CN117440816A - Modified MIR-135, conjugated forms thereof and uses thereof - Google Patents

Abstract

Disclosed are compositions of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 37 and a complementary strand shown in SEQ ID NO. 40. Also disclosed are conjugates comprising a composition of matter comprising a synthetic miR-135 molecule and a cell targeting moiety.

Description

Modified MIR-135, conjugated forms thereof and uses thereof

RELATED APPLICATIONS

The present application claims priority from U.S. provisional patent application Ser. No. 63/138,555, filed on 1 month 18 of 2021, and U.S. provisional patent application Ser. No. 63/272,329, filed on 10 month 27 of 2021, the disclosures of which are incorporated herein by reference in their entireties.

Statement of sequence Listing

An ASCll file created at 2022, month 1, 17, entitled 89938Sequence Listing.txt filed concurrently herewith, which file is incorporated herein by reference, comprises 21,023 bytes.

Technical field and background art

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

Mood disorders, such as major depressive disorder and anxiety disorder, are some of the most common and increasing health problems worldwide, affecting about 10% of the population. Despite decades of research, little is known about the pathogenesis, susceptibility, and mechanisms behind the available therapies for depression. Currently, only about one third of patients respond to available treatments, and therefore, a better understanding of pathology is highly desirable.

Current dogma (dog ma) on the etiology of depression is a complex interaction between environmental factors and genetic susceptibility, suggesting a mechanistic role for the epigenetic process.

Serotonin (5 HT) is a monoamine neurotransmitter produced in the brain by the nucleus of the central office (RN), which is widely projected throughout the brain to regulate various cognitive, emotional and physiological functions. The link between deregulated serotonergic activity and depression has been well documented [ Michelsen ka et al, brain experiment review (Brain Res Rev) (2007) 55 (2): 329-42]. In depression, the level of 5HT, as well as the genetic circuits responsible for its production, secretion, reuptake and inactivation, are deregulated. Furthermore, most currently available antidepressants target the function of 5HT system-related proteins, resulting in increased 5HT levels in the synapse [ KrishnanV and Nestler EJ, nature (2008) 455:894-902]. Existing treatments require a long period of administration to observe relief of symptoms.

Micrornas (mirs) are a subset of endogenous small (about 22 nucleotides) non-coding RNA molecules that inhibit posttranscriptional gene expression. MiR is transcribed into primary-miR molecules, processed into precursor miR with stem-loop structure in the nucleus, exported into the cytoplasm where they are further processed into viable mature miR. Mature mirs are then integrated into RNA-induced silencing complexes and function primarily by binding to the 3 'untranslated regions (3' utrs) of specific mRNA molecules. Binding occurs through a seed sequence, which is a 6 to 8 nucleotide sequence at the 5 'end of the miR, whose bases pair with complementary seed matching sequences on the target mRNA 3' utr. Binding of miR results in direct mRNA destabilization or translational inhibition, ultimately resulting in reduced protein levels of the target gene.

MiR is abundant in the nervous system, and initial research has focused mainly on neurons in developmental, cancer and neurodegenerative diseases, as well as normal processes (e.g., plasticity) [ Kosik KS, journal of 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 R.M. et al, molecular Psychiatry (2012) 17:359-376]. In addition, miR has been suggested to play a role in mental diseases such as schizophrenia, autism, and depression and anxiety in human and mouse models [ Miller BH and Wahlestedt C, brain research (Brain Res) (2010) 1338:89-99 and issler O and Chen A, nature review of the nervous system (Nat Rev Neurosci) (2015) 16:201-212]. Several recent studies have shown that miR is involved in the regulation of 5 HT-related genes [ Millan MJ, contemporary pharmacological opinion (Curr Opin Pharmacol) (2011) 11 (1): 11-22], revealing a novel role for miR in 5HT system regulation and its potential association with depression-related disorders.

PCT application publication No. WO 2013/018060 discloses mirs (e.g., miR-135) for the treatment and diagnosis of serotonin-, epinephrine-, norepinephrine-, glutamate-and corticotropin-releasing hormone related medical conditions, and compositions comprising the same.

PCT application publication No. WO 2015/118537 discloses a method of treating bipolar disorders 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.

U.S. patent application US20170037404 discloses methods and compositions for introducing miRNA activity or function into cells using synthetic nucleic acid molecules. The synthesized nucleic acid molecules may be modified.

