA molecules, RNA molecules and polypeptides, such as, for example, serotonin-related genes, such as the serotonin transporter (i.e., SERT or Slc6a4), serotonin inhibitory receptor 1a (Htr1a), tryptophan hydroxylase 2 (Tph2) and monoamine hydroxylase (MaoA), adrenergic or noradrenergic receptors (adrenergic receptors, such as Adr1), adenylate cyclase type 1 (ADCY1), CRH receptors, such as Crh1R, or other molecules such as FK506 binding protein 5 (FKBP5), translin-related protein X (Tsnax) and cell adhesion molecule L1 (L1cam), or other targets associated with psychiatric disorders, as listed in Table 7 below.

[0105] Additional direct or indirect targets include adenylate cyclase-activating polypeptide 1 (Adcyap1 or PACAP); adenylate cyclase-activating polypeptide 1 receptor 1 (Adcyap1r1); adrenergic receptor, alpha 2a (Adra2a); ankyrin 3 (ANK3); activity-regulated cytoskeleton-associated protein (Arc); Rho GTPase-activating protein 6 (Arhgap6); activating transcription factor 3 (Atf3); beta-site APP cleavage enzyme 1 (Bace1); calcium channel, voltage-dependent, L-type, alpha 1D subunit (Cacna1d); cell adhesion molecule 3 (Cadm3); complexin 1 (Cplx1); complexin 2 (Cplx2); CUB and sushi multiple domain 1 (Csmd1); casein kinase 1, gamma 1 (Csnk1g1); doublecortin (Dcx); DIRAS family, GTP-binding RAS-like 2 (Diras2); disc large homolog 2 (Drosophila) (Dlg2); ELK1 (Elk1), a member of the ETS oncogene family; fyn-related kinase (Frk); fucosyltransferase 9 (alpha(1,3) fucosyltransferase) (Fut9); gamma-aminobutyric acid (GABA-A) receptor, subunit beta 2 (Gabrb2); GATA-binding protein Glutamate receptor, ionotropic, kainate 3 (Grik3);G protein-coupled receptor kinase 5 (Grk5);Glycogen synthase kinase-3 beta (GSK3B);Hyperpolarization-activated cyclic nucleotide-gated potassium channel 1 (Hcn1), hyperpolarization-activated cyclic nucleotide-gated K+2 (Hcn2),5-hydroxytryptamine (serotonin) receptor 1A (Htr1a);Inositol monophosphatase (IMPA1), Kalirin, RhoGEF kinase (Kalrn);Medium / small conductance calcium-activated potassium channel, subfamily N, member 3 (KCNN3);Karyopherin alpha 3 (importin alpha 4) (Kpna3);Myelin transcription factor 1-like (Myt1l);nuclear receptor coactivator 2 (Ncoa2); N-Myc downstream regulated gene 4 (Ndrg4); nitric oxide synthase 1 (neuronal) adaptor protein (NOS1AP); nuclear receptor subfamily 3, group C, member 2 (Nr3c2); netrin G1 (Ntng1); nuclear casein kinase and cyclin-dependent kinase substrate 1 (Nucks1); phosphodiesterase 1A, calmodulin-dependent (Pde1a); phosphodiesterase 4A, cAMP-specific (Pde4a); phosphodiesterase 8B (Pde8b); phospholipase C, beta 1 (Plcb1); prolactin receptor (Prlr); RAB1B, member RAS oncogene family (Rab1b); Ras-related protein Rap-2a (Rap2a); retinoid-related orphan receptor beta (Rorb); sirtuin 1 (silent mating type information regulation 2, homolog 1) (Sirt1); solute carrier family 12, (potassium chloride transporter ) member 6 (Slc12a6); solute carrier family 5 (chloride transporter), member 7 (Slc5a7); solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (Slc6a4); trans-acting transcription factor 1 (Sp1); synaptic vesicle glycoprotein 2b (Sv2b); synaptic nuclear envelope 1 (encoding nesprin-1) (Syne1); synaptotagmin I (Syt1); synaptotagmin II (Syt2); synaptotagmin III (Syt3); transforming growth factor, beta receptor II (Tgfbr2); thyroid hormone receptor, beta (Thrb); transient receptor potential-gated cation channel, subfamily C, member 6 (Trpc6); endoplasmic reticulum-associated membrane protein 2 (Vamp2); wingless-related MMTV integration site 3 (Wnt3); and zinc finger, BED domain containing 4 (Zbed4);

[0106] [Table 1-1]

[0107] [Table 1-2]

[0108] "Nucleic acid" as used herein generally refers to a molecule (single- or double-stranded oligomer or polymer) of RNA or its derivatives, mimetics or analogs that contain nucleobases. Nucleobases include, for example, naturally occurring purine or pyrimidine bases found in RNA (e.g., adenine "A", guanidine "G", uracil "U" or cytosine "C"). The term "nucleic acid" includes "oligonucleotides" and "polynucleotides", each of which is a subgenus of the term "nucleic acid". The term "nucleic acid" further includes synthetic polynucleotides and / or oligonucleotides that are derived from synthetic polynucleotides and / or oligonucleotide molecules and that are composed of naturally occurring bases, sugars and covalent internucleoside linkages (e.g., backbones), as well as synthetic polynucleotides and / or oligonucleotides that have non-natural portions that function similarly to the corresponding naturally occurring portions. Such modified or substituted oligonucleotides may be preferred over native forms due to desirable properties, such as improved cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases, as described in more detail below.

[0109] The term "mimetic" or "analog" refers to a molecule that may or may not have a similar structure to a naturally occurring molecule, but has a similar function.

[0110] The synthetic miR-135 molecules of some embodiments of the present invention are double-stranded nucleic acid molecules comprising miR-135b and a complementary strand. Such double-stranded nucleic acid molecules have a structure similar to that of naturally occurring miRNA precursors and can bind to cellular protein complexes and be processed into active mature miRNAs.

[0111] In certain embodiments, miR-135b (also referred to as the "guide strand" or "active strand") comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology or identity to endogenous mature miR-135b. In certain embodiments, miR-135b comprises a sequence comparable (e.g., identical) to endogenous mature miR-135b.

[0112] "Sequence identity" or "identity" as used herein with respect to two nucleic acid or polypeptide sequences refers to residues that are the same in the two sequences when aligned. When percentages of sequence identity are used with reference to proteins, residue positions that are not identical often differ by conservative amino acid substitutions, where an amino acid residue is replaced by another amino acid residue with similar chemical properties (e.g., charge or hydrophobicity), so that the functional properties of the molecule are not altered. When sequences differ by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Means for making such adjustments are well known to those of skill in the art. Typically, this involves scoring conservative substitutions as partial mismatches rather than complete mismatches, thereby increasing the percentage sequence identity. Thus, for example, conservative substitutions are given a score between 0 and 1, where an identical amino acid has a score of 1 and a non-conservative substitution has a score of 0. Conservative substitution scores are calculated, for example, according to the algorithm of Henikoff S and Henikoff JG. [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. USA 1992, 89(22): 10915-9].

[0113] Identity (eg, percent homology) can be determined using any homology comparison software, such as that provided by the National Center for Biotechnology Information (NCBI), using default parameters.

[0114] According to some embodiments of the invention, the identity is a global identity, i.e., identity to the entire length of a nucleic acid sequence of the invention and not to a portion thereof.

[0115] According to some embodiments of the invention, the term "homology" or "homologous" refers to the identity of two or more nucleic acid sequences, or the identity of two or more amino acid sequences, or the identity of an amino acid sequence with one or more nucleic acid sequences.

[0116] According to some embodiments of the invention, the homology is global homology, ie, homology to the entire length of the nucleic acid sequence of the invention and not to a portion thereof.

[0117] The degree of homology or identity between two or more sequences can be determined using a variety of known sequence comparison tools. The following is a non-limiting description of such tools that can be used with some embodiments of the present invention.

[0118] If one is starting with a polynucleotide sequence and wishes to compare it to another polynucleotide sequence, one can use the EMBOSS-6.0.1 Needleman-Wunsch algorithm (available at emboss(dot)sourceforge(dot)net / apps / cvs / emboss / apps / needle(dot)html).

[0119] According to some embodiments, the determination of the degree of homology further requires use of the Smith-Waterman algorithm (for protein-protein or nucleotide-nucleotide comparisons).

[0120] According to some embodiments of the invention, before determining global homology to the target polypeptide or polynucleotide (e.g., 80% global homology to the full length sequence), global homology is determined to a preselected sequence based on local homology to the target polypeptide or polynucleotide (e.g., 60% identity over 60% of the sequence length). For example, homologous sequences are selected using BLAST software with Blastp and tBlastn algorithms as filters in the first stage, and Needle (EMBOSS package) or Frame+ algorithms for alignment in the second stage. Local identity (Blast alignment) is defined with a generous cutoff value of 60% identity over 60% of the sequence length, since it is only used as a filter for the global alignment stage. This particular embodiment (when using local identity) does not use the default filtering of the Blast package (by setting the parameter "-FF"). In the second stage, homologs are defined based on at least 80% global identity to the polypeptide sequence of the core gene.

[0121] In a specific embodiment, miR-135b has the sequence shown in SEQ ID NO:37.

[0122] In certain embodiments, miR-135b has the sequence set forth in SEQ ID NO: 1, 5, 7, 8, 10, 14, 16, 41, 42, 43, 44, 45 or 46.

[0123] In certain embodiments, the complementary strand of a synthetic miR-135 molecule (also referred to herein as the "passenger strand") comprises a sequence having at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% homology to the mature miR-135b sequence.

[0124] In certain embodiments, the complementary strand is 100% complementary to the guide strand, ie, contains a perfectly matched sequence.

[0125] In a specific embodiment, the complementary strand comprises the sequence shown in SEQ ID NO:40.

[0126] In certain embodiments, the complementary strand comprises the sequence shown in SEQ ID NO: 2, 3, 4, 6, 9, 11, 12, 13, 15 or 47.

[0127] In certain embodiments, the nucleic acid sequence of miR-135b (e.g., as set forth in SEQ ID NO:37) and the nucleic acid sequence of the complementary strand (e.g., as set forth in SEQ ID NO:40) are 100% complementary across the entire nucleic acid sequence (e.g., across the entire length of SEQ ID NO:37 and SEQ ID NO:40).

[0128] In certain embodiments, there is no overhang between the guide and passenger strands of the synthetic miR-135 molecules of some embodiments of the present invention.

[0129] In one embodiment, the synthetic miR-135 molecule comprises the nucleic acid sequence of miR-135b as shown in SEQ ID NO:37.

[0130] In one embodiment, the synthetic miR-135 molecule comprises the complementary strand shown in SEQ ID NO:40.

[0131] The length of the synthetic miR-135 molecules of the invention is optionally 100 nucleotides or less, or optionally 90 nucleotides or less, or optionally 80 nucleotides or less, or optionally 70 nucleotides or less, or optionally 60 nucleotides or less, or optionally 55 nucleotides or less, or optionally 50 nucleotides or less.

[0132] In one embodiment, the synthetic miR-135 molecule comprises 46-50 residues.

[0133] In one embodiment, the synthetic miR-135 molecule comprises between 46 and 55 residues.

[0134] In one embodiment, the synthetic miR-135 molecule comprises between 46 and 60 residues.

[0135] In one embodiment, the synthetic miR-135 molecule comprises between 46 and 70 residues.

[0136] In one embodiment, the synthetic miR-135 molecule comprises between 46 and 80 residues.

[0137] In certain embodiments, synthetic miR-135 molecules comprise at least about 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleic acid residues in length.

[0138] In certain embodiments, synthetic miR-135 molecules comprise no more than 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 nucleic acids in length.

[0139] In certain embodiments, a synthetic miR-135 molecule comprises 52 nucleic acids or less.

[0140] In certain embodiments, a synthetic miR-135 molecule comprises 50 nucleic acids or less.

[0141] In certain embodiments, a synthetic miR-135 molecule comprises 48 nucleic acids or less.

[0142] In certain embodiments, the synthetic miR-135 molecule comprises 52 nucleic acids.

[0143] In certain embodiments, the synthetic miR-135 molecule comprises 50 nucleic acids.

[0144] In certain embodiments, the synthetic miR-135 molecule comprises 48 nucleic acids.

[0145] In certain embodiments, the synthetic miR-135 molecule comprises 46 nucleic acids.

[0146] In one embodiment, the synthetic miR-135 molecule does not include a linker region, in which case the miR-135b region and the complementary region (i.e., the guide strand and passenger strand, respectively) are separate and annealed as a duplex.

[0147] In one embodiment, the nucleic acid sequence of miR-135b shown in SEQ ID NO: 37 and the complementary strand shown in SEQ ID NO: 40 are present in separate nucleic acid sequence molecules that form a double-stranded synthetic miR-135 molecule.

[0148] In one embodiment, the nucleic acid sequence of miR-135b set forth in any one of SEQ ID NOs: 1, 5, 7, 8, 10, 14, 16, 41, 42, 43, 44, 45 or 46 and the complementary strand set forth in any one of SEQ ID NOs: 2, 3, 4, 6, 9, 11, 12, 13, 15 or 47 are present in separate nucleic acid sequence molecules that form a double-stranded synthetic miR-135 molecule.

[0149] In one embodiment, the nucleic acid sequence of miR-135b shown in SEQ ID NO: 37 and the complementary strand shown in SEQ ID NO: 40 form a hairpin loop structure.

[0150] In one embodiment, the nucleic acid sequence of miR-135b shown in any one of SEQ ID NOs: 1, 5, 7, 8, 10, 14, 16, 41, 42, 43, 44, 45 or 46 and the complementary strand shown in any one of SEQ ID NOs: 2, 3, 4, 6, 9, 11, 12, 13, 15 or 47 form a hairpin loop structure.

[0151] In one embodiment, the synthetic miR-135 molecule comprises a linker region between the nucleic acid sequence of miR-135b (e.g., the guide strand) and the nucleic acid sequence of the complementary sequence (e.g., the passenger strand). Such a linker region may form a hairpin loop. Thus, the synthetic miR-135 molecule of some embodiments of the invention can form a hairpin loop structure as a result of binding between the miR-135b region and the complementary region within the molecule.

[0152] In one embodiment, the linker region comprises 2 to 30 residues.

[0153] In one embodiment, the linker region comprises at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 residues in length.

[0154] In one embodiment, the linker region is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 residues in length.

[0155] In one embodiment, a synthetic miR-135 molecule may include sequences adjacent to either the 5' or 3' end of miR-135b (eg, the guide strand) and / or a complementary sequence (eg, the passenger strand).

[0156] In one embodiment, there are at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides flanking one or both sides of miR-135b (eg, the guide strand) and / or the complementary sequence (eg, the passenger strand).

[0157] In one embodiment, miR-135b (eg, the guide strand) and / or the complementary sequence (eg, the passenger strand) do not comprise adjacent sequences.

[0158] In one embodiment, the nucleic acid sequence of a miR-135 molecule (eg, miR-135b and / or the complementary strand) contains one or more modifications.

[0159] In one embodiment, the nucleic acid sequence of a miR-135 molecule (eg, of miR-135b and / or its complementary strand) has a single modification compared to the endogenous mature miR-135b sequence or its complementary strand (also referred to as a native miR-135b sequence).

[0160] According to other embodiments, the nucleic acid sequence of a miR-135 molecule (e.g., miR-135b and / or the complementary strand) has 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more modifications compared to the native miR-135b sequence.

[0161] In one embodiment, the nucleic acid sequence of the miR-135 molecule has one to two modifications compared to the native miR-135b sequence.

[0162] In one embodiment, the nucleic acid sequence of the miR-135 molecule has 3-4 modifications compared to the native miR-135b sequence.

[0163] In one embodiment, the nucleic acid sequence of the miR-135 molecule has 5 to 10 modifications compared to the native miR-135b sequence.

[0164] In one embodiment, the nucleic acid sequence of the miR-135 molecule has 10-15 modifications compared to the native miR-135b sequence.

[0165] In one embodiment, the nucleic acid sequence of the miR-135 molecule has between 16 and 26 modifications compared to the native miR-135b sequence.

[0166] Thus, synthetic miR-135 molecules of the invention can be synthesized to contain modifications that impart desired characteristics. For example, modifications can improve stability, thermodynamics of hybridization with target nucleic acids, targeting to specific tissues or cell types, or cell permeability (e.g., by endocytosis-dependent or -independent mechanisms). Modifications can also increase sequence specificity, thereby decreasing off-site targeting.

[0167] In one embodiment, a synthetic miR-135 molecule contains a modification selected from a nucleic acid insertion, deletion, substitution, or point mutation, so long as the molecule retains at least about 90%, 95%, 99% or 100% of its biological activity (e.g., miR-135 silencing activity).

[0168] In one embodiment, the synthetic miR-135 molecule includes at least one base (e.g., nucleobase) modification or substitution. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). "Modified" bases include other synthetic and natural bases, such as 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyluracil and cytosine; 6-azouracil, cytosine, and thymine; 5- ... 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo, especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Additional modified bases include those disclosed in U.S. Pat. No. 3,687,808; Kroschwitz, JI, ed. (1990), "The Concise Encyclopedia Of Polymer Science and Engineering," pages 858-859, John Wiley & Sons; Englisch et al. (1991), "Angewandte Chemie," International Edition, 30, 613; and Sanghvi, YS, "Antisense Research and Applications," Chapter 15, pages 289-302, ST Crooke and B. Lebleu, eds., CRC Press, 1993.Such modified bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase the stability of nucleic acid duplexes by 0.6-1.2°C (Sanghvi, YS et al. (1993), "Antisense Research and Applications," pages 276-278, CRC Press, Boca Raton) and are currently the preferred base substitution, especially when combined with 2'-O-methoxyethyl sugar modifications. Additional base modifications are described in Deleavey and Damha, Chemistry and Biology (2012) 19: 937-954, the contents of which are incorporated herein by reference.

[0169] In one embodiment, the modification is a chemical modification.

[0170] In one embodiment, the synthetic miR-135 molecules of the invention may have chemical modifications at nucleotides in internal (i.e., non-terminal) regions that do not have complementarity to the target nucleic acid. For example, modified nucleotides may be incorporated into regions of the miRNA that form bulges. The modifications may include, for example, a ligand attached to the miRNA via a linker. The modifications may result in, for example, improved pharmacokinetics or stability of the polynucleotide, or improved hybridization properties (e.g., hybridization thermodynamics) of the polynucleotide to the target nucleic acid.

[0171] In some embodiments, the orientation of the modification or ligand incorporated or tethered to the bulge region of the polynucleotide is such that it occupies the space within the bulge region. For example, the modification may include modified bases or sugars on the nucleic acid strand, or ligands that function as intercalators. These are preferably located within the bulge. The intercalators may be aromatic, for example, polycyclic aromatic or heterocyclic aromatic compounds. Polycyclic intercalators may have stacking capabilities and may include systems with two, three, or four fused rings. In some embodiments, the orientation of the modification or ligand incorporated or tethered to the bulge region of the polynucleotide is such that it occupies the space within the bulge region. This orientation facilitates improved hybridization properties or other desired characteristics of the polynucleotide.

[0172] In one embodiment, the synthetic miR-135 molecule may include an aminoglycoside ligand that may allow the polynucleotide to exhibit improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine; galactosylated polylysine; neomycin B; tobramycin; kanamycin A; and acridine conjugates of aminoglycosides, such as neo-N-acridine, neo-S-acridine, neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. The use of an acridine analog may increase sequence specificity. For example, neomycin B has a higher affinity for RNA than DNA, but less sequence specificity. In some embodiments, a guanidine analog (i.e., a guanidinoglycoside) of the aminoglycoside ligand is tethered to the polynucleotide agent. The guanidinoglycoside amine group of the amino acid is substituted for the guanidine group. The attachment of the guanidine analog increases the cell permeability of the polynucleotide.

[0173] To obtain increased nuclease resistance and / or binding affinity to the target, the synthetic miR-135 molecules of the present invention may contain 2'-O-methyl, 2'-fluorine, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino, and / or phosphorothioate linkages. The inclusion of certain nucleobase modifications, such as locked nucleic acids (LNAs) (e.g., nucleic acid analogs in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom), ethylene nucleic acids (ENAs) (e.g., 2'-4'-ethylene bridged nucleic acids), and 2-amino-A, 2-thio (e.g., 2-thio-U), and G-clamp modifications, may also increase binding affinity to the target. The inclusion of pyranose sugars in the oligonucleotide backbone may also reduce cleavage by endonucleases.

[0174] miR-135 molecules can be further modified by including a 3' cationic group or by replacing the terminal nucleoside with a 3'-3' linkage. Alternatively, the 3' terminus can be blocked with an aminoalkyl group (e.g., 3' C5-aminoalkyl dT). Other 3' conjugates can also inhibit 3'-5' exonuclease cleavage. Without being bound by theory, 3' conjugates such as naproxen or ibuprofen may inhibit exonuclease cleavage by structurally blocking the binding of exonucleases to the 3' end of the oligonucleotide. Small alkyl chains, aryl groups, or even heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5'-exonucleases.

[0175] In one embodiment, the 5'-terminus may be blocked with an aminoalkyl group (e.g., a 5'-O-alkylamino substituent). Other 5'-conjugates may inhibit 5'-3' exonuclease cleavage. Without being bound by theory, 5'-conjugates such as naproxen or ibuprofen may inhibit exonuclease cleavage by structurally blocking the binding of exonucleases to the 5'-terminus of the oligonucleotide. Small alkyl chains, aryl groups, or even heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) may block 3'-5'-exonucleases.

[0176] In one embodiment, the synthetic miR-135 molecule contains modifications that improve targeting. Examples of modifications that target oligonucleotide reagents to specific cells include carbohydrate sugars such as galactose, N-acetylgalactosamine, mannose, vitamins such as folic acid, RGD and other ligands such as RGD mimetics, small molecules including naproxen, ibuprofen, or other known protein-binding molecules (described in more detail below).

[0177] In one embodiment, the modification is selected from the group consisting of a sugar modification, a nucleobase modification, and a modification of an internucleotide linkage, as briefly described below.

[0178] Specific examples of synthetic miR-135 molecules useful in this aspect of the invention include those containing modified backbones (e.g., sugar-phosphate backbones) or non-natural internucleoside linkages. Oligonucleotides or polynucleotides with modified backbones include those that retain a phosphorus atom in the backbone, and are described in U.S. Patent Nos. 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; and 5,406,892. Nos. 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,306, 5,550,111, 5,563,253, 5,571,799, 5,587,361, 5,625,050, and 8,017,763, and U.S. Patent Application Publication No. 20100222413, all of which are incorporated herein by reference.

[0179] In one embodiment, the nucleic acid sequence of a miR-135 molecule (eg, miR-135b and / or the complementary strand) comprises a phospho-modified internucleotide linkage at the 5' or 3' end of the nucleotide sequence.

[0180] Exemplary internucleotide linkage modifications include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, alkylphosphonates (including 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (3'-aminophosphoramidates), aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, boranophosphates (including normal 3'-5' linkages, 2'-5' linked analogs of these, and those in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2' and of opposite polarity), boron phosphonates, phosphodiesters, phosphonoacetic acid (PACE), morpholino, peptide nucleic acid (PNA) and threose nucleic acid (TNA). Various salts, mixed salts, and free acids of the above modifications may also be used. Additional internucleotide bond modifications are described in Deleavey and Damha, Chemistry and Biology (2012) 19: 937-954, incorporated herein by reference.

[0181] In certain embodiments, the internucleotide linkage modification comprises phosphorothioate.

[0182] In one embodiment, the synthetic miR-135 molecule contains at least one phosphorothioate linkage modification in the nucleic acid sequence of miR-135b or its complementary strand.

[0183] In one embodiment, a synthetic miR-135 molecule contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more phosphorothioate linkage modifications in the nucleic acid sequence of miR-135b or its complementary strand.

[0184] In one embodiment, a synthetic miR-135 molecule contains a phosphorothioate internucleotide linkage at the 5' or 3' end of the nucleotide sequence (eg, the last nucleotide at the 5' end of a miR-135b nucleic acid sequence).

[0185] In one embodiment, the synthetic miR-135 molecule contains a phosphorothioate between the last two nucleotides at the 3' end of the nucleic acid sequence of miR-135b or its complementary strand.

[0186] In one embodiment, the synthetic miR-135 molecule contains a phosphorothioate between the last two nucleotides at the 5' end of the nucleic acid sequence of miR-135b or its complementary strand.

[0187] In one embodiment, the synthetic miR-135 molecule comprises a phosphorothioate between the last two nucleotides at the 3' end and between the last two nucleotides at the 5' end of the nucleic acid sequence of miR-135b or its complementary strand.

[0188] In one embodiment, the synthetic miR-135 molecule contains phosphorothioates in both the nucleic acid sequence of miR-135b and the nucleic acid sequence of the complementary strand.

[0189] In one embodiment, a synthetic miR-135 molecule contains a boranophosphate at an internucleotide linkage at the 5' or 3' end of the nucleotide sequence (eg, the last nucleotide at the 5' end of a miR-135b nucleic acid sequence).

[0190] In one embodiment, a synthetic miR-135 molecule contains a methylphosphonate at an internucleotide linkage at the 5' or 3' end of the nucleotide sequence (eg, the last nucleotide at the 5' end of a miR-135 nucleic acid sequence).

[0191] In one embodiment, a synthetic miR-135 molecule contains a phosphodiester internucleotide linkage at the 5' or 3' end of the nucleotide sequence (eg, the last nucleotide at the 5' end of a miR-135b nucleic acid sequence).

[0192] In certain embodiments, a synthetic miR-135 molecule comprises a phosphate at an internucleotide linkage at the 5' or 3' end of the nucleotide sequence (eg, the last nucleotide at the 5' end of a miR-135b nucleic acid sequence).

[0193] Alternatively, synthetic miR-135 molecule backbones that do not contain a phosphorus atom have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamic acid backbones; methyleneimino and methylenehydrazino backbones; sulfonic acid and sulfonamide backbones; amide backbones; and those described in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,9 Specification No. 38, Specification No. 5,434,257, Specification No. 5,466,677, Specification No. 5,470,967, Specification No. 5,489,677, Specification No. 5,541,307 Specification, Specification No. 5,561,225, Specification No. 5,596,086, Specification No. 5,602,240, Specification No. 5,610,289, Specification No. 5,602,240, Other backbones containing mixtures of N, O, S and CH2 moieties are disclosed in US Pat. Nos. 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437 and 5,677,439.

[0194] In one embodiment, a synthetic miR-135 molecule contains at least one sugar modification (eg, a ribose modification).

[0195] In one embodiment, a synthetic miR-135 molecule contains at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more sugar modifications (e.g., modifications of the ribose).

[0196] In one embodiment, at least one nucleic acid of a miR-135 molecule (eg, miR-135b and / or the complementary strand) comprises a modification corresponding to the 2-position of the ribose.

[0197] In one embodiment, the sugar modification is at the last nucleotide at the 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0198] In one embodiment, the sugar modification is at the last nucleotide at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0199] In one embodiment, the sugar modifications are at the last nucleotide at the 3' and 5' ends of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0200] In certain embodiments, the sugar modifications are in the first 1-2 nucleotides at the 5' end of the nucleic acid sequence of the miR-135b strand and / or the last 1-3 nucleotides at the 3' end of the nucleic acid sequence of the miR-135b strand.

[0201] In certain embodiments, sugar modifications are in the first two nucleotides at the 5' end of the nucleic acid sequence on the complementary strand and / or the last nucleotide at the 3' end of the nucleic acid sequence on the complementary strand.

[0202] In one embodiment, sugar modifications are present in both the nucleic acid sequence of miR-135b and the nucleic acid sequence of the complementary strand.

[0203] Exemplary sugar modifications include, but are not limited to, 2'-modified nucleotides (e.g., 2'-deoxy, 2'-fluoro (2'-F), 2'-deoxy-2'-fluoro, 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-fluoroarabinooligonucleotide (2'-F-ANA), 2'-O--N-methylacetamide (2'-O-NMA), 2'-NH2) or locked nucleic acids (LNA). Additional sugar modifications are described in Deleavey and Damha, Chemistry and Biology (2012) 19: 937-954, which is incorporated herein by reference.

[0204] In one embodiment, a synthetic miR-135 molecule contains at least one 2'-O-methyl (2'-O-Me) modified nucleotide.

[0205] In one embodiment, a synthetic miR-135 molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more 2'-O-methyl (2'-O-Me) modified nucleotides.

[0206] In one embodiment, all nucleotides of the synthetic miR-135 molecule have a 2'-O-methyl (2'-O-Me) modification.

[0207] In certain embodiments, the 2'-O-methyl (2'-O-Me) modified nucleotides are at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0208] In certain embodiments, the 2'-O-methyl (2'-O-Me) modified nucleotides are at the 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0209] In certain embodiments, the 2'-O-methyl (2'-O-Me) modified nucleotides are in the last two nucleotides at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand and / or the last nucleotide at the 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0210] In one embodiment, a synthetic miR-135 molecule contains at least one 2'-O-methoxyethyl (2'-O-MOE) modified nucleotide.

[0211] In one embodiment, a synthetic miR-135 molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides.

[0212] In certain embodiments, the 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides are at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0213] In certain embodiments, the 2'-O-methoxyethyl (2'-O-MOE) modified nucleotide is at the 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0214] In certain embodiments, the 2'-O-methoxyethyl (2'-O-MOE) modified nucleotides are in the last two nucleotides at the 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand and / or the last nucleotide at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0215] In certain embodiments, the 2'-O-MOE modification is provided at an adenine (A) nucleotide of the miR-135b strand or its complementary strand (eg, at the 3' end of the nucleic acid sequence of the miR-135b strand).

[0216] In certain embodiments, the 2'-O-MOE modification further comprises methylation of the 5' ribose (referred to as 2'-O-MOE-5'-Me).

[0217] In certain embodiments, the 2'-O-MOE-5'-Me modifications are provided at uracil (U) nucleotides of the miR-135b strand or its complementary strand (eg, at the 5' end of the nucleic acid sequence of the miR-135b strand).

[0218] In one embodiment, a synthetic miR-135 molecule contains at least one 2'-fluoro (2'-F) modified nucleotide.

[0219] In one embodiment, a synthetic miR-135 molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more 2'-fluoro (2'-F) modified nucleotides.

[0220] In certain embodiments, the 2'-fluoro (2'-F) modified nucleotides are at the 5' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0221] In certain embodiments, the 2'-fluoro (2'-F) modified nucleotide is not the terminal nucleotide at the 5' or 3' end of the nucleic acid sequence of the miR-135b strand or its complementary strand.

[0222] In one embodiment, the synthetic miR-135 molecule comprises modified internucleotidic linkages and sugar modifications. Thus, the synthetic miR-135 molecules that may be used in the present invention may have both sugar and internucleoside linkage modifications, i.e., nucleotide units in the backbone are replaced by novel groups. The base units are maintained for complementarity with the appropriate polynucleotide target. Examples of such oligonucleotide mimics include peptide nucleic acids (PNAs). By PNA oligonucleotides is meant oligonucleotides in which the sugar-backbone is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The bases are retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. United States patents that teach how to make PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Other backbone modifications that may be used in the present invention are disclosed in U.S. Patent No. 6,303,374.

[0223] In one embodiment, the synthetic miR-135 molecule contains a phospho-modified internucleotide linkage and at least one sugar modification (eg, a 2'-modified nucleotide).

[0224] In one embodiment, a synthetic miR-135 molecule comprises a phospho-modified internucleotide linkage, at least one phosphorothioate-modified internucleotide linkage, and at least one sugar modification (eg, a 2'-modified nucleotide).

[0225] In certain embodiments, the synthetic miR-135 molecule comprises a phosphate at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence and a 2'-O-Me modification at the last nucleotide at the 3' end of the miR-135b nucleic acid sequence.

[0226] In certain embodiments, the synthetic miR-135 molecule comprises a phosphate at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, comprises a 2'-O-Me modification at the last two nucleotides at the 5' end of the miR-135b nucleic acid sequence, and comprises a 2'-O-Me modification at the last nucleotide at the 3' end of the miR-135b nucleic acid sequence.

[0227] In certain embodiments, the synthetic miR-135 molecule comprises a phosphate at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, a 2'-O-MOE-5'-Me modification at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, and a 2'-O-Me modification at the last nucleotide at the 3' end of the miR-135b nucleic acid sequence.

[0228] In certain embodiments, the synthetic miR-135 molecule comprises a phosphate at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, a 2'-O-MOE-5'-Me modification at the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, and a 2'-O-MOE modification at the last two nucleotides at the 3' end of the miR-135b nucleic acid sequence.

[0229] In certain embodiments, the synthetic miR-135 molecule comprises a 2'-O-Me modification at the last nucleotide at the 3' end of the nucleic acid sequence of the complementary strand and a 2'-O-Me modification at the last two nucleotides at the 5' end of the nucleic acid sequence of the complementary strand.

[0230] In one embodiment, any of the above-described synthetic miR-135 molecules further comprises at least one 2'-F modified nucleotide.

[0231] In one embodiment, any of the above-described synthetic miR-135 molecules further comprises at least one phosphorothioate modified internucleotide linkage.

[0232] Exemplary synthetic miR-135b sequences include, but are not limited to, SEQ ID NOs: 1, 5, 7, 8, 10, 14, 16, 41, 42, 43, 44, 45 and 46.

[0233] Exemplary modified complementary sequences include, but are not limited to, SEQ ID NOs: 2, 3, 4, 6, 9, 11, 12, 13, 15 and 47.

[0234] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:10.

[0235] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:16.

[0236] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:41.

[0237] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:42.

[0238] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0239] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0240] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:37, and the nucleic acid sequence of the complementary strand as shown in SEQ ID NO:40.

[0241] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:10, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0242] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:41, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0243] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:42, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0244] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:43, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0245] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b as shown in SEQ ID NO:44, and the nucleic acid sequence of the complementary strand as shown in SEQ ID NO:13.

[0246] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:45, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0247] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:46, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0248] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:16, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:13.

[0249] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:10, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0250] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:41, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0251] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:42, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0252] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:43, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0253] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:44, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0254] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:45, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0255] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:46, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0256] In a specific embodiment, a synthetic miR-135 molecule of the invention has the nucleic acid sequence of miR-135b shown in SEQ ID NO:16, and the nucleic acid sequence of the complementary strand shown in SEQ ID NO:47.

[0257] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:10 and the complementary strand as set forth in SEQ ID NO:13.

[0258] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:41 and the complementary strand as set forth in SEQ ID NO:13.

[0259] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:42 and the complementary strand as set forth in SEQ ID NO:13.

[0260] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:43 and the complementary strand as set forth in SEQ ID NO:13.

[0261] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:44 and the complementary strand as set forth in SEQ ID NO:13.

[0262] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:45 and the complementary strand as set forth in SEQ ID NO:13.

[0263] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:46 and the complementary strand as set forth in SEQ ID NO:13.

[0264] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:16 and the complementary strand as set forth in SEQ ID NO:13.

[0265] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:10 and the complementary strand as set forth in SEQ ID NO:47.

[0266] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:41 and the complementary strand as set forth in SEQ ID NO:47.

[0267] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:42 and the complementary strand as set forth in SEQ ID NO:47.

[0268] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:43 and the complementary strand as set forth in SEQ ID NO:47.

[0269] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:44 and the complementary strand as set forth in SEQ ID NO:47.

[0270] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:45 and the complementary strand as set forth in SEQ ID NO:47.

[0271] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:46 and the complementary strand as set forth in SEQ ID NO:47.

[0272] According to one aspect of the invention, there is provided a composition comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID NO:16 and the complementary strand as set forth in SEQ ID NO:47.

[0273] Synthetic miR-135 molecules of the invention can be constructed by chemical synthesis and / or enzymatic ligation reactions known in the art (described in more detail below). For example, polynucleotides can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides (e.g., phosphorothioate derivatives and acridine substituted nucleotides) designed to increase the biological stability of the molecule or to increase the physical stability of the duplex formed between the polynucleotide and the target nucleic acid (described in more detail below).

[0274] Synthetic miR-135 molecules designed according to the teachings of the present invention can be produced according to any known oligonucleotide synthesis method, including both enzymatic synthesis and solid-phase synthesis.Synthetic oligonucleotides by various different mechanisms are disclosed in, for example, U.S. Patent Nos. 4,659,774, 4,816,571, 5,141,813, 5,264,566, 4,959,463, 5,428,148, 5,554,744, 5,574,146, and 5,602,244, each of which is incorporated herein by reference.

[0275] In one embodiment, chemical synthesis can be accomplished by the diester method, the triester method, the polynucleotide phosphorylase method, and solid phase chemistry, which are described in more detail below.

[0276] Diester Method: The diester method was the first method developed to be available. The basic step is the linking of two appropriately protected deoxynucleotides to form a dideoxynucleotide containing a phosphodiester bond.

[0277] Triester Method: The main difference between the diester and triester methods is that the latter has an extra protecting group on the phosphate atom of the reactants and products. The phosphate protecting group is usually a chlorophenyl group that makes the nucleotide and polynucleotide intermediates soluble in organic solvents. Thus, purification is carried out in chloroform solution. Other improvements of this method include (i) block coupling of trimers and larger oligomers, (ii) extensive use of high performance liquid chromatography in the purification of both intermediates and final products, and (iii) solid phase synthesis.

[0278] Polynucleotide phosphorylase method: This is an enzymatic method of DNA synthesis that can be used to synthesize many useful oligonucleotides. Under controlled conditions, polynucleotide phosphorylase adds mainly single nucleotides to short oligonucleotides. Chromatographic purification allows obtaining the desired single adducts. At least a trimer is required to start the method, and this primer must be obtained by other methods. The polynucleotide phosphorylase method works and has the advantage that the procedure is familiar to most biochemists.

[0279] Solid-phase methods: focus on techniques for the solid-phase synthesis of polypeptides, where the initial nucleotides are attached to a solid-phase support material and the addition of nucleotides one at a time can proceed. All mixing and washing steps are simplified and the procedure is amenable to automation. These syntheses are now routinely performed on automated nucleic acid synthesizers. Substrates and reagents for carrying out solid-phase synthesis are commercially available, for example from Applied Biosystems.

[0280] Phosphoramidite chemistry is currently the most widely used coupling chemistry for oligonucleotide synthesis. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves the activation of nucleoside phosphoramidite monomer precursors with an activating reagent to form activated intermediates, followed by sequential addition of the activated intermediates to a growing oligonucleotide chain (usually terminated at one end to a suitable solid support) to form the oligonucleotide product.

[0281] Recombinant methods: Recombinant methods for producing nucleic acid in cells are widely known to those skilled in the art and can be used when synthetic miR-135 molecules do not contain chemical modifications. These methods include the use of vectors, plasmids, cosmids, and other vehicles for the delivery of nucleic acid to cells, which may be target cells or simply host cells (to produce desired RNA molecules in large quantities). Alternatively, such vehicles can be used in cell-free systems, as long as the reagents for making RNA molecules are present. Such methods include those described in Sambrook, 2003, Sambrook, 2001, and Sambrook, 1989, which are incorporated herein by reference.

[0282] Any method for such synthesis may be used, and the actual synthesis of oligonucleotides is well within the capabilities of one of ordinary skill in the art and can be accomplished using solid-phase chemistry, e.g., cyanoethyl phosphoramidites followed by deprotection, desalting, and purification (e.g., by automated trityl-on or HPLC), according to established methodologies described in Sambrook, J. and Russell, DW (2001), "Molecular Cloning: A Laboratory Manual", Ausubel, RM et al., eds. (1994, 1989), "Current Protocols in Molecular Biology", Volumes I-III, John Wiley & Sons, Baltimore, Maryland, Perbal, B. (1988), "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York, and Gait, MJ, ed. (1984), "Oligonucleotide Synthesis",

[0283] In one embodiment, the synthetic miR-135 molecule is linked to a ligand (also referred to as a moiety) selected to improve the stability, distribution, cellular uptake, crossing the blood-brain barrier (BBB), or targeting of the synthetic miR-135 molecule to cells of interest. Thus, the synthetic miR-135 molecule may be modified to include a non-nucleotide moiety, as described in more detail below.

[0284] In one embodiment of the invention, a conjugated miR-135 molecule is provided.

[0285] As used herein, the term "conjugate" refers to any compound resulting from the covalent attachment of two or more separate compounds. In the present invention, a conjugate refers to a molecule that includes a synthetic miR-135 molecule and a cell targeting moiety covalently attached thereto.

[0286] The term "cell targeting moiety" as used herein refers to any substance that binds to a molecule expressed or presented, preferably specifically expressed or presented (e.g., not expressed / presented in one cell type but expressed / presented at a higher level in another cell) by a target cell of interest. In a particular embodiment, the molecule is a receptor. This binding specificity allows the delivery of a synthetic miR-135 molecule (bound to the cell targeting moiety) to a cell, tissue or organ that expresses or presents the molecule. In this way, the conjugate bearing the cell targeting moiety is specifically directed to the cell when administered to a subject (e.g., a human) or when contacted in vitro with a population of cells of different types.

[0287] The cell targeting moieties of the present invention have a Kd for a target (a molecule expressed or displayed by a target cell of interest, e.g., a receptor) of at least about 10 -4 M, alternatively at least about 10 -5 M, alternatively at least about 10 -6 M, alternatively at least about 10 -7 M, alternatively at least about 10 -8 M, alternatively at least about 10 -9 M, alternatively at least about 10 -10 M, alternatively at least about 10 -11 M, alternatively at least about 10 -12 It can be M or more.

[0288] The term "receptor" refers to a cell-associated protein that binds to a bioactive molecule called a "ligand."

[0289] In one embodiment, the molecule (e.g., a receptor) expressed or displayed on a target cell of interest is expressed in a cell-specific manner (e.g., in central nervous system cells, bone cells, muscle cells, cancer cells, gastrointestinal cells, etc.).

[0290] Receptors that may be targeted by the cell targeting moieties of the invention include 5-hydroxytryptamine receptors (e.g., 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, 5-HT1D, 5-HT6), adenosine receptors (e.g., A1, A2, A2A), adrenoceptor receptors (e.g., alpha1A-adrenoceptor, alpha1B-adrenoceptor, alpha1D-adrenoceptor), angiotensin receptors (e.g., AT2), bombesin receptors (e.g., BB1, BB2, BB3), bradykinin receptors (e.g., B1, B2), calcitonin receptors (e.g., AM1, AMY1, CGRP, CT-R, AM2, AMY3), chemokine receptors (e.g., CXCR4), cholecystokinin receptors (e.g., CCK2), corticotropin-releasing factor receptors (e.g., CRF1, CRF2), dopamine receptors (e.g., D1, D2), endothelin receptors (e.g., Etα, Etβ), ephrin receptors (e.g., EphA1, EphA2, EphA3, EphA4, EphB1, EphB2, EphB3), formyl peptide receptors (e.g., FPR1, FPR2, FPR3), Frizzled receptors (e.g., FZD2, FZD3, FZD4, FZD 5, FZD6, FZD7, FZD8, FZD9, FZD10), galanin receptors (e.g. GAL1, GAL2, GAL3), growth hormone secretagogue receptors (ghrelin) (e.g. GHS-R1a), kisspeptin receptors, melanocortin receptors (e.g. MC1, MC2, MC3, MC4), melatonin receptors (e.g. MT1, MT2), neuropeptide FF / neuropeptide AF receptors (e.g. NPFF1, NPFF2), neuropeptide S receptors (e.g. NPS), neuropeptide W / neuropeptide B receptors (e.g. NPBW2), neuropeptide Y receptors (e.g. Y1, Y2, Y4, Y6, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17, Y18, Y19, Y20, Y21, Y22, Y23, Y24, Y25, Y26, Y27, Y28, Y29, Y30, Y31, Y32, Y33, Y34, Y35, Y36, Y37, Y38, Y39, Y40, Y41, Y42, Y43, Y44, Y45, Y46, Y47, Y48, Y49, Y50, Y51, Y52, Y53, Y54, Y55, Y56, Y57, Y58, Y59, Y60, Y61, Y62, Y63, Y64, Y65, Y666, Y67, Y68, Y70, Y71, Y72, Y73, Y74, Y75, Y76, Y77, Y78, Y79, Y81, Y8 , Y5), neurotensin receptors (e.g. NTS1, NTS2), opioid receptors (e.g. delta, kappa, mu), orexin receptors (e.g. OX1, OX2), peptide P518 receptors (e.g. QRFP), prostanoid receptors, SLC6 neurotransmitter transporter family (e.g. DAT, NET, SERT, GlyT1), somatostatin receptors (e.g. sst1, sst2, sst3, sst4, sst5), tachykinin receptors (e.g. NK1, NK2, NK3), toll-like receptors (e.g. TLR7), vasopressin and oxytocin receptors (e.g. OT,V1A, V1B, V2), VEGF receptors (e.g., VEGFR1, VEGFR2, VEGFR3), and G-protein coupled receptors (GPCRs).

[0291] In one embodiment, the molecule (e.g., receptor) expressed or presented on the target cell of interest is expressed / presented on cells of the central nervous system (CNS), including but not limited to the hypothalamus, brainstem, cortex, cerebellum, striatum, midbrain, hippocampus, glia and / or spinal cord.

[0292] In one embodiment, the molecule (eg, a receptor) is expressed by or presented to a brain cell.

[0293] In one embodiment, the molecule (eg, a receptor) is expressed by a neuronal cell or presented to a brain cell.

[0294] In one embodiment, the molecule (eg, receptor) is expressed or presented on glial cells.

[0295] In one embodiment, the molecule (eg, a receptor) expressed or displayed on a target cell of interest is a neurotransmitter transporter.

[0296] In one embodiment, the second component of the conjugate of the invention is a cell targeting moiety that specifically binds to a neurotransmitter transporter.

[0297] As used herein, the term "neurotransmitter transporter" refers to a protein belonging to a class of membrane transport proteins that are present across the plasma membrane of nerve cells and whose primary function is to transport neurotransmitters across the membrane and further to specific intracellular locations.

[0298] Neurotransmitter transporters that may be targeted by the cell targeting moieties of some embodiments of the present invention include, but are not limited to, uptake vehicles present in the plasma membrane of neurons and glial cells that drive neurotransmitters from the extracellular space into the cell. + Gradient, especially Na + The transport of neurotransmitters depends on the co-transport of neurotransmitters and Na. Two protein families have been identified. The first family includes transporters for GABA, monoamines (e.g., noradrenaline), dopamine, serotonin, and amino acids (e.g., glycine and proline). Common structural components include 12 putative transmembrane α-helical domains, cytoplasmic N- and C-termini, and a large glycosylated extracellular loop separating transmembrane domains 3 and 4. This family of homologous proteins is involved in the transport of neurotransmitters and Na + ions and Cl - They induce energy by cotransporting ions into cells (Na / Cl neurotransmitter transporters). The second family includes transporters of excitatory amino acids such as glutamate. Common structural components include 6-10 transmembrane domains, cytoplasmic N- and C-termini, and glycosylation of the extracellular loops. Excitatory amino acid transporters are Cl-independent and transport intracellular K + May require ions (Na + / K + neurotransmitter transporters) (Liu, Y. et al. (1999) Trends Cell Biol. 9: 356-363).

[0299] Neurotransmitter transporters that may be targeted by the cell targeting moieties of the invention include, but are not limited to, uptake transporters present in the plasma membrane of neurons and glial cells that pump neurotransmitters from the extracellular space into the cell. + Gradient, especially Na +The transport of neurotransmitters depends on the co-transport of neurotransmitters and Na. Two protein families have been identified. The first family includes transporters for GABA, monoamines (e.g., noradrenaline), dopamine, serotonin, and amino acids (e.g., glycine and proline). Common structural components include 12 putative transmembrane α-helical domains, cytoplasmic N- and C-termini, and a large glycosylated extracellular loop separating transmembrane domains 3 and 4. This family of homologous proteins is involved in the transport of neurotransmitters and Na + ions and Cl - They induce energy by cotransporting ions into cells (Na / Cl neurotransmitter transporters). The second family includes transporters of excitatory amino acids such as glutamate. Common structural components include 6-10 transmembrane domains, cytoplasmic N- and C-termini, and extracellular loop glycosylation. Excitatory amino acid transporters are Cl-independent and transport intracellular K + May require ions (Na + / K + neurotransmitter transporters) (Liu, Y. et al. (1999) Trends Cell Biol. 9: 356-363).