PCT application publication No. WO/2011/131693 discloses a conjugate comprising (i) a nucleic acid that is complementary to a target nucleic acid sequence and whose expression prevents or reduces the expression of a target nucleic acid (e.g., miRNA), and (ii) a selective agent capable of binding with high affinity to a neurotransmitter transporter (e.g., a serotonin reuptake inhibitor). WO/2011/131693 contemplates the use of these conjugates to deliver nucleic acids to cells of interest, for the treatment of diseases and for diagnostic purposes.

Disclosure of Invention

According to an aspect of some embodiments of the invention there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 37 and a complementary strand shown in SEQ ID NO. 40.

According to an aspect of some embodiments of the invention there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in any one of SEQ ID NOS 10, 16 or 41-46 and a complementary strand shown in any one of SEQ ID NOS 13 or 47.

According to an aspect of some embodiments of the invention there is provided a composition of matter comprising a nucleic acid construct of a synthetic miR-135 molecule of some embodiments of the invention.

According to an aspect of some embodiments of the present invention there is provided a conjugate comprising:

(i) A composition of matter comprising a synthetic miR-135 molecule of some embodiments of the invention; and

(b) A cell targeting moiety.

According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising a composition of matter of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, and a pharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present invention there is provided a method of treating a Central Nervous System (CNS) -related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter of some embodiments of the present invention or a conjugate of some embodiments of the present invention, thereby treating the CNS-related disorder.

According to an aspect of some embodiments of the present invention there is provided a method of treating a depression-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter of some embodiments of the present invention or a conjugate of some embodiments of the present invention, thereby treating the depression-related disorder.

According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter of some embodiments of the present invention or a conjugate of some embodiments of the present invention, thereby treating the cancer.

According to an aspect of some embodiments of the present invention there is provided a method of promoting bone regeneration or muscle cell differentiation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter 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.

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a composition of matter of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for use in treating a CNS-related disorder in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a composition of matter of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for use in treating a depression-related disorder in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a composition of matter of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for use in treating cancer in a subject in need thereof.

According to an aspect of some embodiments of the present invention there is provided a therapeutically effective amount of a composition of matter of some embodiments of the present invention, or a conjugate of some embodiments of the present invention, for use in the treatment of a bone-related disease or disorder or a muscle-related disease or disorder in a subject in need thereof.

According to some embodiments of the invention, the miR-135 molecule comprises no more than 50 nucleic acids.

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 located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

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 form a hairpin loop structure.

According to some embodiments of the invention, 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 SEQ ID NO. 37 and SEQ ID NO. 40.

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

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' -fluoroarabinooligonucleotide (F-ANA).

According to some embodiments of the invention, the saccharide in the miR-135b is modified in at least one nucleotide at the 3' end of the nucleic acid sequence.

According to some embodiments of the invention, the saccharide in the miR-135b is modified in at least one nucleotide at the 5' end of the nucleic acid sequence.

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

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

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.

According to some embodiments of the invention, the sugar modifications are 2' -O-methoxyethyl (2 ' -O-MOE) and 5' -ribomethylation (2 ' -O-MOE-5' -Me).

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, methylphosphonates, alkylphosphonates, chiral phosphonates, phosphinates, phosphoramidates, aminoalkyl phosphoramidates, thiocarbonyl phosphoramidates, thionalkyl phosphates, thionalkyl phosphotriesters, borane phosphate, phosphodiester, phosphonoacetate (PACE) and Peptide Nucleic Acids (PNA).

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

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

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

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

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

According to some embodiments of the invention, the nucleic acid sequence comprising the modified miR-135b is shown in any one of SEQ ID NOs 10, 16 or 41-46.

According to some embodiments of the invention, the nucleic acid sequence comprising the modified complementary strand is as shown in any one of SEQ ID NOs 13 or 47.

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.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO. 41, and the complementary strand is shown in SEQ ID NO. 13.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO. 42, and the complementary strand is shown in SEQ ID NO. 13.

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. 47.

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.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO. 41, and the complementary strand is shown in SEQ ID NO. 47.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b is shown in SEQ ID NO. 42, and the complementary strand is shown in SEQ ID NO. 47.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b as shown in any one of SEQ ID NOs 10, 16 or 41-46 and the complementary strand as shown in any one of SEQ ID NOs 13 or 47 are located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b as shown in any one of SEQ ID NOS 10, 16 or 41-46 and the complementary strand as shown in any one of SEQ ID NOS 13 or 47 form a hairpin loop structure.

According to some embodiments of the invention, the nucleic acid sequence of miR-135b as shown in any one of SEQ ID NOS 10, 16 or 41-46 is 100% complementary to the complementary strand as shown in any one of SEQ ID NOS 13 or 47.

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.

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

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.

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

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

According to some embodiments of the invention, the linking group comprises a compound selected from the group consisting of phosphodiester, phosphoramidite, phosphorothioate, carbamate, methylphosphonate, guanidine, sulfamate, sulfonamide, methylal (formatotal), thiomethylal, sulfone, amide, and mixtures thereof.

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

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

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

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-adrenoceptor reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NASSA), noradrenergic Reuptake Inhibitors (NRIs), dopamine Reuptake Inhibitors (DRI), endogenous cannabinoid reuptake inhibitors (ecbris), adenosine reuptake inhibitors (AdoRI), excitatory Amino Acid Reuptake Inhibitors (EAARI), glutamate reuptake inhibitors (GluRI), GABA Reuptake Inhibitors (GRI), glycine reuptake inhibitors (GlyRI) and noradrenergic-dopamine reuptake inhibitors (NDRI).

According to some embodiments of the invention, the Selective Serotonin Reuptake Inhibitor (SSRI) is selected from the group consisting of: sertraline, sertraline structural analogues, fluoxetine, fluvoxamine, paroxetine, indacene, zimeldine, citalopram, dapoxetine, escitalopram and mixtures thereof.

According to some embodiments of the invention, when the cell targeting moiety is house Qu Linshi, the conjugate has the following structure:

according to some embodiments of the invention, when the cell targeting moiety is house Qu Linshi, the conjugate has the following structure:

according to some embodiments of the invention, when the cell targeting moiety is house Qu Linshi, the conjugate has the following structure:

according to some embodiments of the invention, when the cell targeting moiety is house Qu Linshi, the conjugate has the following structure:

according to some embodiments of the invention, the cell targeting moiety specifically binds to a tumor associated antigen.

According to some embodiments of the invention, the cell targeting moiety specifically binds to a molecule expressed or presented on bone cells, muscle cells or cells of the gastrointestinal tract.

According to some embodiments of the invention, the composition of matter or conjugate of some embodiments of the invention further comprises at least one of a cell penetrating moiety or a moiety for transport across the Blood Brain Barrier (BBB).

According to some embodiments of the invention, the composition of matter or conjugate of some embodiments of the invention further comprises a linker between the cell penetrating moiety or the moiety for transport across the BBB and the synthetic miR-135 molecule.

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

According to some embodiments of the invention, the administration is achieved by a mode of administration selected from the group consisting of: intranasal, intraventricular, intrathecal, oral, topical injection, and intravenous.

According to some embodiments of the invention, the composition is for intranasal, intraventricular, intrathecal, oral, topical injection or intravenous administration.

According to some embodiments of the invention, the CNS-related disorder is a psychotic disorder.

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

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

According to some embodiments of the invention, the cancer is selected from the group consisting of ovarian cancer, colorectal cancer, and prostate cancer.

According to some embodiments of the invention, the subject has a bone-related disease or disorder selected from the group consisting of osteoporosis, bone fracture or defect, primary or secondary hyperparathyroidism, osteoarthritis, periodontal disease or defect, osteolytic bone disease, post-plastic surgery, post-orthopedic implant, post-implant dental.

According to some embodiments of the invention, the subject suffers from a muscle-related disease or disorder selected from the group consisting of: muscle degeneration diseases, neuromuscular diseases, spinal muscular atrophy, inflammatory muscle diseases, and metabolic muscle diseases.

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

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be necessarily limiting.

Brief description of the drawings

Some embodiments of the invention are described herein, by way of example, with reference to the accompanying drawings. Referring now in specific detail to the drawings, it is emphasized that the details shown are exemplary and are for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how the embodiments of the present invention may be embodied.

FIG. 1 depicts the synthesis of miR-135 single-stranded oligonucleotides. The synthesis was performed using a typical experimental method of solid phase synthesis on CPG (controlled pore glass) support. Typical oligonucleotide synthesis is performed by a series of cycles consisting of four steps (deprotection, coupling, capping, and oxidation) which are repeated until the majority of nucleotides are ligated (as described in detail in the "general materials and Experimental methods" section below).

FIG. 2 illustrates the synthesis of sertraline conjugated miR-135. The method of adding sertraline and a linker at the 5' end is as follows:

1. carboxyl-C ₁₀ And (3) adding a connector: after the last cycle (DMT-OFF), in the final coupling step, the carboxyl group-C is reacted ₁₀ The linker amide block (in its N-hydroxysuccinimide ester form) is linked to the and sequence to promote derivatization of sertraline (with C ₆ -NH ₂ Conjugated sertraline) to the 5' end of an oligonucleotide.