[0300] Neurotransmitter transporters that may be targeted by the cell targeting moieties of the invention also include neurotransmitter transporters present in intracellular vesicular membranes, typically synaptic vesicles, whose primary function is to concentrate neurotransmitters from the cytoplasm to the endoplasmic reticulum prior to exocytosis of the vesicular contents during synaptic transmission. +-ATPase is used to drive the transport of neurotransmitters into the endoplasmic reticulum, using an electrochemical gradient across the vesicle membrane. Two families of proteins are involved in the transport of neurotransmitters into the endoplasmic reticulum. The first family primarily uses proton exchange to drive transport into the secretory endoplasmic reticulum and includes monoamine and acetylcholine transporters. For example, monoamine transporters exchange two intraluminal protons for each molecule of cytoplasmic transmitter. The second family includes GABA transporters, which rely on positive charges in synaptic vesicles. The two classes of vesicular transporters show no sequence similarity to each other and have structures that are distinct from cell membrane carriers (Schloss, P. et al. (1994) Curr. Opin. Cell Biol. 6: 595-599, Liu, Y. et al. (1999) Trends Cell Biol. 9: 356-363).

[0301] In one embodiment, types of neurotransmitter transporters that can be targeted by the cell targeting moieties of the present invention include, for example, the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET).

[0302] Dopamine transporter (also called DAT or SLC6A3) refers to a molecule that is an integral membrane protein that transports the neurotransmitter dopamine out of the synaptic cleft and deposits it within the surrounding cell, thereby terminating the neurotransmitter signal.

[0303] Serotonin transporter (also referred to as SERT or SLC6A4) refers to a polypeptide that is an integral membrane protein that transports the neurotransmitter serotonin from the synaptic space to the presynaptic neuron.

[0304] Norepinephrine transporter (also called NET or SLC6A2) refers to a molecule that is a transmembrane protein that transports synaptically released norepinephrine back to the presynaptic neuron.

[0305] Specific types of neurotransmitter transporters that may be targeted by the cell targeting moieties of the present invention include glutamate / aspartate transporters such as excitatory amino acid transporter 1 (EAAT1), excitatory amino acid transporter 2 (EAAT2), excitatory amino acid transporter 3 (EAAT3), excitatory amino acid transporter 4 (EAAT4), excitatory amino acid transporter 5 (EAAT5), vesicular glutamate transporter 1 (VGLUT1), vesicular glutamate transporter 2 (VGLUT2) and vesicular glutamate transporter 3 (VGLUT3); GABA transporter type 1 (GAT1), GABA transporter type 2 (GAT2), GABA transporter type 3 (GAT3), Betaine transporter (BGT1) and vesicular GABA transporter ( GABA transporters such as GABA transporter type 1 (GlyTl) and glycine transporter type 2 (GlyT2); monoamine transporters such as dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), vesicular monoamine transporter 1 (VMAT1) and vesicular monoamine transporter 2 (VMAT2); adenosine transporters such as equilibrative nucleoside transporter 1 (ENT1), equilibrative nucleoside transporter 2 (ENT2), equilibrative nucleoside transporter 3 (ENT3), equilibrative nucleoside transporter 4 (ENT4), and vesicular acetylcholine transporter (VAChT), but are not limited thereto.

[0306] In one embodiment, the conjugate of the invention comprises a cell targeting moiety that specifically binds to a tumor-associated antigen.

[0307] In the present application, the phrase "tumor associated antigen" refers to a protein that is common to a particular hyperproliferative disease (such as cancer) and is produced by tumor cells.

[0308] The types of tumor antigens referred to in the present invention include tumor-specific antigens (TSA) or tumor-associated antigens (TAA). "TSA" refers to a protein or polypeptide antigen that is unique to tumor cells and does not occur in other cells in the body. "TAA" refers to a protein or polypeptide antigen that is expressed by tumor cells. For example, TAA can be one or more surface proteins or polypeptides, nuclear proteins or glycoproteins, or fragments thereof, of tumor cells.

[0309] In one embodiment, the tumor-associated antigen is associated with a solid tumor (eg, colon carcinoma, breast carcinoma, prostate carcinoma, renal cell carcinoma (RCC), lung carcinoma, sarcoma, or melanoma).

[0310] In one embodiment, the tumor associated antigen is associated with a hematological malignancy.

[0311] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, overexpressed embryonic antigens such as CEA, overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu, unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A~PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.291 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA~50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA~90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.Further examples of tumor antigens include, but are not limited to, A33, BAGE, Bcl-2, beta-catenin, CA125, CA19-9, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEAc-Met, CS-1, cyclin B1, DAGE, EBNA, EGFR, ephrin B2, estrogen receptor, FAP, ferritin, folate binding protein, GAGE, G250, GD-2, GM2, gp75, gp100 (Pmel 17), HER-2 / neu, HPV E6, HPV E7, Ki-67, LRP, mesothelin, p53, and PRAME. Further tumor antigens are described in van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancer Immun (2013), www(dot)cancerimmunity(dot)org / peptide / , which is incorporated herein by reference.

[0312] In one embodiment, a conjugate of the invention comprises a cell targeting moiety that specifically binds to a molecule (eg, a receptor) expressed or presented on bone cells.

[0313] In one embodiment, the cell targeting moiety targets the skeletal system.

[0314] In one embodiment, the cell targeting moiety is directed to a particular bone cell type (eg, osteoblasts, osteocytes, osteoclasts, osteocyte precursors, osteoclast precursors, or bone lining cells).

[0315] Exemplary bone cell targets that can be targeted by the cell targeting moieties of the present invention include, but are not limited to, hydroxyapatite (HA), osteocalcin, bone sialoprotein, type I collagen, bone alkaline phosphatase, dentin matrix protein 1, and sclerostin.

[0316] In one embodiment, a conjugate of the invention comprises a cell targeting moiety that specifically binds to a molecule (eg, a receptor) expressed or displayed on muscle cells.

[0317] Exemplary muscle cell molecules that may be targeted by the cell targeting moieties of the invention include, but are not limited to, M-cadherin / cadherin-15, caveolin-1, ABCG2, and myogenin.

[0318] In one embodiment, a conjugate of the invention comprises a cell targeting moiety that specifically binds to a molecule (eg, a receptor) expressed or displayed by gastrointestinal cells.

[0319] Exemplary gastrointestinal cell molecules that can be targeted by the cell targeting moieties of the invention include, but are not limited to, the serotonin transporter or serotonin receptors 1-7.

[0320] The choice of cell targeting moiety of the present invention will depend on the particular type of disease being treated (eg, psychiatric disorder, cancer, bone disease, etc.).

[0321] In one embodiment, the cell targeting moiety is a small molecule.

[0322] In one embodiment, cell targeting moiety is small drug.For example, the exemplary small drug that can be used to target 5-HT neuron and post-synaptic neuron includes but is not limited to the ligand of 5-HT1a receptor [11C]DASB, [11C]WAY100635 or [18F]MPPF.

[0323] In one embodiment, the cell targeting moiety is a synthetic moiety.

[0324] In one embodiment, the cell targeting moiety is a nanoparticle capable of binding to an antigen, receptor or other protein, or nonproteinaceous membrane compound of the target cell.

[0325] In one embodiment, the cell targeting moiety is an affinity binding moiety, i.e., a naturally occurring or artificially produced molecule or composition that binds to a specific molecule (e.g., an antigen) with higher affinity than to a non-specific molecule (e.g., an antigen).

[0326] Affinity can be quantified by known methods, such as surface plasmon resonance (SPR) (described in Scarano S, Mascini M, Turner AP, Minunni M. Surface plasmon resonance imaging for affinity-based biosensors. Biosens Bioelectron. 2010, 25: 957-66), for example in the form of a captured or immobilized monoclonal antibody (MAb) to minimize the effects of binding activity, and it should be noted that the dissociation constant Kd can be calculated using, for example, a low Kd reflecting a high affinity. Affinity binding moieties typically have a binding affinity (K) of at least about 2 to about 200 M (i.e., as long as the binding is specific (i.e., there is no background binding)). D In certain embodiments, the affinity binding moiety is an aptamer or a lectin.

[0327] In certain embodiments, the affinity binding moiety is an antibody or an antibody fragment.

[0328] The term "antibody" as used in the present invention includes intact molecules and functional fragments thereof, such as Fab, Fab', F(ab')2, Fv, linear antibodies, scFv antibodies, multispecific antibodies formed from antibody fragments capable of binding to antigens, etc. These functional antibody fragments are defined as follows: (1) Fab: A fragment containing a monovalent antigen-binding fragment of an antibody molecule, which can be obtained by digesting a whole antibody with the enzyme papain to produce an intact light chain and one part of a heavy chain. (2) Fab': A fragment of an antibody molecule that can be obtained by treating a whole antibody with pepsin followed by reduction to produce an intact light chain and a part of a heavy chain. Two Fab' fragments are obtained per antibody molecule. (3) (Fab')2: A fragment of an antibody that can be obtained by treating a whole antibody with the enzyme pepsin followed by no reduction. F(ab')2 is a dimer of two Fab' fragments linked by two disulfide bonds. (4) Fv: defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. (5) Single-chain antibody ("SCA"): a genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain linked by a suitable polypeptide linker as a genetically fused single-chain molecule. (6) CDR peptide: a peptide encoding a single complementarity determining region (CDR). (7) Single-domain antibody (also called nanobody): a genetically engineered single monomeric variable antibody domain that selectively binds to a specific antigen. Nanobodies have a molecular weight of only 12-15 kDa, much smaller than conventional antibodies (150-160 kDa).

[0329] In one embodiment, the cell targeting moiety binds to a neurotransmitter transporter.

[0330] In one embodiment, the cell targeting moiety that specifically binds to a neurotransmitter transporter is a serotonin reuptake inhibitor (SRI), a selective serotonin reuptake inhibitor (SSRI), a serotonin-norepinephrine reuptake inhibitor (SNRI), a noradrenergic-specific serotonergic antidepressant (NASSA), a noradrenaline reuptake inhibitor (NRI), a dopamine reuptake inhibitor (DRI), an endocannabinoid reuptake inhibitor (eCBRI), an adenosine reuptake inhibitor (Ad oRI), excitatory amino acid reuptake inhibitors (EAARI), glutamate reuptake inhibitors (GluRI), GABA reuptake inhibitors (GRI), glycine reuptake inhibitors (GlyRI), norepinephrine-dopamine reuptake inhibitors (NDRI), triple reuptake inhibitors, noradrenaline-dopamine double reuptake inhibitors, serotonin single reuptake inhibitors, noradrenaline single reuptake inhibitors, and dopamine single reuptake inhibitors.

[0331] The term "serotonin reuptake inhibitor" or "SRI" refers to a molecule capable of blocking the uptake of serotonin and includes both selective serotonin reuptake inhibitors (SSRIs) (which specifically block serotonin uptake without affecting other neurotransmitters) as well as non-selective serotonin reuptake inhibitors, such as serotonin-norepinephrine reuptake inhibitors (SNRIs) and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRIs).

[0332] The term "serotonin selective reuptake inhibitor" or "SSRI" refers to a selective inhibitor of serotonin reuptake without substantially affecting other neurotransmitter reuptake or transporter systems. Such compounds act primarily on presynaptic serotonergic cells, resulting in an increase in the extracellular levels of the neurotransmitter serotonin, thereby increasing the levels of serotonin available to bind to presynaptic receptors and reversing the underactivity of this monoamine neurotransmitter system in the brain. Non-limiting examples of SSRIs include, but are not limited to, sertraline (CAS 79617-96-2), sertraline structural analogs, fluoxetine (CAS 54910-89-3), fluvoxamine (CAS 54739-18-3), paroxetine (CAS 61869-08-7), indalpine (CAS 63758-79-2), zimeldine (CAS 56775-88-3), citalopram (CAS 59729-33-8), and escitalopram (CAS 219861-08-2). Whether a compound acts as an SSRI can be determined by any method known in the art, including the rat cardiac intravenous [ 3 3H] norepinephrine uptake without affecting its uptake and the ability to antagonize the serotonin depleting activity of p-chloroamphetamine.

[0333] In certain embodiments, the SSRI is sertraline, or a structural analog thereof, having the following structure (Formula I): [ka] In the formula, R l、R2, R3, R4, R5, and R6 are each independently hydrogen or an optionally substituted C1-C6 alkyl; X and Y are each selected from the group consisting of hydrogen, fluoro, chloro, bromo, trifluoromethyl, C1-C3 alkoxy, and cyano; and W is selected from the group consisting of hydrogen, fluoro, chloro, bromo, trifluoromethyl, nitro, and C1-C3 alkoxy. In some embodiments, the sertraline analog is in the cis isomeric form. The term "cis isomer" refers to the relative arrangement of the NR1R2 and phenyl moieties on the cyclohexene ring (i.e., both are located on the same side of the ring). Because both the 1- and 4-position carbons are asymmetrically substituted, each cis compound has two optically active enantiomeric forms designated (with reference to the I-carbon) as the cis-(lR) enantiomer and the cis-(lS) enantiomer.

[0334] Some useful sertraline analogs are the following compounds and their pharma- ceutically acceptable salts, which are in either the (IS)-enantiomeric form or the (1S)(1R) racemic form: ·cis-N-methyl-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-l-naphthalenamine; cis-N-methyl-4-(4-bromophenyl)-l,2,3,4-tetrahydro-l-naphthalenamine; cis-N-methyl-4-(4-chlorophenyl)-l,2,3,4-tetrahydro-l-naphthalenamine; ·cis-N-methyl-4-(3-trifluoromethyl-phenyl)-l,2,3,4-tetrahydro-l-naphthalenamine; cis-N-methyl-4-(3-trifluoromethyl-4-chlorophenyl)-1,2,3,4-tetrahydro-1-naphthalenamine; ·cis-N,N-dimethyl-4-(4-chlorophenyl)-1,2,3,4-tetrahydro-l-naphthalenamine; cis-N,N-dimethyl-4-(3-trifluoromethyl-phenyl)-l,2,3,4-tetrahydro-l-naphthalenamine; and · cis-N-methyl-4-(4-chlorophenyl)-7-chloro-l,2,3,4-tetrahydro-l-naphthalenamine.

[0335] Of further note is the (lR)-enantiomer of cis-N-methyl-4-(3,4-dichlorophenyl)-l,2,3,4-tetrahydro-l-naphthalenamine.

[0336] Sertraline analogs are also described in U.S. Patent No. 4,536,518, which is incorporated herein by reference. Other related compounds include (S,S)-N-desmethylsertraline, rac-cis-N-desmethylsertraline, (1S,4S)-desmethylsertraline, 1-des(methylamine)-1-oxo-2-(R,S)-hydroxysertraline, (1R,4R)-desmethylsertraline, sertraline sulfonamide, sertraline (reverse) methanesulfonamide, 1R,4R sertraline enantiomer, N,N-dimethylsertraline, nitrosertraline, sertraline aniline, sertraline iodide, sertraline sulfonamide NH2 ... Sertraline sulfonamide ethanol, sertraline nitrile, sertraline-CME, dimethylsertraline reverse sulfonamide, sertraline reverse sulfonamide (CH2 linker), sertraline B-ring ortho methoxy, sertraline A-ring methyl ester, sertraline A-ring ethanol, sertraline N,N dimethylsulfonamide, sertraline A-ring carboxylic acid, sertraline B-ring paraphenoxy, sertraline B-ring para-trifluoromethane, N,N-dimethylsertraline B-ring and para-trifluoromethane, and UK-416244. The structures of these analogs are shown below.

[0337] [Table 2-1]

[0338] [Table 2-2]

[0339] [Table 2-3]

[0340] [Table 2-4]

[0341] [Table 2-5]

[0342] [Table 2-6]

[0343] The term "serotonin-norepinephrine reuptake inhibitors" or "SNRIs" refers to a family of compounds that inhibit the reuptake of serotonin by blocking the serotonin transporter and inhibit the reuptake of norepinephrine by blocking the norepinephrine transporter, including, but not limited to, compounds such as venlafaxine (CAS 93413-69-5), desvenlafaxine (CAS 93413-62-8), duloxetine (CAS 116539-59-4), milnacipran (CAS 92623-85-3), sibutramine (106650-56-0), tramadol (CAS 27203-92-5), and bicifadine (CAS 71195-57-8). Whether a compound acts as an SNRI can be determined by any method known in the art, including, but not limited to, assaying for the ability to inhibit the uptake of serotonin and norepinephrine by brain synaptosomes, essentially as described in Bolden-Watson C, Richelson E. (Life Sci. 1993;52(12):1023-9).

[0344] In one embodiment, the SNRI is a tricyclic antidepressant, which is an SNRI with a basic molecular structure that includes three rings. Prominent among the tricyclic antidepressants are linear tricyclics, such as imipramine, desipramine, amitriptyline, nortriptyline, protriptyline, doxepin, ketipramine, mianserin, dothiepin, amoxapine, dibenzepin, melitrestone, maprotiline, flupentixol, azaphen, tianeptine, and related compounds that exhibit similar activity. Angular tricyclics include indoliline, clodazon, nomifensine, and related compounds. Various other structurally different antidepressants, such as iprindole, wellbutrin, nialamide, milnacipran, phenelzine, and tranylcypromine, have been shown to produce similar activity. These are functional equivalents of tricyclic antidepressants and are therefore intended to be within the scope of the present invention. Thus, the term tricyclic antidepressant is intended by the inventors to encompass the broad range of antidepressants mentioned above, as well as related compounds which all share the common property of having antidepressant activity, including, but not limited to, amitriptyline, amitriptyline oxide, carbamazepine, butyriptiline, clomipramine, demexiptyline, dibenzepine, dimethacrine, dosulepin / dothiepin, doxepin, imipramine, imipramine oxide, iprindole, lofepramine, melitracen, metapramine, nitroxazepine, nortriptyline, noxiptyline, pregabalin, propzepine, protriptyline, quinupramine, and trimipramine.

[0345] The terms "noradrenaline reuptake inhibitors", "NRIs", "NERIs", "adrenergic reuptake inhibitors" or "ARIs" refer to a family of compounds that block the action of the norepinephrine transporter (NET) to block the reuptake of noradrenaline and adrenaline. This family of compounds includes not only selective NRIs, which block only the NET without affecting other monoamine transporters, but also nonselective NRIs, such as SNRIs (see above), which block the norepinephrine and serotonin transporters, norepinephrine-dopamine reuptake inhibitors (NDRIs) (see below), which block the norepinephrine and dopamine transporters, and tricyclic and tetracyclic antidepressants (see above). Selective NRIs suitable for the present invention include, but are not limited to, atomoxetine / tomoxetine (Strattera® or CAS 83015-26-3), mazindol (Mazanor®, Sanorex® or CAS 22232-71-9), reboxetine (Edronax®, Vestra® or CAS 98819-76-2) and viloxazine (Vivalan® or CAS 46817-91-8).

[0346] The term "dopamine reuptake inhibitor" or "DRI" refers to an agent that acts as a reuptake inhibitor of the neurotransmitter dopamine by blocking the action of the dopamine transporter (DAT). This in turn results in an increase in the extracellular concentration of dopamine, and therefore, an increase in dopaminergic neurotransmission. Suitable DRIs include medications such as amineptine, benztropine / benzotropine, bupropion, dexmethylphenidate, esketamine, ethybenztropine / Ethybe, ponalid, fencamfamine, fencamine, ketamine, lefetamine, medifoxamine, mesocarb, methylphenidate, nefopam, nomifensine, pipradrol, prolintane, pyrovalerone, tiletamine, and tripelennamine, as well as other agents. Examples of suitable DRIs include, but are not limited to, research compounds such as altropane, amfonelic acid, benocyclidine, brasofensine, bromantan, DBL-583, diclopropane, diclofensine, dieticyclidine, difluoropine, gacyclidine, GBR-12,935, indatraline, isofulpane, iometopane, manifaxine, ladafaxine, tametraline, tesofensine, troparil, and vanoxerine. Suitable DRIs can be identified by methods known to those skilled in the art, for example, by determining the inhibitory ability of a putative DRI against high affinity dopamine uptake by synaptosomal preparations from rat striatum, according to the method published by Kula et al. (Life Sciences 34: 2567-2575, 1984).

[0347] The term "endocannabinoid reuptake inhibitor" or "eCBRI" as used herein refers to any compound that functions as an endocannabidonoid reuptake inhibitor by blocking the action of the endocannabidonoid transporter. Compounds with this activity can be identified using the methods described in Beltramo, M. et al. (Science, 1997, 277: 1094-1097) based on the ability of putative endocannabinoid reuptake inhibitors to block anandamide uptake by rat neurons and astrocytes, including, but not limited to, AM404, arvanil, and olvanil.

[0348] The term "adenosine reuptake inhibitor" or "AdoRI" refers to compounds that act as reuptake inhibitors for purine nucleosides and the neurotransmitter adenosine by blocking the activity of one or more equilibrative nucleoside transporters (ENTs). This action results in increased extracellular concentrations of adenosine and therefore increased adenosine neurotransmission. Compounds with AdoRI activity can be identified not only in in vitro assays based on the ability of putative AdoRIs to inhibit adenosine uptake by red blood cells, but also in in vivo assays based on the ability of putative AdoRIs to inhibit the vasodilatory effects of adenosine and to prevent adenosine-mediated stimulation of collateral blood vessel growth, which can be performed substantially as described in U.S. Pat. No. 6,984,642, which is incorporated herein by reference. Suitable AdoRIs include, but are not limited to, acadesine, acetic acid, barbiturates, benzodiazepines, calcium channel blockers, carbamazepine, carisoprodol, cilostadil, diclobenzaprine, dilazep, dipyridamole, estradiol, ethanol (alcohol), flumazenil, hexobendine, hydroxyzyne, indomethacin, inosine, KF24345, meprobamate, nitrobenzylthioguanosine, nitrobenzylthioinosine, papaverine, pentoxifylline, phenothiazines, phenytoin, progesterone, propentofylline, propofol, puromycin, R75231, RE 102 BS, solfurazine, toyocamycin, tracazolate, and tricyclic antidepressants.

[0349] The term "excitatory amino acid reuptake inhibitor" or "EAARI" refers to a compound that inhibits the reuptake of excitatory amino acids by blocking excitatory amino acid transporters or EATs. Many compounds are known that bind to EAATs and inhibit transporter function. Inhibitors of EAATs are divided into two classes with different mechanisms of action: non-transport blockers and competitive substrates. Suitable EAARIs include, but are not limited to, DL-threo-beta-benzyloxyaspartic acid, kainite, dihydrokainic acid, 2S4R4MG, threo-P-hydroxyaspartic acid, and L-trans-pyrrolidine-2,4-dicarboxylic acid (t-2,4-PDC). Suitable EEARIs can be identified, for example, using the assay described in Shimamotot et al. (Molecular Pharmacology, 1998, 53: 195-201), which is based on the ability of putative EEARIs to inhibit the uptake of radiolabeled glutamate by Cos-1 cells expressing human excitatory amino acid transporter-1 (EAAT1) or human excitatory amino acid transporter-2 (EEAT2).

[0350] The term "glutamate reuptake inhibitor" or "GluRI" refers to a compound that acts as a reuptake inhibitor for glutamate by blocking the action of one or more glutamate transporters. Suitable inhibitors of glutamate reuptake include, by way of example, any one of the previously known inhibitors including threo-3 hydroxy-DL-aspartic acid (THA), (2S)-transpyrrolidine-2,4-dicarboxylic acid (PDC), aminocaproic acid, and (2S,3S)-3-{3-[4-(trifluoromethyl)benzoylamino]benzyloxy}aspartic acid. Compounds having GluRI activity can be identified, for example, using the assay described by Shimamotot et al. (Molecular Pharmacology, 1998, 53: 195-201), which is based on the ability of putative GluRIs to inhibit the uptake of radiolabeled glutamate by Cos-1 cells expressing human excitatory amino acid transporter 1 (EAAT1) or human excitatory amino acid transporter 2 (EEAT2).