2. Addition of sertraline: derivatized sertraline is bound to 5' -carboxy-C via an amide bond ₁₀ Modified oligonucleotidesAcid conjugation. This condensation was carried out under organic conditions (diisopropylethylamine and dimethylformamide (DIPEA/DMF) at room temperature for 24 hours as discussed in detail in the examples section below.

FIGS. 3A-B depict luciferase reporter results, indicating that Duplex 11 (Duplex 11) significantly targets both the Slc6a43'UTR (FIG. 3A) and the HTR1a 3' UTR (FIG. 3B).

FIGS. 4A-E depict the effect of miR-135 mimics on serotonergic function in vivo. Notably, systemic 8-OH-DPAT dosing (1 mg/kg Body Weight (BW), intraperitoneal injection (i.p.)) did not cause hypothermia in nude (i.e., unconjugated) miR-135 (100 μg) treated mice 24 (fig. 4A), 48 (fig. 4B), 72 (fig. 4C), and 96 (fig. 4D) hours after treatment. Bi-directional analysis of variance showed significant effect P <0.001 compared to the control (n=5-10) group. There was no difference in basal body temperature between groups (fig. 4E).

FIGS. 5A-D illustrate the effect of sertraline-conjugated miR-135 mimetic duplex 11 (miCure-135-1, shown in SEQ ID Nos. 10 and 13) on serotonergic function following direct midback nuclei (dorsal raphe nucleus, DRN) administration. Notably, systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in sertraline-conjugated miR-135 (100 μg) treated mice after 24 hours (fig. 5A), 48 hours (fig. 5B), 96 hours (fig. 5C) and 7 days (fig. 5D) of treatment. Bi-directional analysis of variance showed significant effect P <0.001 compared to the control group (n=10).

FIGS. 6A-H illustrate that acute administration of sertraline conjugated miR-135 (miCure-135-1) at a lower dose (30 μg) can silence 5HT1a and SERT and elicit an antidepressant-like response. Notably, systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in sertraline-conjugated miR-135-treated (30 μg) mice after one day of treatment (fig. 6A), two days (fig. 6B), four days (fig. 7C), and 7 days (fig. 6D). Bi-directional analysis of variance showed significant effect P <0.001 compared to the control group (n=10). There was no difference in basal body temperature between groups (fig. 6E) (n=10-40). During the tail-suspension experiment, an increase in extracellular serotonin was found in the medial prefrontal cortex (mPFC) of mocure-135-1 treated mice. P <0.05 (fig. 6F) was significant compared to the control. In the tail-suspension experiment, single intracerebral miCure-135-1 administration (30 μg) reduced immobility (n=8-9) in mice (FIG. 6G). [3H] Autoradiography of citalopram binding showed a decrease in SERT density of the dorsal nucleus of the middle suture of the treated mice compared to the control group (n=5-7) p <0.05 (fig. 6H).

FIGS. 7A-C illustrate that acute intranasal administration of miCure-135-1 (166 μg) silences 5HT1a and causes an antidepressant-like response. Notably, systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in mice treated with sertraline-conjugated miR-135 (166 μg) delivered nasally after 5 days of treatment. Bi-directional analysis of variance showed significant effect (n=5) P <0.05 compared to the control group (fig. 7A). In the tail-suspension experiment at 4 days post-treatment, single intranasal administration of mecure-135-1 (166 μg) reduced immobility of mice (n=8-9) ×p <0.05 compared to control (fig. 7B). Mice receiving a single intranasal mocure-135-1 administration (166 μg) explored the light chamber longer in the dark/light transfer assay (n=11-12) than the control group (fig. 7C) P <0.05.

FIG. 8 depicts additional miR-135 modifications. The modification is as follows:

capital letters (e.g., A, U, C, G): RNA (ribonucleic acid)

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

Lowercase letter "s": phosphorothioates. The absence of indication means normal phosphodiester linkages.

P: phosphoric acid esters

And (3) underlined: 2 '-fluoro, i.e. 2' -F.

FIGS. 9A-B depict luciferase reporter gene assay results demonstrating that MICure-135-1, MICure-135-2, MICure-135-3, MICure-135-9, MICure-135-10, and MICure-135-11 significantly target Slc6a43'UTR (FIG. 9B) and HTR1A 3' UTR (FIG. 9A). The numbers above the bars represent the number of significant statistical differences found (in 5 experiments).

FIGS. 10A-E depict the immune response of human Peripheral Blood Mononuclear Cells (PBMCs) after treatment with 3 different miR-135 conjugated duplex in vitro. Notably, the miCure-135-10 induced high secretion of TNF- α cytokines, particularly at a concentration of 300nM, whereas the miCure-135-1 moderately activated the cytokine, and the miCure-135-2 induced little secretion (FIG. 10A). IFN- α -2a cytokines have the same secretion pattern (FIG. 10B), wherein the miCure-135-10 induces high levels of secretion, the miCure-135-1 induces medium levels of secretion, and the miCure-135-2 does not induce secretion. None of the tested duplexes induced secretion of IL-10 (FIG. 10C), IL-1β (FIG. 10D) and IL-6 (FIG. 10E). In FIGS. 10A and 10C-E: dir.incubations refer to direct incubations.

FIGS. 11A-F depict the immune response of human Peripheral Blood Mononuclear Cells (PBMCs) after treatment with an additional set of 3 miR-135 conjugated duplex in vitro. Notably, miR-135-11 and miCure-135-9 induced high secretion of IFN-. Alpha. -2a, whereas miCure-135-3 induced little secretion (FIG. 11A). The miscure-135-11 and miscure-135-9 induced low levels of TNF- α secretion, wherein miscure-135-3 did not induce secretion (FIG. 11B). MICure-135-9 showed low levels of IFN-gamma secretion at 300nM concentration, whereas MICure-135-3 and MICure-135-9 did not show their secretion (FIG. 11C). None of the 3 tested duplexes resulted in significant production of cytokines IL-10 (FIG. 11D), IL-1b (FIG. 11E) and IL-6 (FIG. 11F).

FIGS. 12A-G depict the effect of sertraline conjugated miR-135 mimics (miCure-135-2, miCure-135-3 and miCure-135-9) on serotonergic function and their antidepressant-like effect. Systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in miCure-135-2 treated (30 μg) mice after 2 days (FIG. 12A), four days (FIG. 12B) and seven days (FIG. 12C) of treatment. Bi-directional analysis of variance showed significant effect P <0.05, < P <0.001 compared to the control group (n=4-5). 2 days after treatment (FIG. 12D), systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in the miCure-135-9 treated (30 μg) mice. Bi-directional analysis of variance showed significant effect P <0.05 compared to the control group (n=4). There was no difference in basal body temperature between groups (fig. 12E) (n=4-19). The infusion of a locally selective serum reuptake inhibitor (Citalopram) 10 μm) by reverse dialysis resulted in an increase in extracellular 5-hydroxytryptamine (5-HT) in PFC of sertraline conjugated control (100 μg) treated mice compared to mocure-135-3 (100 μg) (fig. 12F). Bi-directional analysis of variance showed significant effect P <0.05 compared to control group (n=6). In the tail-suspension experiment, single intracerebral miCure-135-3 administration (100 μg) reduced immobility in mice p <0.05 (FIG. 12G).

Figures 13A-F depict the effect of intranasal and Intraventricular (ICV) administration of sertraline conjugated miR-135 mimics maintenance on serotonergic function in vivo. Notably, systemic 8-OH-DPAT administration (1 mg/kg BW, intraperitoneal injection) did not cause hypothermia in mice 48 hours after treatment with miCure-135-3 (50 μg) (FIG. 13A), (100 μg) (FIG. 13B) and (200 μg) (FIG. 13C). The same effect was shown by intranasal administration of miCure-135-2 (33 μg/day) for 7 days (FIG. 13D) 24 hours after treatment and ICV administration of miCure-135-3 (200 μg/day) for 7 days (FIG. 13E) 96 hours after treatment. Bi-directional analysis of variance showed significant effect P <0.05 compared to control group (n=6). The increase in extracellular 5-hydroxytryptamine (5-HT) in PFC after infusion of the locally selective serum reuptake inhibitor (citalopram) by reverse dialysis was smaller in mice treated with intranasal administration of miCure-135-3 (200. Mu.g/day) for 7 days compared to mice treated with sertraline conjugated control (200. Mu.g/day) (FIG. 13F). Bi-directional analysis of variance showed significant effect P <0.05 compared to control group (n=6).