[0351] The term "GABA reuptake inhibitor" or "GRI" refers to compounds that act as reuptake inhibitors for the neurotransmitter gamma-aminobutyric acid (GABA) by blocking the action of the gamma-aminobutyric acid transporter (GAT), resulting in an increase in the extracellular concentration of GABA and therefore an increase in GABAergic neurotransmission. Suitable inhibitors of GABA reuptake include, but are not limited to, adhyperforin (discovered in Hypericum perforatum (St. John's wort)) as described by Borden LA (Eur J Pharmacol. 1994, 269: 219-224), CI-966, delamiciclane (EGIS-3886), guvacine (C10149), hyperforin (discovered in Hypericum perforatum (St. John's wort)), nipecotic acid, NNC05-2090, NNC-71 1, SKF-89976A, SNAP-5114, stiripentol, and tiagabine (gabitiril). Methods for detecting whether a given compound is a GABA reuptake inhibitor are known and are described, for example, in U.S. Patent Nos. 6,906,177, 6,225,115, and 4,383,999, as well as Ali, FE, et al. (J. Med. Chem. 1985, 28, 653-660). These methods typically involve contacting cells with radiolabeled GABA and detecting uptake of GABA in the presence or absence of a candidate compound.

[0352] The term "glycine reuptake inhibitor" or "GlyRI" refers to compounds that act as reuptake inhibitors for the neurotransmitter glycine by blocking the action of glycine transporters (GlyTs), and includes compounds that block the glycine transporter (type 1), i.e., GlyTI, which is involved in the removal of glycine from the synaptic cleft, as well as GlyT2, which is required for the reuptake and reloading of glycine into synaptic vesicles (Gomeza et al, (2003) Curr Opin Drug Discov Devel 6(5): 675-82). Glycine reuptake inhibitors suitable for use in the present invention include GlyTl specific inhibitors, non-limiting examples of which include N-methyl-N-[[(lR,2S)-l,2,3,4-tetrahydro-6-methoxy-l-phenyl-2-naphthalenylmethylglycine (MTHMPNM glycine free base), 4-[3-fluoro-4-propoxyphenyl]-2-naphthalenylmethylglycine, as described in International Publication Nos. WO / 0007978 and WO / 0136423, and the like. phenyl]-spiro[2H-1-benzopyran-2,4'-piperidine]-1'-acetic acid (free base of FPPSBPAA), ALX5407, sarcosine, 5,5-diaryl-2-amino-4-pentenoic acid ester or compounds described in International Publication WO / 0208216, and further GlyT2 specific inhibitors include those described in International Publication WO / 05044810A, the contents of which are incorporated herein by reference. Methods for detecting GlyT1-specific or GlyT2-specific reuptake inhibitors are known in the art, such as those described in International Publication WO / 05018676A or WO / 05044810, in which cells expressing the relevant receptor (GlyT1 or GlyT2) are contacted with radiolabeled glycine in the presence of a compound to be tested for reuptake inhibitory activity, and the amount of glycine found in the cells after a period of time is determined.

[0353] As used herein, the term "norepinephrine-dopamine reuptake inhibitor" or "NDRI" refers to compounds that act as reuptake inhibitors for the neurotransmitters norepinephrine and dopamine by blocking the action of the norepinephrine transporter (NET) and dopamine transporter (DAT), respectively. This action results in increased extracellular concentrations of both norepinephrine and dopamine, which in turn increases adrenergic and dopaminergic neurotransmission. Suitable NDRIs for use in the conjugates of the invention include ameptine (Survector®, Maneon®, Directin®), bupropion (Wellbutrin®, Zyban®), dexmethylphenidate (Focalin®), fencamfamin (Glucoenergan®, Reactivan®), fencamine (Altimina®, Sicoclor®), lefetamine (Santenol®), methylphenidate (Ritalin®, Concerta®), nomifensine (Merital®), pipradrol (Meretran®), prolintane (Promotil®, Katovit®), pyrovalerone (Centroton®, Thymergix®), nefopam (Acupan®), (Hypericum®), These include, but are not limited to, adhyperforin (discovered in Hypericum perforatum (St. John's wort)), hyperforin (discovered in Hypericum perforatum (St. John's wort)), cocaine, desoxypipradrol (2-DPMP), diphenylprolinol (D2PM), methylenedioxypyrovalerone (MDPV), cilobamine, manifaxin (GW-320,659), radafaxin (GW-353,162), and tametraline (CP-24,441).

[0354] In one embodiment, a conjugate of the invention comprises a cell targeting moiety that specifically binds to a neurotransmitter transporter that is a selective serotonin reuptake inhibitor (SSRI).

[0355] In certain embodiments, the conjugates of the invention comprise an SSRI selected from the group consisting of sertraline, sertraline-structural analogs, fluoxetine, fluvoxamine, paroxetine, indalpine, zimelizine, citalopram, dapoxetine, escitalopram, and mixtures thereof.

[0356] In a particular embodiment, the conjugate of the invention comprises an SSRI as defined above, namely sertraline, or a structural analogue thereof.

[0357] In certain embodiments, when the cell targeting moiety targets bone cells, the targeting moiety can include a synthetic moiety, such as a tetracycline or a bisphosphonate (BP).

[0358] The synthetic miR-135 molecule and the cell targeting moiety may be directly linked, or indirectly linked through an intervening moiety, such as a linker, bridge, or spacer moiety.

[0359] In one embodiment, the synthetic miR-135 molecule and the cell targeting moiety are directly linked. Alternatively, according to other embodiments, both moieties may be linked by a linking group.

[0360] The terms "linking group", "linker", "linking group" and their grammatical equivalents refer herein to an organic moiety that connects two parts of a compound. The cell targeting moiety can be attached to any nucleotide of the sense (e.g., guide) or antisense (e.g., passenger) strand within the miR-135 molecule, but is preferably attached via the 3'- and / or 5'-terminal nucleotide. Internal conjugates may be attached directly to the 2'-position of the ribose group or to a nucleotide at any other suitable position, or indirectly via a linker.

[0361] As described above, the synthetic miR-135 molecules of the present invention are preferably double-stranded nucleic acid molecules, such that the conjugate moiety (i.e., the cell targeting moiety) can be attached to the 3'-terminal nucleotide of the guide sequence, the 5'-terminal nucleotide of the guide sequence, the 3'-terminal nucleotide of the passenger sequence, and / or the 5'-terminal nucleotide of the passenger sequence.

[0362] Without wishing to be limited by definition or prior art, in this application, the length of a linker is determined by counting the number of atoms to represent the minimum distance between the atom that connects the conjugate moiety (i.e., the cell targeting moiety) to the linker and the oxygen atom of the terminal phosphate moiety associated with the miR-135 oligonucleotide through which the linker is attached to the miR-135 oligonucleotide. When the linker includes one or more ring structures, it is preferable to count the atoms around the ring that shows the shortest path.

[0363] Linker groups suitable for use in the present invention include, but are not limited to, modified or unmodified nucleotides, nucleosides, polymers, sugars, carbohydrates, polyalkylenes such as polyethylene glycol and polypropylene glycol, polyalcohols, polypropylene, mixtures of ethylene and propylene glycol, polyalkylamines, polyamines such as polylysine and spermidine, polyesters such as poly(ethyl acrylate), polyphosphodiesters, aliphatics and alkylenes. Additionally, linkers / linker chemistries based on omega-amino-1,3-diol, omega-amino-l,2-diol, hydroxyprolinol, omega-aminoalkanol, diethanolamine, omega-hydroxy-l,3-diol, omega-hydroxy-l,2-diol, omega-thio-l,3-diol, omega-thio-1,2-diol, omega-carboxy-l,3-diol, omega-carboxy-1,2-diol, co-hydroxy-alkanol, omega-thio-alkanol, omega-carboxyalkanol, functionalized oligoethylene glycol, allylamine, acrylic acid, allyl alcohol, propaginylamine, propaginyl alcohol, and the like, can be adapted in this context to generate linkers of appropriate length.

[0364] The linker may also have other desirable characteristics, such as improved water solubility, optimal separation distance between the conjugate moiety (i.e., the cell targeting moiety) and the miR-135 molecule, flexibility (or its absence), specific orientation, branching, etc.

[0365] In one embodiment, the linking group has the structure: [ka] During the ceremony, m, n and p are selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13; the sum of m+n+p is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18; k is 0 or 1.

[0366] In one embodiment, p is 5, n is 2, k is 1, and m is 6, resulting in a linker having the structure: [ka]

[0367] In one embodiment, p is 5, n and k are 0, and m is 6, resulting in a linker having the structure: [ka]

[0368] In one embodiment, the linker comprises more than one coupling with the cell targeting moiety, hi a preferred embodiment, the linker is a bivalent or trivalent linker, i.e., capable of coupling two or three molecules of a reagent, respectively.

[0369] When more than one molecule of a cell-targeting moiety is coupled to a miR-135 nucleic acid via a linker, the molecules can represent the same or different cell-targeting moieties.

[0370] In one embodiment, the bivalent or trivalent linker has the formula: [ka] m, m', m", n, n', n", p, p', p", r, r', r", s, s', s", t and u are each independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13; k, k', k" and v are each independently selected from 0 and 1; X 1 , X 2 and X 3 are each independently selected from CH2, O, S, NH, CO, C(O)O, and C(O)NH.

[0371] Depending on the valence of the groups, the branched linker may be symmetrical or asymmetrical.

[0372] In certain embodiments, the linker is a bivalent linker described above where p and p' are 5, n and n' are 2, k and k' are 1, and m and m' are 6. In certain embodiments, the linker is a bivalent linker described above where p and p' are 5, n, n', k and k' are 0, and m and m' are 6.

[0373] In certain embodiments, the linker is selected from the group consisting of r and r' being 4, s and s' being 1, t and v being 0, and X 1 and X 2 represents C(O)NH. In another embodiment, the linker is a bivalent linker as described above, where r is 2, r' is 0, s is 1, s' is 0, t and v are 0, and X 1 and X 2 is a bivalent linker representing CH2.

[0374] In certain embodiments, the linker is selected from the group consisting of p and p'=5, n and n'=2, k and k'=1, m and m'=6, r and r'=4, s and s'=1, t and v=0, and X 1 and X 2 is a bivalent linker representing C(O)NH.

[0375] In another embodiment, the linker is selected from the group consisting of p and p'=5, n and n'=2, k and k'=1, m and m'=6, r=2, r'=0, s=1, s'=0, t and v=0, and X 1 and X 2 is a bivalent linker representing CH2.

[0376] In another embodiment, the linker is selected from the group consisting of p and p', n, n', k and k', which are 0, m and m', which are 6, r and r', which are 4, s and s', which are 1, t and v, which are 0, and X 1 and X 2 is a bivalent linker representing C(O)NH.

[0377] In another embodiment, the linker is selected from the group consisting of p and p', n, n', k and k', which are 0, and m and m', which are 6, r is 2, r' is 0, s is 1, s' is 0, t and v are 0, and X 1 and X 2 is a bivalent linker representing CH2.

[0378] In certain embodiments, the linker is a trivalent linker as described above where p, p' and p" are 5, n, n' and n" are 2, k, k' and k" are 1, and m, m' and m" are 6. In certain embodiments, the linker is a trivalent linker where p, p' and p" are 5, n, n', n", k, k' and k" are 0, and m, m' and m" are 6.

[0379] In certain embodiments, the linker is selected from the group consisting of r, r′, and r″ being 3, s, s′, and s″ being 1, t being 1, v being 0, and X 1 , X 2 and X 3 represents O. In another embodiment, the linker is a trivalent linker in which r, r', and r" are 3, s, s', and s" are 1, t is 1, u is 3, v is 1, and X 1 , X 2 and X 3 represents O,

[0380] In certain embodiments, the linker is selected from the group consisting of p, p', and p" being 5, n, n', and n" being 2, k, k', and k" being 1, m, m', and m" being 6, r, r', and r" being 3, s, s', and s" being 1, t being 1, v being 0, and X 1 , X 2 and X 3 represents O,

[0381] In another embodiment, the linker is selected from the group consisting of p, p', and p" being 5, n, n', and n" being 2, k, k', and k" being 1, m, m', and m" being 6, r, r', and r" being 3, s, s', and s" being 1, t being 1, u being 3, v being 1, and X 1 , X 2 and X 3represents O,

[0382] In another embodiment, the linker is selected from the group consisting of p, p', and p" being 5; n, n', n", k, k', and k" being 0; m, m', and m" being 6; r, r', and r" being 3; s, s', and s" being 1; t being 1; v being 0; and X 1 , X 2 and X 3 represents O,

[0383] In another embodiment, the linker is selected from the group consisting of p, p', and p" being 5; n, n', n", k, k', and k" being 0; m, m', and m" being 6; r, r', and r" being 3; s, s', and s" being 1; t being 1; u being 3; v being 1; and X 1 , X 2 and X 3 represents O,

[0384] In one embodiment, the linking compound is selected from phosphodiesters, phosphorothioates, carbamates, methylphosphonates, guanidiniums, sulfamates, sulfamides, formacetals, thioformacetals, sulfones, amides, and mixtures thereof.

[0385] In one embodiment, the linking compound is a C10 N-hydroxysuccinic acid ester linker (ie, a C10 linker).

[0386] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0387] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0388] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0389] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0390] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0391] In certain embodiments, the conjugate of the invention has the structure: [ka]

[0392] The conjugates of the present invention can be prepared by techniques known to those skilled in the art. The synthesis of the conjugates may include selective protection and deprotection of functional groups. Suitable protecting groups are known to those skilled in the art. For example, a general description of protecting groups in organic chemistry is provided by Wuts, PGM and Greene TW in Protecting Groups in Organic Synthesis (4th Ed. Wiley-Interscience), and Kocienski PJ in Protecting Groups (3rd Ed. Georg Thieme Verlag).

[0393] For the purposes of the present invention, a "protecting group" is to be understood as a chemical modification introduced at either end of a miR-135 oligonucleotide. Non-limiting examples of 5'-caps include an inverted abasic residue (moiety), 4',5'-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted nucleotide moiety; 3'-2'-inverted abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3'-phosphorothioate; phosphorodithioate; or a bridged or non-bridged methylphosphonate moiety. Further details are described in WO97 / 26270, which is incorporated herein by reference.3'-caps include, for example, 4',5'-methylene nucleotides; 1-(beta-D-erythrofuranosyl) nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5'-amino-alkyl phosphates; l,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotides; L-nucleotides; alpha-nucleotides; modified base nucleotides; phosphorodithioates; threo-pentanol nucleotides. Furanosyl nucleotides; acyclic 3',4'-seco nucleotides; 3,4-dihydroxybutyl nucleotides; 3,5-dihydroxypentyl nucleotides; 5'-5'-inverted nucleotide moieties; 5'-5'-inverted abasic moieties; 5'-phosphoramidates; 5'-phosphorothioates; 1,4-butanediol phosphate; 5'-amino; bridged and / or unbridged 5'-phosphoramidates, phosphorothioates and / or phosphorodithioates, bridged and / or unbridged methylphosphonates and 5'-mercapto moieties. See Beaucage and Iyer, 1993, Tetrahedron 49, 1925, which is incorporated herein by reference.

[0394] In one embodiment, the synthetic miR-135 molecule or conjugate of the invention may be further modified by chemically linking one or more moieties or conjugates to the nucleic acid or protecting groups of miR-135 to increase the activity, cellular distribution or cellular uptake of the miR-135 nucleic acid.

[0395] In one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the present invention further comprises at least one cell membrane permeable moiety. Such moieties include lipid moieties (i.e., naturally occurring or synthetically produced lipids), such as cholesterol moieties (Letsinger et al, Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), choline acids (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers (e.g., beryl-S-tritylthiol (Manoharan et al, Ann. NY Acad. Sci., 1992, 660, 306-309; Manoharan et al, Biorg. Med. Chem. Let., 1993, 3, 2765-2770)), thiocholesterols (Oberhauser et al, Nucl. Acids Res., 1992, 20, 2765-2770), and the like. 533-538), fatty chains (e.g. dodecanediol or undecyl residues (Saison-Behmoaras et al, EMBO J, 1991, 10, 11 11-1118; Kabanov et al, FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54)), phospholipids (e.g. di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H phosphonate (Manoharan et al, Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al, Nucl. Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides and Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmityl moiety (Mishra et al, Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).

[0396] Additional lipid moieties that can be used in accordance with the present invention include, but are not limited to, fatty acids, fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins such as vitamins A, D, E and K, monoglycerides, diglycerides, and phospholipids. In one embodiment, fatty acids include, for example, lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), docosanoic acid (C22), and a hybrid of lithocholic acid and oleylamine (lithocholine oleylamine, C43).

[0397] In certain embodiments, the lipid moiety is palmitoyl.

[0398] In certain embodiments, the lipid moiety is cholesteryl.

[0399] In one embodiment, the lipid moiety is C18-C18 (i.e., C18-phosphodiester-C18). For example, a C18 is provided as a phosphoramidite and added to the 5' end of an oligonucleotide in a coupling reaction, and then a second C18 is added to the previous C18 in the same coupling reaction.

[0400] In one embodiment, the cell membrane penetrating moiety is a peptide or protein.

[0401] In certain embodiments, the cell membrane permeable moiety is an oxytocin peptide or a compound derived therefrom (eg, recombinant or synthetic oxytocin).

[0402] In certain embodiments, the cell membrane permeable moiety is human serum albumin or other plasma protein or a partial peptide thereof.

[0403] In certain embodiments, the cell membrane permeable moiety is a peptide shuttle. Exemplary peptide shuttles include, but are not limited to, angiopep-2, RGV29, and THR.

[0404] In one embodiment, cell membrane permeable moiety is small drug.For example, the exemplary small drug that can be used for targeting 5-HT neuron and post-synaptic neuron includes but is not limited to the ligand of 5-HT1a receptor [11C]DASB, [11C]WAY100635 or [18F]MPPF.

[0405] Alternatively, moieties capable of increasing cellular distribution may be low molecular weight compounds or polypeptides capable of specific movement across biological barriers by the use of receptor-mediated endocytosis using specific transporters present in the biological barrier. A wide array of uptake receptors and vectors and an even larger number of receptor-specific ligands are known in the art. Preferred ligands for receptors mediating endocytosis and / or transcytosis that can be used according to the invention include, for example, the thiamine transporter, the folate receptor, the vitamin B12 receptor, the asialoglycoprotein receptor, the alpha(2,3)-sialoglycoprotein receptor (for example with the FC5 and FC44 nanobodies consisting of llama single domain antibodies (sdAbs) as receptor-specific ligands), the transferrin-1 and -2 receptors, the scavenger receptors (class A or B, type I, type II or III), the scavenger receptors (class A or B, type I, type II or III), the scavenger receptors (class A or B, type I, type II or III), the scavenger receptors (class B ... type B, or CD36 or CD163), low density lipoprotein (LDL) receptor, LDL-related protein 1 receptor (LRP1, type B), LRP2 receptor (also known as megalin or glycoprotein 330), diphtheria toxin receptor (DTR, the membrane-bound precursor of heparin-binding epidermal growth factor-like factor (HB-EGF)), insulin receptor, insulin-like growth factor (IGF) receptor, leptin receptor, substance P receptor, glutathione receptor, glutamate receptor, and mannose 6-phosphate receptor.

[0406] Exemplary ligands that bind to these receptors for use in accordance with the present invention include, for example, ligands selected from the group consisting of lipoprotein lipase (LPL), alpha2-macroglobulin (alpha2M), receptor-associated protein (RAP), lactoferrin, desmoteplus, tissue and urokinase-type plasminogen activator (tPA / uPA), plasminogen activator inhibitor (PAI-I), tPA / uPA:PAI-I complex, melanotransferrin (or P97), thrombospondin 1 and 2, hepatic lipase, factor VIIa / tissue factor pathway inhibitor (TFPI), factor VIIIa, factor IXa, avetal-40, amyloid beta precursor protein (APP), C1 inhibitor, complement C3, apolipoprotein E (apoE), Pseudomonas exotoxin A, CRM66, HIV-ITat protein. , rhinovirus, matrix metalloproteinase 9 (MMP-9), MMP-13 (collagenase-3), sphingolipid activator protein (SAP), pregnancy zone protein, antithrombin III, heparin cofactor II, alpha l-antitrypsin, heat shock protein 96 (HSP-96), platelet-derived growth factor (PDGF), apolipoprotein J (apoJ or clusterin), ABETA bound to apoJ and apoE, aprotinin, angio-pepl, very low density lipoprotein (VLDL), transferrin, insulin, leptin, insulin-like growth factor, epidermal growth factor, lectins, peptidomimetics and / or humanized monoclonal antibodies or receptor-specific peptides, hemoglobin, non-toxic portions of the diphtheria toxin polypeptide chain, all or part of the diphtheria toxin B chain (including DTB-His (Spilsberg et al., 2005, Toxicon., 46(8):900-6), all or part of a non-toxic mutant of diphtheria toxin CRM197, apolipoprotein B, apolipoprotein E (e.g., after binding to a polysorb-80 coating on nanoparticles), vitamin D binding protein, vitamin A / retinol binding protein, vitamin B12 / cobalamin plasma transport protein, glutathione, and transcobalamin-B12.

[0407] In certain embodiments, the conjugates of the invention further comprise a group that facilitates transport of the conjugate across a biological membrane. In one embodiment, the group is amphipathic. Exemplary reagents include, but are not limited to, penetratin, a Tat protein fragment containing 48-60 amino acids, a peptide based on the signal sequence, PVEC, transportan, amphipathic model peptides, Arg9, a recent cell wall penetrating peptide, LL-37, cecropin P1, α-defensin, β-defensin, bactenectin, PR-39, and indolicidin. When the reagent is a peptide, it can be modified to include peptidyl mimetics, invertomers, non-peptide or pseudo-peptide bonds, and D-amino acids can be used. The helical reagent is preferably an alpha helical reagent, and preferably has a lipophilic and lipophobic phase.

[0408] In one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the invention further comprises at least one transport moiety across the BBB.

[0409] In one embodiment, the moiety for transport across the BBB is a peptide or protein.

[0410] In one embodiment, the moiety for transport across the BBB is a neurotropic or neurotoxin-derived peptide or variant thereof.

[0411] Exemplary peptides include, but are not limited to, EPO fusion proteins fused to, for example, the human insulin receptor, anti-transferrin receptor (TfR) monoclonal antibodies, peptidomimetic antibodies with affinity for rabies virus glycoproteins (e.g., chimeric RVG fragment peptides), as disclosed in Razzak et al. Int. J. Mol. Sci. (2019), 20: 3108, which is incorporated herein by reference.

[0412] In one embodiment, the moiety for transport across the BBB is a BBB shuttle (also called a Trojan horse antibody). Exemplary BBB shuttles include Angiopep-2, D-Angiopep-2, ApoB, ApoE, THR, THRre, RVG29, TGN, D Additional BBB shuttles include, but are not limited to, CDX, apamin, TGN, and TAT(47-57). Additional BBB shuttles are disclosed in Oller-Salvia et al., Chem. Soc. Rev. (The Royal Society of Chemistry) 2016, which is incorporated herein by reference.

[0413] In other specific embodiments of the present invention, the conjugate of the present invention may further comprise an endosomolytic ligand. The endosomolytic ligand promotes the lysis of endosomes and / or the transport of the composition of the present invention or its components from endosomes to the cytoplasm of cells. The endosomolytic ligand may be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In certain embodiments, the endosomolytic ligand exhibits an active conformation at endosomal pH. Exemplary endosomolytic ligands include, for example, GAL4 peptides (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptides (Vogel et al., J. Am. Chem. Soc, 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68), INF-7 peptides, Inf HA-2 peptides, diINF-7 peptides, diINF3 peptides, GLF peptides, GALA-INF3 peptides, and INF-5 peptides.

[0414] One skilled in the art can select any of the above ligands or portions thereof (e.g., cell membrane penetrating moieties or moieties for transport across the BBB) taking into consideration the target tissue, the mode of administration to the target cells, the route the oligonucleotide is expected to take, etc.

[0415] In one embodiment, when the ligand or moiety (e.g. a cell membrane penetrating moiety or a moiety for transport across the BBB) is a peptide or peptide product, it can be subjected to in vitro modifications (e.g. PEGylation, lipid modification, etc.) to confer stability (e.g. against protease activity) and / or solubility (e.g. in biological fluids such as blood, digestive juices, etc.) to the amino acid sequence of the peptide, while still maintaining its biological activity and extending its half-life.