FIGS. 14A-G illustrate that acute intranasal administration of miCure-135-3 (2500 μg) affects serotonergic function and causes an antidepressant-like response. Immunoblots and quantification showed that p was compared to control (n=6-7) <0.05 reduced protein levels for SERT (fig. 14A-B) and HTR1AR (fig. 14C-D) for the dorsal nucleus of the midsuture in treated mice; the control group had reduced extracellular serotonin in the mPFC, but was administered 8-OH-DPAT (1 mg kg ^-1 Intraperitoneal injection), the effect is remarkable (P<0.05 (fig. 14E); the infusion of a locally selective serum reuptake inhibitor (citalopram 10 μm) by reverse dialysis resulted in an increase in extracellular 5-hydroxytryptamine (5-HT) in PFC of sertraline conjugated control (2500 μg) treated mice compared to mocure-135-3 (2500 μg) (fig. 14F); two-wayAnalysis of variance showed a P value of 0.05 compared to the control. In the tail-suspension experiment (n=8-14), single intranasal administration of mecure-135-3 (2500 μg) reduced immobility of mice p<0.05 (FIG. 14G).

FIG. 15 illustrates that acute intranasal administration of miCure-135-3 (2500 μg) elicits an antidepressant-like response in mice that undergo a depressive-like behavior induction. Mice received a 28 day regimen, induced depression-like behavior (as described in detail in the "general materials and Experimental methods" section below), followed by a single intranasal administration of control or miCure-135-3. The results show that single intranasal administration of mecure-135-3 (2500 μg) reduced immobility (n=8-9) P <0.05 in the tail-suspension experiment 3 days after treatment compared to the control group.

FIGS. 16A-D illustrate the positioning of miCure-135-3 in the dorsal aspect of the central slit following intranasal administration. Following intranasal administration (1000 μg), alexa 488-labeled miCure-135-3 (green) selectively accumulated in tryptophan hydroxylase 2-positive (TPH 2-positive) neurons. Confocal images showed co-localization of alexa 488-labeled miCure-135-3 (yellow) in the dorsal aspect of the central slit (DR) 5-HT neurons (TPH 2-positive, red). Nuclei were stained with DAPI (4, 6-diamidino-2-phenylindole; blue). Each row represents a different mouse (n=3, fig. 16A); a high magnification micrograph of the frame depicted in "example 1" of fig. 16A is provided to show co-localization of alexa 488-labeled miCure-135-3 in the dorsal aspect of the central slit (DR) 5-HT neurons (fig. 16B-C). Ruler: left = 100 μm, right = 25 μm. In contrast, alexa 488-labeled miCure-135-3 was absent from cells in brain regions near the site of application (olfactory bulb) or ventricles (hippocampus and striatum) (FIG. 16D).

FIGS. 17A-D illustrate immunohistochemical assessment of nuclear cell viability in the middle gap after acute administration of miCure-135-3 (100. Mu.g). The conjugate control, miCure-135-3, or the previously reported positive control with no effect on cell viability was delivered acutely directly to the dorsal aspect of the middleslit. Adjacent 30 μm thick sections through the midbrain middle suture nuclei were stained with neuronal NeuN (fig. 17A), microglial Iba1 (fig. 17B) or serotonergic TPH (fig. 17C) markers. Representative images of midbrain midstitch nuclei stained with NeuN, iba1 and TPH under different treatments are shown in fig. 17D. Notably, no differences were found in any of the markers between all of the experimental groups. Representative pictures are shown in fig. 17C, scale bar: 100 μm.

Detailed Description

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

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

Before explaining 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 by the examples. The invention is capable of other embodiments or of being practiced or of being 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.

Micrornas (mirs) are a subset of small RNA molecules that regulate gene expression post-transcriptionally, in a large number of tissues including the brain.

While putting the specific embodiments of the invention into practice, the inventors have synthesized novel synthetic miR-135 molecules for use in therapy. Furthermore, the inventors have discovered that miR-135 molecules can be targeted to a cell of interest by coupling the miR-135 molecule to another molecule that is capable of specifically binding to the cell of interest.

Specifically, the present inventors designed and synthesized miR-135 mimetic molecules comprising two chains (i.e., a guide chain and a passenger chain) based on endogenous miR-135, with minor modifications aimed at improving stability and cell penetration (see tables 1 and 6 below, and fig. 8). miR-135 mimetic molecules (designated as duplex 1-17 in Table 1 and duplex 1-16 in Table 6) were then screened in vitro on known miR-135 targets (e.g., htr1a and Slc6a 4). One of the synthesized mock miR-135 molecules (referred to as duplex 11 in Table 1 and duplex 1 in Table 6, as shown in SEQ ID Nos. 10 and 13) showed significantly better binding to miR-135 targets (e.g., htr1a and Slc6a 4) than the other molecules synthesized and tested in the human cell line HEK293T (see FIGS. 3A-B and example 1, below). The miR-135 mimetic molecules (i.e., duplex 11 in Table 1 and duplex 1 in Table 6) include a guide sequence for miR-135b (shown in SEQ ID NO: 37) and a complementary sequence that is different from the native sequence (shown in SEQ ID NO: 40). Furthermore, the synthesized miR-135 molecules (i.e., duplex 11 in table 1 and duplex 1 in table 6) were shown to contain in vivo serotonergic function in animal models (see figures 4A-E and example 2 below).