[0416] The conjugate of the present invention is typically synthesized by standard procedures of organic synthesis.Those skilled in the art know that the exact steps of synthesis depend on the exact structure of the conjugate that must be synthesized.For example, when the conjugate comprises a single nucleic acid chain that is conjugated to cell targeting moiety via its 5' end, synthesis is usually carried out by contacting amino-activated oligonucleotide with reactive activated cell targeting moiety.

[0417] In one embodiment, when the conjugate comprises a double-stranded miR-135 nucleic acid, the sense strand (e.g., guide strand) and the antisense strand (e.g., passenger strand) are synthesized separately and annealed in vitro using standard molecular biology techniques (described in detail above). In a typical conjugate, the first nucleic acid strand carries a cell targeting moiety and the second nucleic acid strand.

[0418] In one embodiment, the cell targeting moiety is coupled to the 5' end of the first nucleic acid strand and / or the protecting group is attached to the 5' end of the second nucleic acid strand, although attachment of the cell targeting moiety or protecting group can also be performed at the 3' end of the nucleic acid strand.

[0419] In certain embodiments, a cell targeting moiety is coupled to the 5' end of the passenger strand and / or a protecting group is attached to the 5' end of the guide strand.

[0420] It is understood that when the cell targeting moiety is coupled to the 5' or 3' end of the sense strand (e.g., guide strand) or antisense strand (e.g., passenger strand) of the synthetic miR-135 molecule, the cell membrane penetrating moiety and / or the moiety for transport across the BBB may be coupled to either of the remaining ends (5' or 3') of the double-stranded molecule.

[0421] In one embodiment, a cell membrane penetrating moiety and / or a moiety for transport across the BBB may be attached to a cell targeting moiety.

[0422] In certain embodiments, a lipid moiety (eg, cholesterol) is attached to a cell targeting moiety (eg, an SSRI such as sertraline).

[0423] In one embodiment, when the synthetic miR-135 molecule is not conjugated to a cell targeting moiety, the cell membrane penetrating moiety and / or the transport moiety across the BBB may be coupled to either end (5' or 3') of the sense strand (e.g., guide strand) or antisense strand (e.g., passenger strand) of the synthetic miR-135 molecule.

[0424] In one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the invention and the cell membrane penetrating moiety and / or the transport moiety across the BBB may be directly coupled. Alternatively, according to other embodiments, both moieties may be linked by a linking group.

[0425] Linker groups suitable for use in the present invention are described above.

[0426] In certain embodiments, the linker is a palmitoyl-modified linker having the following structure, where the ASO is an oligonucleotide: [ka]

[0427] In certain embodiments, the linker is a phosphodiester unit for conjugating palmitic acid to an oligonucleotide (eg, a phosphodiester (PO)-trinucleotide linker and a hexylamino spacer).

[0428] In certain embodiments, the linker is a cholesterol linker. Exemplary linkers include triethyl glycol [TEG] linkers and 2-aminobutyl-1-3-propanediol [C7] linkers.

[0429] In a particular embodiment, the conjugates of the invention are synthesized by the following steps. (i) Activation of a cell membrane penetrating moiety (e.g., that specifically binds to a neurotransmitter transporter). In certain embodiments, the activating group in the reagent is a succinimide or amino group. (ii) Activation of the 5' end of the passenger strand (or guide strand): In certain embodiments, the activated group in the oligonucleotide is an amino group (when the reagent is activated with a succinimide group) or a carboxyl group (when the reagent is activated with an amine group). (iii) contacting the activated cell membrane-permeable moiety with an activated passenger strand (or an activated guide strand) under conditions suitable for reaction of the two activating groups.

[0430] In one embodiment, the following steps may be further carried out. (iv) Addition of protecting groups to the guide strand (or passenger strand). This step may be performed using oligonucleotides whose reactive groups are blocked by acetylation or benzylation (furanose groups), 2-cyanoethylation (phosphodiester bonds) and FMOC (exocyclic amino groups).

[0431] In one embodiment, the following steps may be further carried out. (v) Annealing of the guide and passenger strands.

[0432] In one embodiment and as described above, when the synthetic miR-135 molecule does not contain chemical modifications, it may be administered to a target cell (e.g., a brain cell, such as a glial cell, an oligodendrocyte, a choroid plexus (CP) cell, a stem cell, or a differentiated stem cell) as part of an expression construct, in which the synthetic miR-135 molecule is linked in the nucleic acid construct under the control of a cis-element (e.g., a promoter) that can direct the expression of the microRNA constitutively or inducibly in the target cell (e.g., a brain cell, such as a glial cell, an oligodendrocyte, a CP cell, a stem cell, or a differentiated stem cell).

[0433] The expression constructs of the present invention may further comprise additional sequences (e.g., shuttle vectors) that optimize replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals). The expression constructs of the present invention may further comprise enhancers adjacent to or distal to the promoter sequence that can function to upregulate transcription therefrom. Polyadenylation sequences can also be added to the expression constructs of the present invention to increase expression efficiency.

[0434] In addition to the embodiments already described, the expression constructs of the present invention may typically contain other specialized elements aimed at increasing the levels of expression of the cloned nucleic acid or to facilitate identification of cells harboring the recombinant DNA.

[0435] An expression construct of the present invention may or may not contain a eukaryotic replicon.

[0436] The nucleic acid construct can be introduced into the target cells of the invention (eg, brain cells, such as glial cells) using an appropriate gene delivery vehicle / method (transfection, transduction, etc.) and an appropriate expression system. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995); Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995); Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et al. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. See also US Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0437] To circumvent the blood-brain barrier, constructs of the invention can be administered directly to the brain (via the ventricles), to the olfactory bulb (by intranasal administration), to the spinal cord (e.g., by an epidural catheter), or expressed in the choroid plexus as described below, with additional modes of administration being discussed in detail below.

[0438] Additionally or alternatively, lipid-based systems may be used to deliver constructs or conjugated miR-135 molecules to target cells of the invention (eg, brain cells, such as glial cells).

[0439] Liposomes include any synthetic (i.e., non-natural) structure that is composed of a lipid bilayer and has an internal volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, etc. Liposomes may be prepared by methods known in the art (Monkkonen, J. et al., 1994, J. Drug Target, 2:299-308; Monkkonen, J. et al., 1993, Calcif. Tissue Int., 53:139-145; Lasic D., Liposome Technology Inc., Elsevier, 1993, 63-105. (chapter 3); Winterhalter M, Lasic DD, Chem Phys Lipids, 1993 September;64(1-3):35-43). MicroRNA (e.g., synthetic miR-135 molecule) can be introduced into liposomes by any method known in the art. For example, microRNA polynucleotide reagent (e.g., synthetic miR-135 molecule) can be encapsulated in liposomes. Alternatively, it can be adsorbed on the surface of liposomes. Other methods for introducing pharmaceutical ingredients into liposomes of the present invention are those described in Alfonso et al. [The science and practice of pharmacy, Mack Publishing, Easton Pa 19th ed., (1995)] and Kulkarni et al. [J. Microencapsul.1995, 12 (3) 229-46].

[0440] The liposomes used in the method of the present invention may cross the blood barrier.Accordingly, according to one embodiment, the liposomes of the present invention do not contain polysaccharides (e.g., mannose) in their membrane portion that target the blood barrier.A screening assay can be carried out to determine the liposomes that are particularly suitable for the present invention, for example, the assay described in US Patent Application Publication Nos. 20040266734 and 20040266734, and Danenberg et al., Journal of cardiovascular pharmacology 2003, 42:671-9; Circulation 2002, 106:599-605; Circulation 2003, 108:2798-804.

[0441] In one embodiment, dual-targeted liposomes are used. Exemplary dual-targeted liposomes include, but are not limited to, angiopep-2-oligoarginine, T7-TAT, THR-transportan, Tf-TAT, Tf-penetratin, or Tf-mastoparan.

[0442] Additionally or alternatively, non-lipid-based vesicles that can be used in this aspect of the invention include exosomes, e.g., modified exosomes such as EVOX, that can cross the BBB and release therapeutic compositions into the brain, e.g., cerebrospinal fluid (CSF).

[0443] Other non-lipid based vesicles that can be used in this aspect of the invention include, but are not limited to, polylysine, dendrimers, and gagomers.

[0444] Regardless of the method or construct used, isolated cells are provided that contain a nucleic acid construct encoding a synthetic miR-135 molecule, as described in detail above, or that contain a synthetic miR-135 molecule or a conjugated form thereof.

[0445] In certain embodiments, the cell is a neuroglial cell (ie, a neuronal cell or a glial cell, such as an oligodendrocyte or an astrocyte).

[0446] In certain embodiments, the neuroglial cells are neuronal cells, such as serotonergic neuronal cells.

[0447] In certain embodiments, the cell is a cancerous cell.

[0448] In certain embodiments, the cells are bone cells.

[0449] In certain embodiments, the cell is a muscle cell.

[0450] In certain embodiments, the cell is a gastrointestinal cell.

[0451] The synthetic miR-135 molecules of the invention are provided to cells, i.e., target cells of the invention (e.g., glial cells), either in vivo (i.e., in an organism or subject) or ex vivo (e.g., in cultured tissue). When cells are treated ex vivo, the method preferably includes a step of returning such cells to the individual (ex vivo cell therapy).

[0452] For ex vivo therapy, cells (e.g., brain cells such as glial cells (e.g., oligodendrocytes), CP cells, stem cells and / or differentiated stem cells) are preferably treated with a composition of the invention (e.g., a synthetic miR-135 molecule or a conjugated form thereof) and then administered to a subject in need thereof.

[0453] Administration of the ex vivo treated cells of the invention can be by any suitable route of introduction, such as, for example, intravenous, intraperitoneal, intrarenal, intragastrointestinal, subcutaneous, transdermal, intramuscular, intradermal, intrathecal, epidural, and rectal, as described in more detail below. In a currently preferred embodiment, the ex vivo treated cells of the invention can be introduced into an individual by intravenous, intrarenal, intragastrointestinal, and / or intraperitoneal administration.

[0454] The cells of the invention (e.g., glial cells such as oligodendrocytes, CP cells, stem cells, differentiated stem cells and / or cardiac cells) can be derived from autologous or allogeneic sources such as human cadavers or donors. Because non-autologous cells tend to induce immune responses when administered to the body, several approaches have been developed to reduce the propensity of non-autologous cells to be rejected. These approaches include either suppressing the recipient's immune system or encapsulating the non-autologous cells in an immunoisolation semi-permeable membrane prior to transplantation.

[0455] Encapsulation techniques are generally classified into microencapsulation, which involves small spherical vesicles, and macroencapsulation, which involves larger, flat sheet and hollow fiber membranes (Uludag, H. et al. (2000). Technology of mammalian cell encapsulation. Adv Drug Deliv Rev 42, 29-64).

[0456] Methods for producing microcapsules are known in the art and are disclosed, for example, in: Lu, MZ et al. (2000). Cell encapsulation with alginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng 70, 479-483; Chang, TM and Prakash, S. (2001) Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol 17, 249-260; and Lu, MZ, et al. (2000). A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). J Microencapsul 17, 245-521.

[0457] For example, microcapsules have been produced using modified collagen complexed with ter-polymer shells of 2-hydroxyethyl methyl acrylate (HEMA), methacrylic acid (MAA), and methyl methacrylate (MMA) to yield capsules 2-5 μm thick. Such microcapsules can be encapsulated in an additional 2-5 μm ter-polymer shell to impart a negatively charged smooth surface and allow for the absorption of plasma proteins (Chia, SM et al. (2002). Multi-layered microcapsules for cell encapsulation. Biomaterials 23, 849-856).

[0458] Other microcapsules are based on the marine polysaccharide alginate (Sambanis, A. (2003). Encapsulated islets in diabetes treatment. Diabetes Thechnol Ther 5, 665-668) or its derivatives. For example, microcapsules can be produced by polyelectrolyte complexation of the polyanions sodium alginate and sodium cellulose sulfate, and the polycation poly(methylene-co-guanidine hydrochloride) in the presence of calcium chloride.

[0459] It should be understood that cell encapsulation can be improved by using smaller capsules. Thus, for example, by reducing the size of capsules from 1 mm to 400 μm, the quality control, diffusion properties, and in vitro activity of encapsulated cells have been improved (Canaple, L. et al. (2002). Improving cell encapsulation through size control. J Biomater Sci Polym Ed 13, 783-96). Furthermore, nanoporous biocapsules with precisely controlled pore sizes down to 7 nm, tailored surface chemistry, and detailed microarchitecture have been found to be successful in immune isolation of cell microenvironments (see Williams, D. (1999). Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol 10, 6-9, and Desai, TA (2002). Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther 2, 633-646).

[0460] Examples of immunosuppressants that may be used in combination with ex vivo therapy include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporine A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazopyrine), gold salts, D-penicillin, leflunomide, azathioprine, anakinra, infliximab (REMICADE®), etanercept, TNFα inhibitors, biologic agents targeting inflammatory cytokines, and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, and tramadol.

[0461] For in vivo therapy, the composition (eg, a synthetic miR-135 molecule or a conjugated form thereof) is administered to a subject either directly or as part of a pharmaceutical composition.

[0462] As used herein, a "pharmaceutical composition" refers to a preparation that consists of one or more active ingredients described herein and other chemical components, such as physiologically suitable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0463] As used herein, the term "active ingredient" refers to a molecule that produces a biological effect (eg, a synthetic miR-135 molecule or a conjugated form thereof).

[0464] The terms "physiologically acceptable carrier" and "pharmacologically acceptable carrier", used interchangeably hereinafter, refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound administered. Such terms include adjuvants.

[0465] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.

[0466] Techniques for drug formulation and administration are described in the latest edition of "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, which is incorporated herein by reference.

[0467] Suitable routes of administration can include, for example, oral, rectal, mucosal, particularly nasal, intranasal, intraocular, intestinal or parenteral delivery, including, for example, intramuscular, subcutaneous and intramedullary injection, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injection.

[0468] Alternatively, the pharmaceutical composition may be administered locally rather than systemically, for example, by injection of the pharmaceutical composition directly into a tissue area of ​​the patient (eg, localized injection into diseased tissue).

[0469] In certain embodiments, the composition is for oral administration.

[0470] Traditional approaches for drug delivery to the central nervous system (CNS) include neurosurgical strategies (e.g., intracerebral injection or intraventricular infusion), molecular engineering of the agent to exploit one of the endogenous transport pathways of the BBB (e.g., generation of synthetic miR-135 molecules linked to cell targeting moieties with affinity for brain cell surface molecules, as described above), pharmacological strategies designed to increase the lipid solubility of the agent (e.g., conjugation of synthetic miR-135 molecules to lipid or cholesterol carriers), and temporary disruption of the integrity of the BBB by hyperosmotic disruption (resulting from infusion of mannitol solutions into the carotid artery or use of bioactive agents such as angiotensin peptides).

[0471] Methods of drug delivery behind the BBB include intracerebral implants (e.g., by needle) and convection-enhanced drug delivery methods. Mannitol can be used to circumvent the BBB. Mucosal (e.g., nasal) administration can also be used to circumvent the BBB.

[0472] In certain embodiments, the composition is for intranasal administration.

[0473] Intranasal administration may be used to deliver therapeutic agents to the central nervous system (CNS). Delivery occurs through the olfactory epithelium, located at the superior posterior part of the nasal cavity. The nerve cells of the olfactory epithelium project to the olfactory bulb in the brain, thus allowing a direct connection between the brain and the external environment. Movement of drugs to the brain is thought to occur either by slower olfactory neuronal transport or by faster movement through the perineural space around the olfactory neuronal cells to the cerebrospinal fluid in the brain. This is a non-invasive administration and is thought to allow larger molecules that cannot cross the BBB to access the CNS. This route of administration reduces systemic exposure and therefore also reduces undesirable systemic side reactions. Delivery from the nose to the CNS typically occurs in minutes and does not require binding to any receptors or axonal transport.

[0474] In certain embodiments, the compositions are for intrathecal (IC), intracerebroventricular (ICV), intraocular or intravenous (IV) administration, allowing the compositions to cross the blood-brain barrier (BBB).

[0475] In one embodiment, the pharmaceutical composition is administered intrathecally, ie, into the spinal canal or into the subarachnoid space, to reach the cerebrospinal fluid (CSF).

[0476] In one embodiment, the pharmaceutical composition is administered in an intraocular dosage form.

[0477] In one embodiment, the pharmaceutical composition is administered in an intracerebroventricular (ICV) form, ie, by injection directly into the cerebrospinal fluid within the ventricles of the brain.

[0478] In one embodiment, the pharmaceutical composition is administered by intravenous (IV) dosage form.

[0479] The pharmaceutical compositions of the present invention can be manufactured by processes that are well known in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, pulverizing, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0480] Thus, pharmaceutical compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliaries, which facilitate processing of the active ingredient into a pharma- ceutically usable preparation. The appropriate formulation depends on the route of administration chosen.

[0481] For injection, the active ingredients of the pharmaceutical composition can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0482] For oral administration, pharmaceutical compositions can be easily formulated by combining the active compounds with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient. Pharmacological preparations for oral use can be made using solid excipients, the resulting mixture can be milled if necessary, and the mixture of granules can be processed to obtain tablets or dragee cores, after adding suitable auxiliaries if necessary. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carbomethylcellulose, and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof (eg, sodium alginate).

[0483] Dragee cores are provided with suitable coatings.For this purpose, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and concentrated sugar solutions, which may contain suitable organic solvents or solvent mixtures as required, can be used.Dyes or pigments can be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0484] Orally available pharmaceutical compositions include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. In the push-fit capsules, the active ingredient may be mixed with a filler such as lactose, a binder such as starch, a lubricant such as talc or magnesium stearate, and, if necessary, a stabilizer. In the soft capsules, the active ingredient may be dissolved or suspended in a suitable liquid, such as fatty oils, liquid paraffin, or liquid polyethylene glycol. Stabilizers may also be added. All formulations for oral administration should be in dosage forms suitable for the chosen route of administration.

[0485] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0486] For administration by nasal inhalation, the active ingredients for use according to the invention are conveniently delivered in the form of an aerosol spray from pressurized packs or nebulizers using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide). In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges, for example of gelatin, for use in a dispenser can be formulated to contain a powder mix of the compound and a suitable powder base (e.g., lactose or starch).

[0487] The pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion. Preparations for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers, optionally with the addition of preservatives. The compositions can be suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0488] Pharmaceutical compositions for parenteral administration include aqueous solutions of water-soluble active agents. Additionally, suspensions of the active ingredients can be prepared as appropriate oil- or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. If necessary, the suspension can also contain suitable stabilizers or agents that increase the solubility of the active ingredients, allowing for the preparation of highly concentrated solutions.

[0489] Alternatively, the active ingredient can be in powder form for constitution extemporaneously with a suitable vehicle, such as a sterile, pyrogen-free, water-based solution.

[0490] Pharmaceutical compositions of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, eg, conventional suppository bases such as cocoa butter or other glycerides.

[0491] Pharmaceutical compositions suitable for use in the context of the present invention include compositions containing an effective amount of active ingredient to achieve its intended purpose. More specifically, a therapeutically effective amount refers to an amount of active ingredient (e.g., a synthetic miR-135 molecule or a conjugated form thereof) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., a CNS-related condition (e.g., a psychiatric disorder such as a mood disorder) or to prolong the survival of the subject being treated.

[0492] According to one embodiment of the present invention, administration of a synthetic miR-135 molecule or its conjugated form has antidepressant and anti-stress effects.

[0493] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0494] For any preparation used in the methods of the present invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in an animal model to achieve a desired concentration or potency. Such information can be used to more accurately determine useful doses in humans.

[0495] The toxicity and therapeutic effects of the active ingredients described herein can be confirmed by standard pharmaceutical procedures in vitro in cell cultures or experimental animals. Data obtained from such in vitro and cell culture assays and animal studies can be used to formulate dosages in the range for use in humans. Dosages can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.1).

[0496] Dosage and interval can be individualized to obtain plasma levels of active ingredient sufficient to induce or inhibit the biological effect (minimal effective concentration, MEC). The MEC varies from preparation to preparation but can be estimated from in vitro data. The dose required to obtain the MEC depends on individual characteristics and route of administration. Plasma concentrations can be confirmed by detection assays.

[0497] Depending on the severity and responsiveness of the condition being treated, dosages may be administered in single or multiple doses, and the course of treatment may last from a few days to a few weeks, or until a cure is effected or symptoms are alleviated.

[0498] The amount of composition administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc. The amount and timing of administration will be dependent on close and continuous monitoring of changes in a particular condition.

[0499] It is understood that there are animal models for testing the synthetic miR-135 molecules of the present invention before using them for human therapy. For example, the learned helplessness model (LH), chronic mild stress (CMS), social defeat stress (SDS) and maternal deprivation and sleep deprivation models of depression, stress, anxiety, etc. may be used. For example, animal models of bipolar disease may use, for example, transgenic mice exhibiting neuronal-specific expression of mutant Polg(D181A) (as taught in Kato et al., Neuroscience and Biobehavioral Reviews (2007) 6 (31):832-842, which is incorporated herein by reference), as well as well-established manic rat models of amphetamine-induced hyperactivity (as taught in, e.g., U.S. Pat. No. 6,555,585) and ketamine-induced hyperactivity (as taught in, e.g., Ghedim et al., Journal of Psychiatric Research (2012) 46: 1569-1575), which are incorporated herein by reference.

[0500] The compositions of the invention may be provided in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient, if desired. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also contain a notice associated with the container in a format prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, reflecting approval by the agency of the composition's form or administration to humans or animals. Such notice may, for example, be labeling approved by the U.S. Food and Drug Administration for prescription drugs, or an approved product insert. Compositions comprising the formulations of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition, as described in more detail above.

[0501] It is understood that the therapeutic composition of the present invention may contain, in addition to the synthetic miR-135 molecule or its conjugated form, known pharmaceuticals for the treatment of CNS-related conditions (e.g., psychiatric disorders such as depression, stress, anxiety, sleep deprivation, etc.). Such pharmaceuticals include, but are not limited to, selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NaSSAs), norepinephrine (noradrenaline) reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors, selective serotonin reuptake enhancers, norepinephrine-dopamine disinhibitors, tricyclic antidepressants (e.g., imipramine), and monoamine oxidase inhibitors (MAOIs). These pharmaceuticals may be included in a single package in the product or in separate packages.

[0502] In one embodiment, the therapeutic compositions of the invention may include, in addition to a synthetic miR-135 molecule or a conjugated form thereof, a pharmaceutical or combination of pharmaceuticals, including, but not limited to, lithium (e.g., lithium carbonate, lithium citrate, lithium sulfate), antipsychotics (e.g., typical and atypical antipsychotics, as described below), mood stabilizers (e.g., valproic acid (VPA, valproate), minerals, anticonvulsants, antipsychotics), and antidepressants.

[0503] Exemplary typical antipsychotics that can be used in accordance with the present teachings include: Low potency medicaments: chlorpromazine (Largactil®, Thorazine®), chlorprothixene (Truxal®), thioridazine (Mellaril®), mesoridazine, and levomepromazine; Medium potency medicaments: loxapine (Loxapac®, Looxitane®), molindone (Moban®), perphenazine (Trilafon®), and thiothixene (Navane®); High potency medicaments: Medications: include, but are not limited to, haloperidol (Haldol®, Serenace®), fluphenazine (Prolixin®), droperidol, zuclopenthixol (Clopixol®), flupenthixol (Depixol®), prochlorperazine and trifluoperazine (Stelazine®). Additionally, prochlorperazine (Compazine®, BucCAS tem®, Stemetil®) and pimozide (Orap®) may be used.

[0504] Exemplary atypical antipsychotics (also referred to as second generation antipsychotics) that can be used in accordance with the present teachings include amisulpride (Solian®), aripiprazole (Abilify®), asenapine (Saphris®), blonanserin (Lonasen®), bitopertine (RG1678), brexpiprazole (OPC-34712), caprylamine (Prazinil®), clocapramine (Clofekton®), clozapine (Clozaril®), cariprazine (RGH-188), iloperidone (Fanapt®), lurasidone (Latuda®), LY2140023, mesylate (Mesylate®), methadone ... These include, but are not limited to, luperone (Buronil®), mosapramine (Cremin®), olanzapine (Zyprexa®), paliperidone (Invega®), perospirone (Lullan®), pimavanserin (ACP-103®), quetiapine (Seroquel®), remoxipride (Roxiam®), risperidone (Risperdal®), sertindole (Serdolect®), sulpiride (Sulpirid®), babicaserin (SCA-136®), ziprasidone (Geodon®), zotepine (Nipolept®), and diclonapine (Lu 31-130).

[0505] Exemplary mood stabilizers that can be used in accordance with the present teachings include, but are not limited to, minerals (e.g., lithium); anticonvulsant mood stabilizers including valproic acid (Depakine®), divalproex sodium (Depakote®), and sodium valproate (Depacon®, Epilim®), lamotrigine (Lamictal®), carbamazepine (Tegretol®), oxcarbazepine (Trileptal®), topiramate (Topamax®), riluzole (Rilutek®), and gabapentin (Neurontin®); antipsychotics (as discussed above); and food additives (e.g., omega-3 fatty acids).

[0506] Exemplary antidepressants that can be used in accordance with the present teachings include selective serotonin reuptake inhibitors (SSRIs such as citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline); serotonin-norepinephrine reuptake inhibitors (SNRIs such as desvenlafaxine, duloxetine, milnacipran, and venlafaxine); noradrenergic and specific serotonergic antidepressants (such as mianserin and mirtazacin); norepinephrine (noradrenaline); These include, but are not limited to, norepinephrine reuptake inhibitors (NRIs such as alomoxetine, mazindol, reboxetine, and viloxazine); norepinephrine-dopamine reuptake inhibitors (such as bupropion); selective serotonin reuptake enhancers (such as tianeptine); norepinephrine-dopamine disinhibitors (NDDIs such as agomelatine); tricyclic antidepressants (including tertiary amine tricyclic antidepressants and secondary amine tricyclic antidepressants); and monoamine oxidase inhibitors (MAOIs).

[0507] In one embodiment, antidepressants include selective serotonin reuptake inhibitors (SSRIs), tricyclic antidepressants and noradrenaline reuptake inhibitors (NRIs).

[0508] In certain embodiments, the antidepressant comprises a selective serotonin reuptake inhibitor (SSRI).

[0509] It is understood that the present teachings can also be combined with additional non-pharmacological treatment measures, including, but not limited to, clinical psychology, psychoconvulsive therapy, involuntary hospitalization, light therapy, psychotherapy, transcranial magnetic stimulation, and cognitive behavioral therapy.

[0510] In another aspect of the invention, there is provided a method for treating a CNS-related condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition or conjugate of some embodiments of the invention, thereby treating the CNS-related condition.

[0511] In another aspect of the invention, there is provided a therapeutically effective amount of some embodiment compositions or conjugates of the invention for treating a CNS-related condition in a subject in need of such treatment.

[0512] The term "treating" means inhibiting or halting the onset of a disease, disorder or condition and / or causing the reduction, remission or regression of a disease, disorder or condition, or preventing the onset of a disease, disorder or condition (i.e., preventing) in a subject who has not yet been diagnosed with the disease, disorder or condition but is at risk for the disease, disorder or condition. One of skill in the art will appreciate that a variety of methodologies and assays are available for assessing the onset of a disease, disorder or condition, and similarly, a variety of methodologies and assays may be used to assess the reduction, remission or regression of a disease, disorder or condition.

[0513] As used herein, the term "subject" or "subject in need of" refers to a mammal, preferably a human, of any age, male or female, suffering from or at risk of developing a pathology.

[0514] As used herein, the phrase "CNS-related condition" or "central nervous system-related condition" includes psychiatric disorders (e.g., panic syndrome or panic attacks, anxiety disorders (e.g., generalized anxiety disorder), phobic disorders of all kinds (e.g., social phobia), mania, manic depression (e.g., bipolar disorder), hypomania, depression of all forms and / or kinds (e.g., unipolar depression), stress disorders, PTSD, somatoform disorders, personality disorders, obsessive-compulsive behaviors, psychoses, and schizophrenia), addiction or substance-related disorders (e.g., drug dependence [e.g., alcohol, stimulants (e.g., crack, cocaine, speech impulses, etc.), and disorders of the nervous system (e.g., encephalopathy ...). These disorders include, for example, psychiatric disorders such as psychiatric disorders ...

[0515] In certain embodiments, the CNS-related condition is a psychiatric disorder.

[0516] In certain embodiments, the psychiatric disorder is a mood disorder.

[0517] Non-limiting examples of mood disorders include, but are not limited to, depression (i.e., depressive disorder), bipolar disorder, substance-induced mood disorder, alcohol-induced mood disorder, benzodiazepine-induced mood disorder, mood disorder due to general health conditions, and other mood disorders. See, e.g., DSM-5 (mentioned above).

[0518] In certain embodiments, the psychiatric disorder is a depressive disorder.

[0519] Non-limiting examples of depressive disorders include, but are not limited to, major depressive disorder (MDD), atypical depression, melancholic depression, psychotic major or psychotic depression, catatonic depression, postpartum depression, seasonal affective disorder (SAD), acute depression, chronic depression (dysthymia), dual depression, depressive disorder not otherwise specified, depressive personality disorder (DPD), recurrent brief depression (RBD), minor depressive disorder (minor depression), premenstrual syndrome, premenstrual dysphoric disorder, depression due to chronic health conditions (e.g., cancer, chronic pain, chemotherapy, chronic stress), and combinations thereof. Various subtypes of depression are described, for example, in the DSM-5.

[0520] In certain embodiments, the psychiatric disorder is bipolar disorder.

[0521] Non-limiting examples of bipolar disorder include, but are not limited to, mania, acute mania, severe mania, hypomania, depression, moderate depression, dysthymia, severe depression, manic and / or depressive episodes, psychotic / psychotic symptoms (e.g., hallucinations, delusions), mixed bipolar state, bipolar disorder type 1 (mania with or without major depression), bipolar disorder type 2 (hypomania with major depression), rapid cycling bipolar disorder, cyclothymia, and / or bipolar disorder not otherwise specified (BD-NOS). See, e.g., DSM-5 (mentioned above). Bipolar disorder is also known as manic depression.

[0522] In certain embodiments, the psychiatric disorder is schizophrenia.

[0523] In one embodiment, schizophrenia refers to a mental disorder involving an individual's escape from reality. The symptoms include the following symptoms for at least part of a month, two months or more: delusions, hallucinations, disorganized speech, severely disorganized or nervous behavior, or negative symptoms (i.e., affective flattening, aphasia, or amotivation). Schizophrenia includes disorders such as, for example, schizoaffective disorder. Diagnosis of schizophrenia is described, for example, in the DSM-5. Types of schizophrenia include, but are not limited to, paranoid, anaphylactic, catatonic, nontypable, and residual. See, for example, the DSM-5 (mentioned above).

[0524] In certain embodiments, the CNS-related condition is an autism spectrum disorder.

[0525] In one embodiment, autism spectrum disorder refers to a spectrum of neurodevelopmental disorders characterized by impaired social interaction and communication disorders with repetitive and stereotyped behaviors. Autism includes a spectrum of social interaction disorders and communication disorders, but the disorders can be broadly classified as "high-functioning autism" or "low-functioning autism" depending on the degree of social interaction and communication impairment. Individuals diagnosed with "high-functioning autism" have low but identifiable social interaction and communication impairments (e.g., Asperger's syndrome). Additional information regarding autism spectrum disorders can be found, e.g., in the DSM-5; Sicile-Kira and Grandin, Autism Spectrum Disorders: The Complete Guide to Understanding Autism, Asperger's Syndrome, Pervasive Developmental Disorder, and Other ASDs, 2004, Perigee Trade; and Duncan et al., Autism Spectrum Disorders [Two Volumes]: A Handbook for Parents and Professionals, 2007, Praeger.

[0526] In certain embodiments, the CNS-related condition is a neuroimmune-based psychiatric disorder, non-limiting examples of which include mood disorders such as depression (e.g., major depressive disorder) and bipolar disorder, schizophrenia, autism spectrum disorder, childhood acute-onset neuropsychiatric syndrome (PANS), and childhood autoimmune neuropsychiatric disorders (PANDAS).

[0527] In another aspect of the invention, there is provided a method for treating a depression-related disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a composition or conjugate of some embodiments of the invention, thereby treating the depression-related disorder.

[0528] In another aspect of the invention, there is provided a therapeutically effective amount of a composition or conjugate of some embodiments of the invention for treating a depression-related disorder in a subject in need of such treatment.

[0529] In one embodiment, the depression-related disorder is selected from the group consisting of major depression, obsessive-compulsive disorder (OCD), pervasive developmental disorder (PDD), post-traumatic stress disorder (PTSD), anxiety disorder, bipolar disorder, eating disorders, and chronic pain.

[0530] In one embodiment, the treatment of a CNS-related condition (e.g., a psychiatric disorder) or a depression-related disorder may be further carried out by administering to the subject an additional agent (or any combination of agents) for the treatment of a CNS-related condition (e.g., a psychiatric disorder) or a depression-related disorder.

[0531] Exemplary agents for the treatment of psychiatric or depression-related disorders that can be used in accordance with the present teachings include, but are not limited to, selective serotonin reuptake inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NaSSA), norepinephrine (noradrenaline) reuptake inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors, selective serotonin reuptake enhancers, norepinephrine-dopamine disinhibitors, tricyclic antidepressants (e.g., imipramine), and monoamine oxidase inhibitors (MAOIs).

[0532] In one embodiment, the treatment of the mental disorder or depression-related disorder may be further performed by administering to the subject an additional drug (or any combination of drugs) for treating the mental disorder or depression-related disorder, including, but not limited to, lithium (e.g., lithium carbonate, lithium citrate, lithium sulfate), antipsychotics (e.g., the typical antipsychotics and typical antipsychotics described above), mood stabilizers (e.g., valproic acid (VPA, valproic acid), minerals, anticonvulsants, antipsychotics), and antidepressants. Additional drugs that may be used in accordance with the present teachings are described in detail above.

[0533] In one embodiment, effective treatment (eg, treatment of a psychiatric disorder such as antidepressant / mood disorder treatment) is determined by obtaining significantly higher miR-135 expression levels after treatment compared to pre-treatment miR-135 expression levels.

[0534] The miR-135 expression level in a subject following treatment may be about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher compared to the subject prior to treatment.

[0535] Monitoring of treatment may further be performed by assessing the patient's health status and, additionally or alternatively, by subjecting the subject to behavioral testing, MRI, or other methods well known to those of skill in the art.

[0536] In another aspect of the invention, there is provided a method for treating a cancerous disease in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition or conjugate of some embodiments of the invention, thereby treating the cancerous disease.

[0537] In another aspect of the invention, there is provided a therapeutically effective amount of a composition or conjugate of some embodiments of the invention for treating a cancerous disease in a subject in need of such treatment.

[0538] Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of cancerous diseases include myeloid leukemias such as chronic myeloid leukemia, acute myeloid leukemia with a tendency to mature, acute promyelocytic leukemia, acute nonlymphocytic leukemia with basophilia, acute monocytic leukemia, and acute myelomonocytic leukemia with eosinophilia; malignant lymphomas such as Burkitt's non-Hodgkin's lymphoma; lymphocytic leukemias such as acute lymphoblastic leukemia and chronic lymphocytic leukemia; solid tumors, benign meningiomas, and mixed tumors of the salivary glands. myeloproliferative disorders such as colorectal adenoma; adenocarcinomas such as small cell lung cancer, kidney, uterine, prostate, bladder, ovarian, colon, sarcoma, liposarcoma, myxoid, periosteal sarcoma, rhabdomyosarcoma (alveolar), extraskeletal myxoid chondrosarcoma, Ewing's tumor; others include, but are not limited to, testicular and ovarian germinopathies, retinoblastoma, Wilms' tumor, neuroblastoma, malignant melanoma, mesothelioma, breast, skin, prostate and ovarian.

[0539] In certain embodiments, the cancer comprises ovarian cancer, colorectal cancer, or prostate cancer.

[0540] In one embodiment, the treatment of the cancerous disease may be further carried out by administering to the subject an additional agent (or any combination of agents) for the treatment of the cancerous disease.

[0541] Such treatments include, but are not limited to, radiation therapy, chemotherapy, biological therapy, such as immunotherapy, or bone marrow transplantation.

[0542] In one embodiment, effective anti-cancer treatment is determined by at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more reduction in tumor volume, or cessation of tumor growth, compared to a subject not treated with a composition of the invention or compared to the pre-treatment state of the same subject treated with the composition.

[0543] Those skilled in the art will appreciate that a variety of methods and assays are available for assessing the efficacy of cancer treatments, such as CT scans, MRI, X-rays, ultrasound, blood tests, and the like.

[0544] The compositions of some embodiments of the present invention may further be used to promote muscle cell differentiation and bone regeneration.

[0545] Thus, a therapeutically effective amount of a composition or conjugate of some embodiments of the invention may be used to treat a bone-related disease or condition or a muscle-related disease or condition in a subject in need of such treatment.

[0546] In one embodiment, there is provided a method for promoting bone regeneration or muscle cell differentiation in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a composition or conjugate of some embodiments of the invention, thereby promoting bone regeneration or muscle cell differentiation.

[0547] Exemplary bone-related diseases or conditions that may be treated according to the present teachings include, but are not limited to, injuries including bone damage, fractures such as closed, open and nonunion fractures; growth failure; osteolytic diseases such as cancer; periodontal disease and defects and other dental repair processes; bone abnormalities or any condition that benefits from the promotion of bone formation, such as senile osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced or disuse osteoporosis, and arthritis, osteoarthritis, etc. The compositions of the present invention are useful in the treatment of congenital, trauma-induced or surgical bone resections (e.g., cancer treatment), and in orthopedics.

[0548] Exemplary muscle-related diseases or conditions that may be treated according to the present teachings include, but are not limited to, muscle degenerative diseases, neuromuscular diseases, spinal muscular atrophy, inflammatory myopathy, and metabolic myopathy. Exemplary muscle diseases include, but are not limited to, muscular dystrophies (e.g., Ducienne muscular dystrophy (DMD), Becker muscular dystrophy, facioscapulohumeral muscular dystrophy, and myotonic dystrophy); amyotrophic lateral sclerosis (ALS), myasthenia gravis, Lambert-Eaton myasthenic syndrome, botulism, and cerebrovascular accidents.

[0549] In one embodiment, effective treatment (e.g., bone regeneration and muscle cell differentiation) is determined by at least about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more increase in bone regeneration (e.g., bone cell mass) or muscle cell differentiation (e.g., muscle cell mass) compared to a subject not treated with a composition of the invention or compared to a pre-treatment state of the same subject receiving treatment.

[0550] One of skill in the art will appreciate that a variety of methods and assays are available for assessing the promotion of bone regeneration and muscle cell differentiation, for example, CT scans, MRI, x-rays, ultrasound, blood tests, and the like.

[0551] Because it is anticipated that numerous related miRNA modifications will be developed during the life of any patent resulting from this application, it is intended that all such new technologies be included a priori within the scope of the term modification.

[0552] As used herein, the term "about" means ±10%.

[0553] The terms "comprises," "comprising," "includes," "including," "having" and conjugations thereof mean "including but not limited to."

[0554] The term "consisting of" means "including and limited to."

[0555] The term "consisting essentially of" means that a composition, method, or structure may include additional ingredients, steps, and / or moieties, provided that the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0556] As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly indicates otherwise. For example, "a compound" or "at least one compound" includes a plurality of compounds, and may also include mixtures thereof.

[0557] Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity, and is not an inflexible limitation of the scope of the invention. Thus, the description of a range should be considered to specifically disclose all of the possible subranges, as well as each individual numerical value within that range. For example, the description of a range such as 1 to 6 is intended to specifically disclose not only the subranges 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., but also each individual numerical value within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the magnitude of the range.

[0558] Whenever a numerical range is given herein, it is intended to include any recited number (fractional or integer) within the range given. The phrases "range between" a first designated number and a second designated number and "range from" a first designated number to a second designated number are used interchangeably herein and are intended to include the first designated number and the second designated number, and all fractional and integer numbers therebetween.

[0559] As used herein, the term "method" means manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, those known to practitioners in the chemical, pharmacological, biological, biochemical, and medical fields or that can be readily developed by practitioners from known manners, means, techniques, and procedures.

[0560] It will be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, may also be provided in a single embodiment in any combination of those features. Conversely, several features of the invention that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or with respect to other described embodiments as appropriate. Certain features described in the context of various embodiments should not be construed as essential to that embodiment, unless the particular embodiment is inoperable without that element.

[0561] As noted above, various embodiments and aspects of the present invention as described and claimed herein find experimental support in the following examples.

[0562] It is understood that any SEQ ID NO disclosed in this application, even if the SEQ ID NO is expressed only in DNA sequence format or RNA sequence format, may refer to either a DNA sequence or an RNA sequence depending on the context in which the SEQ ID NO is referred to. For example, SEQ ID NO: 10 is expressed in RNA sequence format (e.g., uracil is designated as U) but may refer to either an RNA sequence corresponding to the miR-135b nucleic acid sequence or a DNA sequence of the miR-135b molecule nucleic acid sequence. Furthermore, some sequences are expressed in RNA sequence format (e.g., uracil is designated as U) but may refer to either a sequence of an RNA molecule, including dsRNA, or a sequence of a DNA molecule corresponding to the indicated RNA sequence depending on the actual type of molecule being described. In any case, both DNA molecules and RNA molecules having the disclosed sequences are contemplated interchangeably. EXAMPLES

[0563] Reference is now made to the following examples which, together with the above descriptions, illustrate the invention without limiting it.

[0564] In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are fully explained in the literature, see, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York. (1998), the methods described in U.S. Pat. Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994), "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994), Stites et al.(eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are described extensively in the patent and scientific literature, e.g., U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; The disclosures of US Pat. Nos. 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521, "Oligonucleotide "Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, Eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986), "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996), all of which are incorporated herein by reference. Other general references are provided throughout this document. The procedures described therein are believed to be well known in the art and are provided for the convenience of the reader. The information contained therein is incorporated herein by reference in its entirety.

[0565] General materials and experimental procedures Animals and housing conditions Adult C57BL / 6 male mice, 9–11 weeks of age, were used. Mice were housed in a temperature-controlled room (22 ± 1 °C) with a reversed 12-h light / dark cycle. Food and water were available ad libitum. All experimental protocols were performed with male mice and were approved by the Animal Care and Use Committee of the Weizmann Institute of Science.

[0566] Microdissection and RNA preparation Immediately after decapitation, the brains were removed and placed in a 1 mm metal matrix (cat# 51380) (Stoelting Co., Wood Dale, IL). The brains were cut into 1 mm or 2 mm sections with a standard razor blade (GEM, 62-0165) and quickly frozen on dry ice. Brain regions were extracted from the sections removed from the matrix using blunt-tipped needles of different diameters and then stored at -80°C. RNA extraction was performed using the NucleoSpin™ RNA XS kit (Macherey-Nagel™, Düren, Germany). RNA was reverse transcribed to cDNA using the High Capacity RNA to cDNA kit (Applied Biosystems, Foster City, CA). The cDNA was then analyzed by quantitative real-time PCR (RT-PCR).

[0567] MicroRNA purification and quantitative real-time PCR expression analysis mRNA, including microRNAs, was isolated using miRNeasy® Mini Kit (Qiagen) according to the manufacturer's instructions and treated with miScript® Reverse Transcription Kit to generate cDNA. Samples were then analyzed using SYBR™ Green PCR Kit (Qiagen) on an Applied Biosystems™ 7500 Thermocycler (Applied Biosystems) according to the manufacturer's guidelines. Specific primers for each miR were used together with a commercially available universal primer, and U6 snRNA was used as an internal control. For mRNA quantification, specific primers were designed for each transcript using the software Primer Express 2 (Applied Biosystems), and expression was tested using real-time PCR.

[0568] Cloning of the 3'UTR of the target transcript into the psiCHEK-2 luciferase expression plasmid The 3'UTR sequences of Slc6a4 and Htr1a were amplified by PCR from mouse genomic DNA. The 3'UTR PCR fragments were ligated into pGEM®-T easy vector (Promega) according to the manufacturer's guidelines and further subcloned into the single NotI site at the 3' end of luciferase in the psiCHECK-2 reporter plasmid (Promega). The cloning orientation was verified by diagnostic cleavage and sequencing.

[0569] Transfection and luciferase assay HEK293T cells were grown to 70–85% confluence on poly-L-lysine in 84-well plates and transfected with the following plasmids using polyethylenimine: psiCHECK-2 plasmids containing wild-type or mutated 3'UTRs and overexpression vectors for specific miRNAs, or empty overexpression plasmids. miRs overexpression plasmids were taken from the miR-Vec library. Cells were thawed 24 h after transfection and luciferase reporter activity was analyzed as previously described [Kuperman Y. et al., Mol Endocrinol (2011) 25: 157-169]. Renilla luciferase values ​​were normalized to control firefly luciferase levels transcribed from the same vector but with an unaffected 3'UTR, and the average of six replicates for each condition was obtained for each subsequent test.

[0570] [Table 3]

[0571] [Table 4]

[0572] [Table 5]

[0573] Synthesis of miR-135 Single-Stranded Oligonucleotides (Optionally Conjugated to Sertraline) The oligonucleotide sequences were assembled using standard 2'-deoxy, 2'-O-Me or 2'-MOE phosphoramidite blocks, such as: [ka]

[0574] The synthesis was carried out using a typical experimental procedure for solid phase synthesis on a CPG (Controlled Pore Glass) support. Briefly, a typical oligonucleotide synthesis proceeds by a series of four-step cycles (deprotection, coupling, capping and oxidation) (shown in Figure 1) that are repeated until the 5'-most nucleotide is attached.

[0575] (a) Deprotection / detritylation The acid-labile 5'-dimethoxytrityl protecting group was cleaved from the solid-support-anchored base (either of the starting nucleoside or of the growing oligostrand) to give a free reactive hydroxy function using di- or tri-chloroacetic acid in dichloromethane as the cleavage reagent.

[0576] (b) Coupling The free 5'-OH group is now available to react with the added phosphoramidite. As a result, both nucleosides are linked with a phosphite bridge. The phosphoramidite must first be activated with a weak acid (e.g. 1H-tetrazole).

[0577] (c) Capping To prevent the remaining free OH groups (approximately 1%) from reacting during the synthesis cycle and generating non-specific sequences, the free reactive groups were blocked by acetylation in a capping step, excluding them from being reactive partners in further steps of the synthesis.

[0578] (d) Oxidation The internucleotide phosphite groups generated in the coupling step were oxidized to phosphates using an iodine solution.

[0579] A new synthesis cycle was repeated starting from step (a). These reactions were repeated until the desired oligonucleotide sequence was produced. The cycle could also be stopped to obtain support-bound oligonucleotides bearing a free 5'-OH group or a protected 5'-OH group (DMT).

[0580] Introducing phosphorothioate bonds: The difference between the synthesis of normal oligos with full-length phosphodiester backbones and those with partial or full-length phosphorothioate backbones is based on the choice of oxidizing agent used for the oxidation in step (d).

[0581] Phosphodiester bonds were created using iodine and water to add a fourth oxygen to the phosphate. Phosphorothioate bonds were created using Beaucage's reagent to add sulfur to the phosphate. Once sulfur or oxygen is added to the phosphate, the bond is stabilized and not susceptible to subsequent chemical cycles. Chimeric backbones may be constructed by alternating between the two oxidizing agents.

[0582] Synthesis of sertraline-conjugated miR-135 The guide and passenger RNA strands were synthesized on an automated synthesizer using phosphoramidite chemistry. Bz , rC Ac , rG iBu , rU and 2’OMe A Bz , 2’OMe C Ac , 2’OMe The sequence was extended using U phosphoramidite monomers. After chain extension, the RNA strand was cleaved from the solid support and deprotected using a mixture of ammonium hydroxide and methylamine. The 2'OH position was deprotected using a solution containing fluoride ions. The RNA strand was then purified by anion-exchange HPLC.

[0583] The sertraline-modified passenger RNA strand was synthesized on an automated synthesizer using phosphoramidite chemistry. The sequence was: Bz , rC Ac , rG iBu , rU and 2’OMe A Bz , 2’OMe C Ac , 2’OMe The sertraline-modified passenger RNA strand was then cleaved from the solid support and deprotected using a mixture of ammonium hydroxide and methylamine. The 2'OH position was deprotected using a solution containing fluoride ions. The conjugated RNA strand was then purified by reversed-phase HPLC.