The present inventors further designed and synthesized sertraline-conjugated miR-135 molecules for non-invasive delivery to the brain. In vivo studies with sertraline conjugated miR-135 mimetic duplex 11 (referred to herein as miCure-135-1) showed that a single administration to the dorsal mid-slit nucleus (DRN) was sufficient to eliminate hypothermia induced by the selective HTR1a agonist 8-OH-DPAT for up to 7 days in naive mice (FIGS. 5A-D and 6A-E, and examples 3-4 below). Administration of sertraline conjugated miR-135 molecules into DRN was further shown to induce antidepressant effects and better response mechanisms in treated mice (FIG. 6F-G and example 4 below). Sertraline conjugated miR-135 molecules were also shown to silence SERT genes when administered into DRN (FIG. 6H and example 4 below). In addition, non-invasive intranasal administration of sertraline conjugated miR-135 silences 5HT1a, as shown by the lower low temperature response (FIG. 7A and example 5 below), and causes an antidepressant/anxiolytic-like response in the treated mice (FIGS. 7B-C and example 5 below).

In further experiments, the inventors revealed that other sertraline conjugated miR-135 mimics, i.e., miCure-135-1, miCure-135-2, miCure-135-3, miCure-135-9, miCure-135-10 and miCure-135-11 (see Table 6 below), comprising various chemical modifications, were effective and significantly altered 5HTR1A and SLC6a4 levels (using 3' UTR luciferase constructs, FIGS. 9A-B and example 6 below). Conjugated miR-135 mimics were tested for their effect on immune activation. As is evident from the results (see FIGS. 10A-E and 11A-F, and example 7 below), different conjugated miR-135 mimics induce different immune responses, with miCure-135-1, miCure-135-2 and miCure-135-3 inducing the PBMCs to secrete the lowest cytokines. In addition, acute administration of sertraline-conjugated miR-135 mimics miCure-135-2, miCure-135-3 and miCure-135-9 to the dorsal central office nuclei (DRN) affected serotonergic function and induced antidepressant-like responses (see FIGS. 12A-G and example 8 below). In addition, intranasal and intraventricular administration of sertraline-conjugated miR-135 mimics successfully reduced serotonergic autoreceptors (5 HT1 a) and serotonin transporters (SERT) in the dorsal nucleus of the central slit (see FIGS. 13A-F and example 9 below).

In further experiments, the inventors demonstrated that acute intranasal administration of the miR-135mimetic miCure-135-3 (the miR-135 mimetic-135-3) reduced the protein levels of SERT and 5-HT1A-auto receptor (HTR 1 AR) and had an antidepressant effect (see examples 10 and 11 below). Intranasal administration of miCure-135-3 resulted in accumulation in the dorsal aspect of the midgut, particularly in midbrain 5-HT neurons (see example 12 below). Furthermore, intranasal administration of the miR-135mimetic miCure-135-3 was found to be safe for in vivo administration (see example 13 below).

Thus, the synthetic miR-135 molecules of the invention, and conjugated forms thereof, are useful in therapy, e.g., for the treatment of CNS-related disorders, including psychotic disorders.

Thus, according to one aspect of the present invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 37 and a complementary strand shown in SEQ ID NO. 40.

According to one aspect of the present invention, there is provided a synthetic miR-135 molecule, which comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 37 and a complementary strand shown in SEQ ID NO. 40.

"synthetic" as used herein refers to non-natural molecules.

According to one embodiment, the non-naturally occurring miR-135 comprises a naturally-occurring mature miR-135b sequence, and comprises a synthetic backbone and/or side chain. Typically, the synthetic miR-135 molecules act as naturally-occurring miRNAs (e.g., miR-135 b) in cells or under physiological conditions.

As used herein, the term "miR-135b" refers to microrna molecules that are involved in posttranscriptional gene regulation, and includes a miR-135b 5 primer (i.e., miR-135b, also known as miR-135b-5 p) or a 3 primer (i.e., miR-135b, also known as miR-135b-3 p). Exemplary mature miR-135b includes, but is not limited to, miR-135b as shown in accession number MIMAT0000758 (SEQ ID NO: 37). Exemplary mature miR-135b include, but are not limited to, miR-135b as shown in accession number MIMAT0004698 (SEQ ID NO: 38).