[0584] The guide and passenger RNA strands were quantified by UV spectroscopy with extinction coefficients calculated based on the nearest neighbor method. Duplexes combined equal molar amounts of guide and passenger strands. Annealing was performed in water. Duplexes were not further purified.

[0585] The oligonucleotide primers, the RNA strands conjugated with sertraline, and the RNA duplexes were manufactured by Axolabs, GmbH, according to the specifications and designs of this application.

[0586] structure: Naked miR-135 duplex 1 (duplex 11 in Table 1 and duplex 1 in Table 6 below) [ka]

[0587] Sertraline conjugated miR-135 duplex 1 (miCure-135-1 in Table 6 below) [ka] Sertraline-Acyl C6-NHCO-C9-5'miR-135 sequence 3'-OH

[0588] Bare control (duplex 16 in Table 1 below, and duplex 8 in Table 6) [ka]

[0589] Sertraline conjugate control (miCure-135-8 in Table 6 below) [ka]

[0590] Naked miR-135 duplex 2 (Table 6 below) Guide strand: 5'-P / UmUAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 41) Passenger strand: 5'-ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13)

[0591] Sertraline-conjugated miR-135 duplex 2 (miCure-135-2 in Table 6 below) [ka]

[0592] Naked miR-135 duplex 3 (Table 6 below) Guide strand: 5'-P / UmUAUGGCUUUUCAUUCCUAUGUGaAmsAm (SEQ ID NO: 42) Passenger strand: 5'-ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13)

[0593] Sertraline-conjugated miR-135 duplex 3 (miCure-135-3 in Table 6 below) [ka]

[0594] Naked miR-135 duplex 9 (Table 6 below) Guide strand: 5'-P / UAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 10) Passenger strand: 5'-uscsACAUAGGAAUGAAAAGCCAS Usa (SEQ ID NO: 47)

[0595] Sertraline-conjugated miR-135 duplex 9 (miCure-135-9 in Table 6 below) [ka]

[0596] Naked miR-135 duplex 10 (Table 6 below) Guide strand: 5'-P / UmUAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 41) Passenger strand: 5'-uscsACAUAGGAAUGAAAAGCCAS Usa (SEQ ID NO: 47)

[0597] Sertraline-conjugated miR-135 duplex 10 (miCure-135-10 in Table 6 below) [ka]

[0598] Naked miR-135 duplex 11 (Table 6 below) Guide strand: 5'-P / UmUAUGGCUUUUCAUUCCUAUGUGaAmsAm (SEQ ID NO: 42) Passenger strand: 5'-uscsACAUAGGAAUGAAAAGCCAS Usa (SEQ ID NO: 47)

[0599] Sertraline-conjugated miR-135 duplex 11 (miCure-135-11 in Table 6 below) [ka]

[0600] In all duplexes, Uppercase letters (e.g. N,A,U,C,G): RNA Lowercase letters (e.g. a,u,c,g): 2'-O-Me modification Um: 2'-O-MOE-5'-Me uracil modification Am: 2'-O-MOE adenine modification Lower case 's': phosphorothioate. No modification means normal phosphodiester bond. P: Phosphate Underscore: 2'-fluoro, i.e., 2'-F (C10): Carboxy-modified C10 (Glen Research: 10-1935)

[0601] Intranasal Mice were lightly anesthetized with inhalation of 2% isoflurane and placed in supine position. One drop of 5 μl of conjugated control or conjugated miR-135 was administered alternately into each nostril once daily. A total of 10 μl of solution containing 166 μg was delivered.

[0602] Intracerebral injection For stereotaxic surgery and compound delivery, a computer-guided stereotaxic device and a motorized nanoinjector were used (Angle Two™ stereotaxic device, myNeurolab). Mice were placed in the stereotaxic instrument under general anesthesia and lentiviral formulations were delivered to coordinates defined by the Franklin and Paxinos brain coordinates: DR:ML 1mm; AP -4.5mm; DV -4.2mm at a tilt of 20°. Injections were performed at a rate of 0.2 μl / 1 min.

[0603] Chronic administration in the lateral cerebroventricular The right lateral ventricle was stereotactically drilled (coordinates from bregma: posterior, -0.7 mm; lateral, -1.5 mm; anterior, -2.0 mm) using a Brain Infusion Kit 3 (ALZET) (DURECT Corporation, Cupertino, CA). The infusion cannula was connected to a mini-osmotic pump (ALZET Pump Model 1007D) that was subcutaneously implanted on the animal's back just behind the scapula. The osmotic pump was primed for approximately 8 hours prior to implantation and filled with duplex / control (0.5 nmol / day). Once implanted, the pump released compound or negative control continuously at a rate of 0.5 μl / h for 7 days.

[0604] Brain microdialysis Extracellular 5-HT concentrations were measured by in vivo microdialysis. Briefly, one concentric dialysis probe (cuprophan; 1 mm long) was implanted in the mPFC (AP, 2.2; ML, -0.2; DV, -3.4) of mice anesthetized with pentobarbital. Experiments were performed 48–72 h after surgery. 1 mM citalopram (SSRI) (Valve, Copenhagen, Lundbeck A / S) was added to the artificial CSF. 6 μl of the artificial CSF was added to the -1(WPI model sp220i) was used to deliver the fluid and collect samples for 6 min. A tail suspension test was performed while collecting the eighth fraction. Analysis of 5-HT concentrations was performed by high performance liquid chromatography with amperometric detection (Hewlett-Packard 1049; 0.6V) (Palo Alto, CA, USA) with a detection limit of 1.5 fmol sample. Baseline 5-HT levels were calculated as the average of the four pre-drug samples. Correct probe placement was verified by cresyl violet staining.

[0605] Behavioral assessment Mice were housed in the testing room 2 hours before the start of each test, and all behavioral assessments were performed during the dark phase. The examiner performing the test was blinded to the mouse group.

[0606] Dark-Light Transfer (DLT) test: The DLT test apparatus consisted of a polyvinyl chloride box divided into a black dark chamber (14 x 27 x 26 cm) connected to a white light chamber (30 x 27 x 26 cm) illuminated at 1200 lux. During a 5-min test period, the time spent in the light chamber, the distance traveled in the light, and the number of light-dark transitions were quantified with a video tracking system (VideoMot2) (TSE Systems, Bad Homburg, Germany).

[0607] Tail suspension test: Mice were suspended 30 cm above a bench by adhesive tape attached approximately 1 cm from the tip of their tail. Mice were monitored and recorded using a video camera system (Smart) (Panlab) and the time spent immobile was recorded over a 6 min period.

[0608] 8-OH-DPAT-induced hypothermia Body temperature was measured rectally using a lubricated probe inserted at B2 cm and a digital thermometer (AZ9882) (Panlab, Barcelona, ​​Spain). Twenty minutes before measurement, mice were individually placed in clean cages and two baseline temperatures were then measured. Ten minutes later, animals received an intravenous injection of 1 mg 8-OH-DPAT per kg and body temperature was measured every 15 minutes for a total of 120 minutes. Data were expressed as the change from the mean baseline measurement.

[0609] In experiments testing the effects of various miR-135 mimics (described in the Results section below), body temperature was measured with a microchip. A programmable subcutaneous microchip transponder (IPTT-300 Extended Accuracy Calibration) (Bio Medic Data Systems, Seaford, DE) was implanted subcutaneously in mice according to the manufacturer's instructions. Briefly, the procedure involves rapid insertion of a large-bore needle-type delivery device containing the microchip and depression of the plunger on the device, expelling the microchip from the delivery device. Temperature measurements from the microchip transponder were obtained with a handheld reader (catalog number WRS6007, model IPTT-300, Bio Medic Data Systems). The reader was held 5-6 cm from the back of the mouse as per the manufacturer's instructions. A beep (after 1-3 seconds) signaled the completion of the measurement, and the displayed temperature was recorded. Duplicate temperatures were obtained by performing two consecutive measurements.

[0610] Tissue preparation for in situ hybridization and receptor autoradiography Mice were killed with an overdose of pentobarbital and the brains were rapidly removed, frozen on dry ice and stored at −20° C. Tissue sections (14 mm thick) were cut with a microtome-cryostat (HM500OM) (Microm, Walldorf, Germany), thaw-mounted on 3-amino-propyltriethoxysilane-coated slides (Sigma-Aldrich, Madrid, Spain) and stored at −20° C. until use.

[0611] Receptor autoradiography Autoradiographic binding assays of 5-HT1A and 5-HT1B receptors and the serotonin transporter (SERT) were carried out using the following radioligands: (a) [ 3 H]-8-OH-DPAT(233Cimmol -1 ), (b) [ 125 I]-Cyanopindole (2200Cimmol -1 ) and (c) [ 3 H]-Citalopram (70Cimmol -1 ) (Amersham-GE Healthcare, Barcelona, ​​Spain and Perkin-Elmer, Madrid, Spain). 8-OH-DPAT, isoproterenol, pargyline and 5-HT were from Sigma-Aldrich and fluoxetine was from Tocris. The experimental conditions are summarized in Table 5 below.

[0612] [Table 6]

[0613] The tissues were then transferred to Biomax® MR film (Kodak) 3 H-microscale standard (Amersham-GE Health-care) All experimental and control brains within a group were processed in duplicate and exposed together to film.

[0614] Films were analyzed by microdensitometry using a computer-assisted image analyzer (AIS, Imaging Research, St. Catharines, Ontario, Canada). 5-HT1AR mRNA and 5-HT1B binding sites in selected brain regions were measured from the respective autoradiograms, and relative optical densities were obtained. 5-HT1AR and SERT binding systems were 3 Calibrated with H-microscale standards and calculated from relative optical density data in fmol mg -1Equivalent amounts of protein were obtained. The AIS system was also used to obtain pseudocolor images. Black and white pictures were taken from the autoradiograms using a Wild 420 microscope (Leica, Heerbrugg, Germany) equipped with a Nikon DXM1200 F digital camera and ACT-1 Nikon software (Soft Imaging System Gmbh, Münster, Germany). Images were processed in Photoshop (Adobe Systems, Mountain View) with identical values ​​of contrast and brightness, respectively.

[0615] Peripheral blood mononuclear cell (PBMC) research The study was carried out by Axolabs (www(dot)axolabs(dot)com). Briefly, human PBMCs were isolated from buffy coats of healthy donors (obtained from the Blood Bank, Institute for Transfusion Medicine, Suhl, Germany). Buffy coats were approximately 28-32 ml in volume and delivered in sterile, sealed transfusion bags approximately 19 hours after donation. Human PBMCs (huPBMCs) were isolated by Ficoll density gradient centrifugation.

[0616] huPMBCs were treated by transfection or direct incubation (3 concentrations for 24 h) with different mimetics (described in Results below). Transfection was performed with Lipofectamine 2000. The presence of cytokines in the supernatants was analyzed with a multiplex assay (U-Plex®) implemented on the Mesoscale Discovery (MSD) platform. All procedures were performed by Axolabs (www(dot)axolabs(dot)com).

[0617] Corticosterone-chronic restraint stress model For the chronic restraint stress protocol, mice were isolated and handled starting 3 days before the start of the experiment. Once per day for 28 days, mice were gently placed into a 50 mL Falcon tube with holes fixed to a plastic tray and left in a dark, quiet room for 2 hours. Control mice were not isolated and were handled for only 1 min each day.

[0618] Corticosterone (Cortico) (Sigma-Aldrich, Madrid, Spain) was dissolved in commercial mineral water and adjusted to pH 7.0–7.4 with HCl. During the 28 days of the chronic restraint stress protocol, isolated mice were given Corticosterone solution: 30 μg ml -1 for 15 days, then 15 μg ml -1 for 3 days, and 7.5 μg ml -1 The Cortico solution was used within 3 days and stored in opaque bottles to protect it from light. Control mice were given mineral water only.

[0619] Chronic social defeat stress Nine-week-old C57BL / 6J mice were subjected to a chronic social defeat stress (CSDS) protocol as previously described [Krishnan V.et al., Cell (2007) 131:391-404]. Briefly, mice were placed in a cage housing aggressive ICR (CD1) outbred mice (Harlan) and allowed to have physical contact for 5 min. During this time, the ICR mice attacked the intruder mice, which assumed a subordinate posturing posture. A porous transparent Plexiglas® divider was then placed between the animals, and the mice were left in the same cage for 24 h, allowing sensory contact. This procedure was repeated consecutively with unfamiliar ICR mice every day for 10 days. Control mice were housed in the same room as the socially defeated mice, but were removed from the room for the 5 min of contact with the ICR mice. Control mice were handled daily and housed two per cage, separated by a clear Plexiglas® divider.

[0620] immunohistochemistry Mice were anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) solution in sodium phosphate buffer (pH 7.4). Brains were extracted and then fixed in PFA at 4 °C for 24 h and placed in a 10–30% density gradient of sucrose solution at 4 °C for 3 days. After cryopreservation, 30 μm-thick serial sections were cut to obtain the prefrontal cortex (PFC), neostriatum (CPu), hippocampus (HPC), and dorsal raphe nucleus (DRN). Brain sections were washed and incubated in 1x PBS / Triton 0.2% solution containing normal serum from the secondary antibody host. The primary antibodies used were NeuN (anti-NeuN 1:1000; ref: MAB377, Millipore), Iba1 (anti-Iba1 1:1000; ref: 019-197741, Wako), and TPH (anti-TPH1 1:2500; ref: AB1541, Millipore). Briefly, the primary antibodies were incubated overnight at 4°C, followed by incubation with the corresponding biotinylated anti-mouse IgG1 for NeuN (1:200; ref: A-10519, Life Technologies), biotinylated anti-rabbit IgG1 for anti-Iba1 (1:200; ref: BA-1000, Vector Laboratories), and biotinylated anti-sheep IgG1 for anti-TPH1 (1:200; ref: BA-6000, Vector Laboratories). Colorimetric reactions were performed by incubation with diaminobenzidine tetrahydrochloride (DAB) (ref: 18865.02, Quimigen) solution. Sections were mounted and embedded in Entellan (Electron Microscopy Sciences). Software (v1.51s) (NIH, Bethesda, MD, USA) was used to determine the numbers of NeuN-, Iba1- and TPH-positive cells in the DRN from sections corresponding to different anterior-posterior levels from -4.24 mm to -4.84 mm from the bregma. All labeled cells whose nuclei were included in the counting frame were counted in three consecutive DRN sections.

[0621] Immunofluorescence Mice were anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde (PFA) solution in sodium phosphate buffer (pH 7.4). Brains were extracted and then fixed in PFA at 4°C for 24 h and placed in a 10–30% density gradient of sucrose solution at 4°C for 3 days. After cryopreservation, 30 μm-thick serial sections were cut to obtain the olfactory bulb (OB), CPu, HPC and DRN. Brain sections were washed and incubated in 1× PBS / Triton 0.2% solution containing normal serum from the secondary antibody host. Alexa488 (anti-Alexa488 1:1000; ref.:A11094, Invitrogen) and TPH (anti-TPH 1:1541; ref.:ab1541, Abcam) were used as primary antibodies. Briefly, primary antibodies were incubated overnight at 4°C, rinsed, and treated with secondary antibodies A555 anti-sheep (1:500; ref.: A-21436, Life Technologies) and Alexa488 anti-rabbit (1:500; ref.: A-21206, Life Technologies) for 120 min. Nuclei were stained with Hoechst (1:10.000, ref.: H3570, Life Technologies).

[0622] TPH of Alexa488-conjugated miR-135 +Subcellular localization in neurons was observed and imaged using an inverted Nikon Eclipse Ti2-E microscope (Nikon Instruments) attached to a spinning disk unit Andor Dragonfly (Oxford Instruments). In all experiments, an oil immersion objective (Plan Apochromat Lambda blue 60×, numerical aperture (NA) 1.4, oil) was used. Samples were excited with 405 nm, 488 nm and 561 nm laser diodes. The laser light was coupled into a multimode fiber through an Andor Borealis unit that reshapes the laser light from a Gaussian profile to a uniform flat top. From there it was passed through a 40 μm pinhole disk. Tissue sections were imaged with a high-resolution Scientific complementary metal-oxide semiconductor (sCMOS) camera (Zyla 4.2, 2.0 Andor, Oxford Instruments Company). Fusion software (Andor, Oxford Instruments) was used for acquisition and ImageJ / Fiji (1.51s, open source software) for image processing.

[0623] Western blot Tissue samples of OB, PFC, CPu, HPC, DRN and cerebellum (Cb) were dissected from brain sections and homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5 mM EDTA, 0.1% SDS, 50 mM Tris, pH 8.0) with protease and phosphatase inhibitors. Protein was quantified using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Protein lysates (10–15 μg) were separated using 4–15% SDS-PAGE and electrotransferred to nitrocellulose membranes. Protein blots were probed with primary antibodies against SERT (1:1000, ref.:ab130130, Abcam) and 5HT1AR (1:1000, ref.:ab85165, Abcam) and anti-β-actin (1:50000, ref.:A3854, Sigma-Aldrich) as a loading control, followed by incubation with HRP-conjugated anti-goat IgG corresponding to SERT (1:20000, ref.:P0449, Dako) and HRP-conjugated anti-rabbit IgG corresponding to 5HT1AR (1:10000; ref.:NA934, GE Healthcare Life). Detection was by chemiluminescence using the SuperSignal™ Chemiluminescent ECL Substrate Kit (Thermo Fisher Scientific) and photographed using a ChemiDoc™ Imaging System (Bio-Rad). Images were analyzed with ImageLab™ software (BioRad).

[0624] statistical analysis Data are expressed as mean ± SEM. Data were analyzed by Student's t test, one-way or two-way ANOVA, as appropriate, followed by post-hoc tests (Newman-Keuls). The level of significance was set at P < 0.05 (two-tailed).

[0625] Example 1 In vitro design, synthesis and validation of miR-135 mimetic oligos miR-135 mimics (oligos) were designed to mimic the antidepressant effects exhibited by overexpression of endogenous miR-135 in transgenic mouse models and viral manipulation in mouse 5HT neuronal cells or DRN, respectively. Based on endogenous miR-135, the oligos (miR-135 mimics) were slightly modified to improve stability and cell permeability. The modified oligonucleotides were synthesized by BioSpring (www(dot)biospring(dot)de), an oligonucleotide manufacturer, based on known chemistry, and each oligo was used as a duplex for improved stability. Seventeen different mimics (see Table 1) were designed, synthesized, and screened in vitro for their effects on the Htr1a and Slc6a4 target transcripts of interest using a luciferase assay. The human cell line HEK293T and the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) were used for luciferase assay screening.

[0626] Duplex 11 was found to be the most potent miR-135 mimetic oligo, significantly affecting both 5HTR1A and SLC6a4 levels (using the 3'UTR luciferase construct; Figure 3, A-B). The design of the duplex 11 mimic of miR-135 is as follows. Guide strand: 5Ph / UAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 10) Passenger strand: ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13) Lowercase letters (e.g. a,u,c,g): 2'-O-Me modification Ph=phosphate, the 5 indicates that this is the 5' end of the sequence.

[0627] Mimetics identified to consistently decrease the target transcripts (Htr1a and Slc6a4) regardless of preparation were further used for in vivo experiments.

[0628] [Table 7-1]

[0629] [Table 7-2]

[0630] [Table 7-3]

[0631] Example 2 Effects of miR-135 mimics on serotonergic function in vivo To test the effect of miR-135 mimics on the in vivo serotonin system, miR-135 mimics (duplex 11) were delivered directly into the dorsal raphe nucleus (DRN) of wild-type mice using stereotaxic surgery. Naked (i.e., unconjugated) mimics were administered acutely (100 μg) and the physiological events of 5-HT1A autoreceptor (HTR1a) silencing were tested using hypothermic responses induced by the selective 5-HT1AR agonist 8-OH-DPAT, an effect mediated exclusively by presynaptic 5-HT1AR in mice. While miR-135 mimic (duplex 11)-treated mice did not exhibit 8-OH-DPAT-induced hypothermia, both control groups (mice treated with artificial cerebrospinal fluid (aCSF) or mice treated with control miR (i.e., bare control)) presented the expected hypothermic response (Figure 4A-D). No differences in baseline temperatures were seen between groups (Figure 4E). The dynamics of this effect were demonstrated at four different time points (24, 48, 72, and 96 hours) following acute dosing (Figure 4A-D).

[0632] Example 3 In vivo effects of sertraline-conjugated miR-135 mimics on serotonergic function Following in vitro and in vivo validation of the efficiency of miR-135 mimics, we used a previously published approach (Ferres-Coy et al., Mol. Psychiatr. (2016) 21(3): 328-38) for non-invasive delivery of oligonucleotides to the brain using intranasal administration of sertraline conjugates. Ferres-Coy et al. (2016, supra) have previously reported that intranasal administration of sertraline-conjugated small interfering RNA (siRNA) silenced SERT expression / function in mice. After passing through the permeable olfactory epithelium, sertraline-conjugated siRNA was absorbed and transported to serotonin cell bodies by deep Rab-7-associated endomembrane endoplasmic reticulum.

[0633] Modified miR-135 mimics and control oligos were conjugated to non-functional sertraline (purchased from NEDKEN SOLUTIONS, SL, Barcelona, ​​Spain) and administered acutely into the dorsal raphe nucleus (DRN) of naive mice. Mice treated with a 100 μg dose of sertraline-conjugated miR-135 mimic duplex 11 (designated miCure-135-1, shown in SEQ ID NOs: 10 and 13) showed no hypothermic response following 8-OH-DPAT administration. This effect persisted up to 7 days after acute administration (Figure 5A-D).

[0634] Example 4 Acute intracerebral administration of sertraline-conjugated miR-135 mimic (30 μg) silences 5HT1a and SERT and produces antidepressant-like responses To further explore the efficiency of sertraline-conjugated miR-135 in downregulating gene expression of the serotonin system, which has already been shown to be directly controlled by miR-135, a lower dose of miCure-135-1 (30 μg) was acutely administered to the DRN of adult mice. First, physiological events associated with silencing of the 5-HT1A autoreceptor (HTR1a) were examined using the hypothermia response induced by 8-OH-DPAT, as performed with the higher dose (100 μg). The results showed that a single administration of miCure-135-1 at a dose of 30 μg was sufficient to eliminate hypothermia induction by the selective HTR1a agonist without altering baseline temperature up to 7 days after treatment (Figure 6A-E).

[0635] Next, a microdialysis probe that allows for real-time CSF collection was fixed to the medial prefrontal cortex (mPFC) of mice, followed by measuring 5-HT levels using liquid chromatography. Mice were placed in cages individually and 15 fractions of 6 min each were collected. The eighth fraction was collected during the tail suspension test (a test to evaluate depression-like behavior in mice). The results revealed that mice treated with miCure-135-1 showed a significant decrease in immobility time compared to the control group (Figure 6G), indicating the antidepressant-like effect of sertraline-conjugated miR-135. Of note, consistent with the behavioral results, mPFC 5-HT levels of treated mice were significantly higher (Figure 6F), suggesting a better adaptive mechanism for sertraline-conjugated miR-135-treated mice. Next, the inventors evaluated whether silencing of the SERT gene by sertraline-conjugated miR-135 is possible. Histological examination 1–4 days after administration revealed a significant decrease in SERT binding density in the dorsal raphe (75% of control; Fig. 6H ) at 24 and 96 h after injection.

[0636] Example 5 Acute intranasal administration of sertraline-conjugated miR-135 mimic (166 μg) silences 5HT1a and produces antidepressant / anxiety-like responses The ability of miCure-135-1 to function as an antidepressant following a non-invasive delivery method was tested by intranasal delivery, where anesthetized mice received 5 μl of miCure-135-1 through each nostril for a total of 166 μg. 8-OH-DPAT-induced hypothermia was examined over a 5-day period following treatment, revealing that the miCure-135-1-treated mice group exhibited a chemically reduced hypothermic response, which is a physiological event of HTR1a silencing (Figure 7A). Furthermore, an antidepressant-like effect was demonstrated by the reduced immobility time of miCure-135-1-treated mice compared to controls in the tail suspension test (Figure 7B), and an anxiolytic effect was demonstrated by treated mice spending more time in the light compartment compared to controls in the dark / light transfer test (Figure 7C).

[0637] Example 6 Design, synthesis and validation of 16 highly chemically modified miR-135 mimetic oligos in vitro As mentioned above, based on endogenous miR-135, additional miR-135 mimetics (oligos) were designed (see Table 1 above), which had advanced chemical modifications aimed at improving stability and reducing inherent immunotoxicity without compromising potency.

[0638] Sixteen different mimetics (see Figure 8 and Table 6 below) were designed and synthesized using known chemistry (as described above in "General Materials and Experimental Procedures"), with each oligo used as a duplex to improve stability. Sixteen different miR-135 mimetics were screened in vitro for their effect on the validated Htr1a and Slc6a4 target transcripts using a luciferase assay. The human cell line HEK293T and the transfection reagent Lipofectamine 2000 (Thermo Fisher Scientific) were used for luciferase assay screening.

[0639] miCure-135-1, miCure-135-2, miCure-135-3, miCure-135-9, miCure-135-10 and miCure-135-11 were the most potent miR-135 mimetic oligos, significantly affecting both 5HTR1A and SLC6a4 levels (using the 3'UTR luciferase construct; Figures 9A-B).

[0640] Mimetics identified as consistently decreasing the target transcripts (Htr1a and Slc6a4) regardless of preparation were further used in additional experiments.

[0641] [Table 8]

[0642] Example 7 Advanced chemical modification of miR-135 oligos alters cytokine secretion patterns by PBMCs in vitro Oligonucleotides that are not recognized as self by the body typically induce an immune response through the release of pro-inflammatory cytokines. Chemical modification of oligonucleotides aims to increase their potency and exert their potential to decrease innate immune activity. To test the immune activation induction pattern of novel modifications, human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation starting from fresh buffy coats of healthy volunteers (obtained from the transfusion center as described above under "General Materials and Experimental Procedures"). PBMCs were treated with the different mimetics for 24 hours and analyzed for the presence of cytokines in the supernatants in a multiplex assay carried out on a Mesoscale Discovery (MSD).

[0643] The results of this assay demonstrated that the two novel duplexes, miCure-135-2 and miCure-135-3, did not induce secretion of the tested cytokines (Figures 10A-E and 11A-F, respectively), miR-135-1 showed moderate activation of TNF-alpha (Figure 10A) and IFN-alpha-2a (Figure 10B), miR-135-9 induced high secretion of IFN-alpha-2a (Figure 11A) and low secretion of TNF-alpha (Figure 11B), and miR-135-11 induced high secretion of IFN-alpha-2a (Figure 11A) and low secretion of TNF-alpha (Figure 11B) and IFN-gamma (Figure 11C). Results with MiCure-135-10 revealed very high secretion of TNF-alpha and IFN-alpha-2a (Figures 10A and 10B, respectively). The duplexes identified as inducing the lowest levels of secretion (i.e., miCure-135-2 and miCure-135-3) were further tested in vivo. The relatively high cytokine secretion induction by miCure-135-9, miCure-135-10 and miCure-135-11 may be related to their common passenger strand consisting of two phosphorothioate bonds replacing the normal phosphodiester at one of the 5' and 3' termini, which was also tested.

[0644] Example 8 Acute DRN administration of a sertraline-conjugated miR-135 mimic affects serotonergic function and produces antidepressant-like responses To further test the effect of miR-135 mimics on the serotonin system in vivo, three miR-135 mimics conjugated to non-functional sertraline, namely, miCure-135-2 (shown in SEQ ID NOs: 41 and 13), miCure-135-3 (shown in SEQ ID NOs: 42 and 13) and miCure-135-9 (shown in SEQ ID NOs: 10 and 47), were delivered directly into the dorsal raphe nucleus (DRN) using stereotaxic surgery.

[0645] miCure-135-2 and miCure-135-9 were administered acutely (30 μg) to test the physiological consequences of 5-HT1A autoreceptor (HTR1a) silencing using the hypothermic response induced by the selective 5-HT1AR agonist 8-OH-DPAT, an effect mediated exclusively by the presynaptic 5-HT1AR in mice. As illustrated in Figure 12A-D, sertraline-conjugated miR-135-treated mice (both mimics miCure-135-2 and miCure-135-9) did not exhibit hypothermia, whereas both control groups, i.e., mice treated with artificial cerebrospinal fluid (aCSF) or mice treated with control miR, presented the expected hypothermic response. No differences in baseline temperature were found between the groups (Figure 12E). The dynamics of this effect were shown at three different time points following acute administration.

[0646] Next, miCure-135-3 and sertraline conjugate control were administered acutely (100 μg) to mouse DRN to examine the functional events of serotonin transporter (SERT) silencing. To do so, a microdialysis probe that allows real-time collection of CSF was fixed to the medial prefrontal cortex (mPFC) of mice, followed by measurement of 5-HT levels using liquid chromatography. Mice were individually caged and 12 fractions of 20 min each were collected. A locally selective serotonin reuptake inhibitor (10 μM citalopram) was infiltrated from the seventh fraction by retrodialysis, resulting in an increase in extracellular 5-hydroxytryptamine (5-HT). The increase in 5-HT levels in miCure-135-3-treated mice was significantly lower than that in the control group (Figure 12F), suggesting that miCure-135-3 results in a decrease in available serotonin transporters. The same experimental group, i.e., mice injected once with 100 μg miCure-135-3 or conjugate control in the DRN, was tested in the tail suspension test. The results revealed that compared with the control group, mice treated with miCure-135-3 showed a significant decrease in immobility time (Figure 12G), indicating the antidepressant-like effect of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3).

[0647] Example 9 Silencing of 5HT1a and SERT by intranasal and intracerebroventricular administration of sertraline-conjugated miR-135 mimics The potential ability of sertraline-conjugated miR-135 mimics to function as antidepressants with clinically accepted drug administration methods was demonstrated using a non-invasive administration method (i.e., intranasal). Mice were acutely treated with miCure-135-3 and hypothermia induced 48 hours after 8-OH-DPAT administration was examined. Administration of 200 μg (FIG. 13C) and 100 μg (FIG. 13B) of miCure-135-3 significantly reduced hypothermia in treated mice compared to control-treated mice. Acute intranasal administration of 50 μg miCure-135-3 showed a clear trend (p=0.054) for reduced hypothermic response (FIG. 13A). A similar response was seen 7 days after intranasal administration of miCure-135-2 (200 μg / day) (FIG. 13D).

[0648] The intracerebroventricular (ICV) delivery method is intended to model intrathecal administration, in which the drug is administered directly into the CSF. In this experiment, a subcutaneous osmotic minipump was connected to a cannula placed in the mouse ventricle. miCure-135-3 was delivered continuously at a rate of 200 μg / day into the second ventricle for 7 days. A smaller decrease in body temperature was observed in the miCure-135-2 treatment group compared to the control (Figure 13E). Taken together, the hypothermia results (Figure 13A-E) suggest that the sertraline-conjugated miR-135 mimic was successful in decreasing serotonergic autoreceptor (5HT1a).

[0649] Next, to investigate the effect of sertraline-conjugated miR-135 mimics on serotonin transporter (SERT) levels, a microdialysis probe that allows real-time CSF collection was fixed to the medial prefrontal cortex (mPFC) of mice, followed by measurement of 5-HT levels using liquid chromatography. Mice were individually placed in cages and 18 fractions of 20 min each were collected. A locally selective serotonin reuptake inhibitor (10 μM citalopram) was infiltrated from the seventh fraction by retrodialysis, resulting in an increase in extracellular 5-hydroxytryptamine (5-HT). The increase in 5-HT levels in the miCure-135-3-treated group was significantly lower than that in the control group (Figure 13F), suggesting that intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) results in a decrease in the serotonin transporter in the dorsal raphe nucleus.

[0650] Example 10 Acute intranasal administration of miCure-135-3 affects serotonin function and produces antidepressant-like responses To further explore the effect of non-invasively administered sertraline-conjugated miR-135 mimics on serotonin function, wild-type mice were delivered with miCure-135-3 via the intranasal route. To assess whether sertraline-conjugated miR-135 could reduce protein levels of SERT and 5-HT1A autoreceptor (HTR1AR), mice were acutely treated with miCure-135-3, and immunoblot analysis of samples taken 3 days after administration revealed that protein levels of SERT and HTR1Ar were significantly reduced in the dorsal raphe 72 hours after injection (both proteins at 75% of control; Figure 14, A-D).

[0651] Next, to investigate the effect of sertraline-conjugated miR-135 mimics on serotonin transporter (SERT) levels, a microdialysis probe that allows for real-time CSF collection was fixed to the medial prefrontal cortex (mPFC) of mice, followed by measurement of 5-HT levels using liquid chromatography. 48 hours after administration, mice were placed individually in cages and 12 fractions of 20 min each were collected. Infiltration of a locally selective serotonin reuptake inhibitor (10 μM citalopram) from the seventh fraction by retrodialysis led to an increase in extracellular 5-hydroxytryptamine (5-HT). The increase in 5-HT levels in the miCure-135-3-treated group was significantly lower than that in the control group (Figure 14F), suggesting that intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) led to a decrease in the serotonin transporter in the dorsal raphe nucleus.

[0652] The same group of mice was subjected to additional microdialysis experiments aimed at investigating the effect of acute intranasal administration of miCure-135-3 on HTR1AR levels, using the same probe fixed in the mPFC. Real-time CSF was collected 72 h after administration to measure 5-HT levels using liquid chromatography. Mice were individually caged and 12 fractions of 20 min each were collected. In the sixth fraction, a selective HTR1a agonist (8-OH-DPAT, 1 mg kg -1Intraperitoneal injection of miCure-135-3 (ip) resulted in an increase in extracellular 5-hydroxytryptamine (5-HT). The decrease in 5-HT levels in the miCure-135-3-treated group was significantly shorter than that in the control group (Figure 14E), suggesting a decrease in 5-HT1A autoreceptors in the dorsal raphe nucleus by intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3). The same experimental groups, i.e., mice receiving a single intranasal administration of 2500 μg miCure-135-3 or conjugate control, were tested in the tail suspension test. The results showed that mice treated with miCure-135-3 showed a significant decrease in immobility time compared to the control group (Figure 14G), indicating an antidepressant-like effect of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3).

[0653] Example 11 Acute intranasal administration of sertraline-conjugated miR-135 mimic reduces depressive-like behavior in mice subjected to a depression-like induction protocol The potential of sertraline-conjugated miR-135 mimics to act as antidepressants was demonstrated in a depression-like mouse model. Mice were treated with corticosterone in drinking water and placed under restraint stress for 2 hours daily for 28 days. Mice subjected to the paradigm showed increased depression-like behavior. Following the protocol, mice were treated with a dose of miCure-135-3 (2500 μg). Antidepressant-like effects were demonstrated by reduced immobility time in the tail suspension test in miCure-135-3-treated mice 3 days after treatment compared to controls (Figure 15).

[0654] Example 12 Selective accumulation of miR-135 mimics in the dorsal raphe nucleus following intranasal administration To investigate the brain distribution of miR-135 mimics following intranasal administration, alexa488-labeled miCure-135-3 was synthesized. The labeled molecule was delivered to mice by intranasal administration, and brain tissue was harvested 6 hours after administration. Confocal fluorescence microscopy revealed that alexa488-labeled miCure-135-3 was detected intracellularly in TPH2-positive midbrain 5-HT neurons after intranasal administration (Figure 16A-C). Confocal analysis showed the absence of alexa488-labeled miCure-135-3 in cells in brain regions near the administration site (olfactory bulb) or ventricles (hippocampus and striatum) (Figure 16D), supporting that surface SERT expression is essential for oligonucleotide uptake and internalization and that miCure-135-3 selectively accumulates in the DRN.

[0655] Example 13 miR-135 mimics do not affect cell viability in the brain To further test the safety of the miR-135 mimics, mice were injected directly into the dorsal raphe nucleus with a single injection of the conjugate control, miCure-135-3, or a positive control previously reported to have no effect on cell viability (sertraline-siRNA conjugated to SERT as taught in Ferres-Coy et al., Mol. Psychiatr. (2016) 21(3): 328-38). Midbrain raphe nucleus sections were stained for neuronal (NeuN positive), serotonergic neuronal (TPH positive), or microglial (Iba-1 positive) markers. Immunohistochemical analysis showed that miCure-135-3 did not induce neuronal degeneration (Figure 17A), serotonergic neuronal degeneration (Figure 17C), or immune responses (Figure 17B). These are also seen in representative images of the midbrain raphe nucleus stained with NeuN, Iba1, and TPH under various treatments (FIG. 17D). These data support the specificity and safety of miCure-135-3.

[0656] Example 14 Evaluating the effects of acute intranasal administration of sertraline-conjugated miR-135 in a chronic social defeat stress depression-like protocol The efficacy of sertraline-conjugated miR-135 mimics as antidepressants was demonstrated using a chronic social defeat stress protocol in aggressive ICR mice [Krishnan V. et al., Cell (2007) 131:391-404]. Mice subjected to this paradigm exhibited increased depression-like behavior. Mice were treated with a dose of miCure-135-3 (2500 μg) and the antidepressant-like effects were tested by measuring the immobility time in the tail suspension test 2 and 3 days after treatment and comparing control and miCure-135-3 treated mice.

[0657] While the present invention has been described in conjunction with specific embodiments thereof, many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0658] Applicants intend that all publications, patents, and patent applications mentioned in this specification are incorporated herein in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application should not be construed as an admission that such reference is available as prior art to the present invention, nor should it necessarily be construed as limiting, to the extent that section headings are used. Additionally, the priority documents of this application are hereby incorporated herein in their entirety as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated herein by reference. [Sequence List Free Text]

[0659] SEQ ID NO:1: in vitro miR-135 mimic oligo; 5' phosphorylation at position 1; 2-OMe modification at position 23 SEQ ID NO:2: In vitro miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 22 is 2-OMe modified SEQ ID NO:3: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 22 is 2-OMe modified SEQ ID NO: 4: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 23 is 2-OMe modified SEQ ID NO: 5: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-OMe modification at positions 10-11; 2-OMe modification at position 23 SEQ ID NO: 6: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 10 is 2-OMe modified; position 22 is 2-OMe modified SEQ ID NO: 7: miR-135 mimic oligo; position 1 is 5' phosphorylated; positions 1-2 are 2-OMe modified; position 4 is 2-OMe modified; position 23 is 2-OMe modified SEQ ID NO: 8: miR-135 mimic oligo; position 1 is 5' phosphorylated; positions 1-2 are 2-OMe modified; position 4 is 2-OMe modified; position 23 is 2-OMe modified SEQ ID NO: 9: miR-135 mimic oligo; 2-OMe modification at position 1; 2-OMe modification at position 23 SEQ ID NO: 10: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-OMe modification at position 23 SEQ ID NO: 11: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 22 is 2-OMe modified SEQ ID NO: 12: miR-135 mimic oligo; positions 1-2 are 2-OMe modified SEQ ID NO: 13: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; position 1 can be conjugated to sertraline or other cell targeting moieties at the 5' end; position 23 is 2-OMe modified SEQ ID NO: 14: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-OMe modification at positions 11-13; 2-OMe modification at position 23 SEQ ID NO: 15: miR-135 mimic oligo; positions 1-2 are 2-OMe modified; positions 9-10 are 2-OMe modified; position 22 is 2-OMe modified SEQ ID NO: 16: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-OMe modification at positions 1-2; 2-OMe modification at position 4; 2-OMe modification at position 23

[0660] SEQ ID NO: 17: single stranded DNA oligonucleotide SEQ ID NO: 18: single stranded DNA oligonucleotide SEQ ID NO: 19: single stranded DNA oligonucleotide SEQ ID NO: 20: single stranded DNA oligonucleotide SEQ ID NO: 21: Single stranded DNA oligonucleotide SEQ ID NO: 22: single stranded DNA oligonucleotide SEQ ID NO: 23: single stranded DNA oligonucleotide SEQ ID NO: 24: single stranded DNA oligonucleotide SEQ ID NO: 25: single stranded DNA oligonucleotide SEQ ID NO: 26: single stranded DNA oligonucleotide SEQ ID NO: 27: single stranded DNA oligonucleotide SEQ ID NO: 28: single stranded DNA oligonucleotide SEQ ID NO: 29: single stranded DNA oligonucleotide SEQ ID NO: 30: single stranded DNA oligonucleotide SEQ ID NO: 31: single stranded DNA oligonucleotide SEQ ID NO: 32: single stranded DNA oligonucleotide SEQ ID NO: 33: single stranded DNA oligonucleotide SEQ ID NO: 34: single stranded DNA oligonucleotide SEQ ID NO: 35: single stranded DNA oligonucleotide SEQ ID NO: 36: single stranded DNA oligonucleotide SEQ ID NO: 37: Mature miR-135b SEQ ID NO: 38: mature miR-135b* SEQ ID NO: 39: miR-135b microRNA precursor SEQ ID NO: 40: RNA oligonucleotide complementary to miR-135b

[0661] SEQ ID NO: 41: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-O-MOE-5-Me at position 1; 2-OMe modification at position 24 SEQ ID NO: 42: miR-135 mimic oligo; 5' phosphorylation at position 1; 2-O-MOE-5-Me at position 1; 2-OMe modification at position 24; 2-OMe modification at positions 25-26; phosphorothioate linkage at positions 25-26 SEQ ID NO: 43: miR-135 mimic oligo; position 1 is 5' phosphorylated; position 1 is 2-O-MOE-5-Me; position 2 is 2'-fluoro; position 2 is 2'-fluoro; position 3 is 2'-O-Me; position 4 is 2'-fluoro; position 5 is 2'-O-Me; position 6 is 2'-fluoro; position 7 is 2'-O-Me; position 8 is 2'-fluoro; position 9 is 2'-O-Me; position 10 is 2'-fluoro; position 11 is 2'-O-Me; position 12 is 2'-fluoro 13 position is 2'-O-Me; 14 position is 2'-fluoro; 15 position is 2'-O-Me; 16 position is 2'-fluoro; 17 position is 2'-O-Me; 18 position is 2'-fluoro; 19 position is 2'-O-Me; 20 position is 2'-fluoro; 21 position is 2'-O-Me; 22 position is 2'-fluoro; 23-24 positions are 2'-O-Me; 25-26 positions are 2-OMe modified; 25-26 positions are phosphorothioate bonds SEQ ID NO: 44: miR-135 mimic oligo; position 1 is 5' phosphorylated; position 1 is 2-O-MOE-5-Me; positions 1-3 are phosphorothioate; position 2 is 2'-fluoro; position 2 is 2'-fluoro; position 3 is 2'-O-Me; position 4 is 2'-fluoro; positions 4-5 are phosphorothioate; position 5 is 2'-O-Me; position 6 is 2'-fluoro; positions 6-7 are phosphorothioate; positions 7-7 are 2'-O-Me; position 8 is 2'-fluoro; positions 8-9 are phosphorothioate; position 9 is 2'-O-Me; position 10 is 2'-fluoro; position 10- Position 11 is phosphorothioate; position 11 is 2'-O-Me; position 12 is 2'-fluoro; positions 12-13 are phosphorothioate; position 13 is 2'-O-Me; position 14 is 2'-fluoro; positions 14-26 are phosphorothioate; position 15 is 2'-O-Me; position 16 is 2'-fluoro; position 17 is 2'-O-Me; position 18 is 2'-fluoro; position 19 is 2'-O-Me; position 20 is 2'-fluoro; position 21 is 2'-O-Me; position 22 is 2'-fluoro; positions 23-24 are 2'-O-Me; positions 25-26 are 2-OMe modified SEQ ID NO: 45: miR-135 mimic oligo; position 1 is 5' phosphorylated; position 1 is 2-O-MOE-5-Me; positions 1-3 are phosphorothioate; position 2 is 2'-fluoro; positions 4-5 are phosphorothioate; positions 6-7 are phosphorothioate; positions 8-9 are phosphorothioate; positions 10-11 are phosphorothioate; position 10 is 2'-fluoro; position 11 is 2'-O-Me; Positions 12-13 are phosphorothioate bonds; positions 14-26 are phosphorothioate bonds; position 15 is 2'-O-Me; position 16 is 2'-fluoro; position 17 is 2'-O-Me; position 18 is 2'-fluoro; position 19 is 2'-O-Me; position 20 is 2'-fluoro; position 21 is 2'-O-Me; position 22 is 2'-fluoro; positions 23-24 are 2'-O-Me; positions 25-26 are 2-OMe modified SEQ ID NO: 46: miR-135 mimic oligo; position 1 is 5' phosphorylated; position 1 is 2-O-MOE-5-Me; positions 1-2 are phosphorothioate; position 2 is 2'-fluoro; positions 10-11 are phosphorothioate; position 10 is 2'-fluoro; position 11 is 2'-O-Me; positions 15-26 are phosphorothioate; position 15 is 2'-O-Me; position 16 is 2'-fluoro; position 17 is 2'-O-Me; position 18 is 2'-fluoro; position 19 is 2'-O-Me; position 20 is 2'-fluoro; position 21 is 2'-O-Me; position 22 is 2'-fluoro; positions 23-24 are 2'-O-Me; positions 25-26 are 2-OMe modified SEQ ID NO: 47: miR-135 mimic oligo; positions 1-2 are 2'-O-Me; position 1 can be conjugated to sertraline or other cell targeting moiety at the 5' end; positions 1-2 are phosphorothioate linkages; positions 22-23 are phosphorothioate linkages; position 23 is 2'-O-Me

Claims

1. A composition comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b shown in SEQ ID NO:37 and the complementary strand shown in SEQ ID NO:

40.

2. The composition of claim 1, wherein the miR-135 molecule comprises 50 or fewer nucleic acids.

3. The composition of claim 1 or 2, wherein the nucleic acid sequence of miR-135b shown in SEQ ID NO: 37 and the complementary strand shown in SEQ ID NO: 40 are 100% complementary over the entire length of each of SEQ ID NO: 37 and SEQ ID NO:

40.

4. The composition according to any one of claims 1 to 3, wherein the nucleic acid sequence of the miR-135b and / or the complementary strand comprises one or more modifications selected from the group consisting of a sugar modification, a nucleobase modification, and an internucleotide bond modification.

5. 5. The composition of claim 4, wherein the sugar modification is selected from the group consisting of 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-fluoro (2'-F), locked nucleic acid (LNA) and 2'-fluoroarabino oligonucleotide (FANA).

6. A composition comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b as set forth in any one of SEQ ID NOs: 10, 16 or 41-46, and the complementary strand as set forth in any one of SEQ ID NOs: 13 or 47.

7. The composition of claim 6, wherein the nucleic acid sequence of the miR-135b is set forth in SEQ ID NO:42 and the complementary strand is set forth in SEQ ID NO:

13.

8. The composition of any one of claims 1 to 7, comprising a nucleic acid construct of a synthetic miR-135 molecule.

9. (i) a composition according to any one of claims 1 to 7, comprising a synthetic miR-135 molecule; (ii) a cell-targeting moiety; and A conjugate comprising:

10. The conjugate of claim 9 , wherein the cell targeting moiety is conjugated to the 5′ end of the nucleic acid sequence of the complementary strand.

11. The conjugate of claim 9 or 10, wherein the synthetic miR-135 molecule and the cell targeting moiety are linked by a linking group.

12. 10. The conjugate of claim 9, wherein the cell targeting moiety is a selective serotonin reuptake inhibitor (SSRI) selected from the group consisting of sertraline, sertraline-structural analogs, fluoxetine, fluvoxamine, paroxetine, indalpine, zimelidine, citalopram, dapoxetine, escitalopram, and mixtures thereof.

13. 13. The conjugate of claim 12, wherein when the cell targeting moiety is sertraline, the conjugate has any of the following structures: 【Chemistry 1】 【Chemistry 2】

14. A therapeutically effective amount of the composition of any one of claims 1 to 8 for treating a CNS-related condition, or a depression-related disorder, or a cancer disease in a subject in need of such treatment.

15. A therapeutically effective amount of the composition described in claim 14, wherein the CNS-related condition is an anxiety disorder.

16. A therapeutically effective amount of the conjugate of any one of claims 9 to 13 for treating a CNS-related condition, or a depression-related disorder, or a cancerous disease in a subject in need of such treatment.

17. A therapeutically effective amount of the conjugate described in claim 16, wherein the CNS-related condition is an anxiety disorder.

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