Micrornas are typically processed from pre-miR (pre-microRNA precursors, precursor micrornas). Pre-miR is a group of precursor miRNA molecules transcribed by RNA polymerase III, which are efficiently processed into functional miRNAs, e.g., when transfected into cultured cells. Pre-miR can be used to elicit specific miRNA activity in cell types that do not normally express such miRNA, thereby addressing the function of its target by down-regulating its expression in "(miRNA) function acquisition" experiments. The Pre-miR design is present in all known mirnas listed in the miRNA database (miRNA Registry) and can be readily designed for any study. As described further below, micrornas can be administered to cells themselves or linked into nucleic acid constructs.

It is to be appreciated that micrornas of the present teachings (e.g., miR-135) can bind, attach, modulate, process, interfere with, enhance, stabilize, and/or destabilize any microrna direct or indirect target (e.g., miR-135 target). Such targets may be any molecule, including but not limited to DNA molecules; RNA molecules and polypeptides, such as, but not limited to, serotonin-related genes, such as serotonin transporter (i.e. SERT or Slc6a 4), serotonin-inhibiting receptor 1a (Htr 1 a), tryptophan hydroxylase 2 (Tph 2) and monoamine hydroxylase (MaoA); adrenergic or noradrenergic receptors (adrenergic receptors such as Adr 1); adenylate cyclase type 1 (ADCY 1); CRH receptors, such as CRH1R; or any other molecule, such as FK506 binding protein 5 (FKBP 5), transferrin-related protein X (Tsnax), and cell adhesion molecule L1 (L1 cam); as well as other targets associated with mental disorders, including those listed in table 7 below.

Additional direct or indirect targets include, but are not limited to: adenylate cyclase activating polypeptide 1 (adacycle 1 or PACAP); adenylate cyclase activates polypeptide 1 receptor 1 (adapter 1r 1); alpha 2a adrenergic receptor (Adra 2 a); ankyrin 3 (ANK 3); cell backbone-related proteins (Arc) with modulated activity; rho gtpase activator protein 6 (Arhgap 6); activating transcription factor 3 (Atf 3); beta-site APP cleavage enzyme 1 (Bace 1); an L-type voltage-dependent calcium ion channel alpha 1D subunit (Cacna 1D); cell adhesion molecule 3 (wdm 3); complex 1 (Cplx 1); complex 2 (Cplx 2); CUB and Sushi diverse domain 1 (Csmd 1); casein kinase 1 γ1 (Csnk 1g 1); biscorticoids (Dcx); DIRAS family GTP binds RAS-like 2 (DIRAS 2); dis, large homolog 2 (drosophila) (Dlg 2); ELK1, ETS oncogene family member (ELK 1); fyn-related kinase (Frk); fucosyltransferase 9 ((α (1, 3) fucosyltransferase) (Fut 9); γ -aminobutyric acid (GABA-A) receptor β2 subunit (Gabrb 2); GATA binding protein 3 (GATA 3); growth hormone secretagogue receptor (Ghsr); G protein coupled receptor 3 (Gpr 3); AMPA3 (. Alpha.3) ionogenic glutamate receptor 3 (GRIA 3); rhodopsin ionogenic glutamate receptor 3 (Grik 3); G protein coupled receptor kinase 5 (Grik 5); glycogen synthase kinase-3 β (GSK 3B); hyperpolarized activated cyclic nucleotide gated potassium ion channel 1 (Hcn 1), hyperpolarized activated cyclic nucleotide gated k+2 (Hcn 2), 5-hydroxytryptamine receptor 1A (Htr 1A); inositol monophosphate (IMPA 1), potassium protein rhorn f kinase (kal), potassium intermediate/small calcium activated channel subfamily N member 3 (nn 3); nuclear transport protein α3 (Grik 3); nuclear transport protein input 4 (. Alpha.4) (nuclear protein 1); nuclear transcription factor 1 (mylar 1, nuclear 2 receptor 1, nuclear transcription 1-like 2 receptor 1 (mylar 1), member 2 (Nr 3c 2); spindle protein G1 (Ntng 1); nucleocasein kinase and cyclin-dependent kinase substrate 1 (Nucks 1); calmodulin-dependent phosphodiesterase (Pde 1 a); cAMP-specific phosphodiesterase 4A (Pde 4A); phosphodiesterase 8B (Pde 8B); phospholipase cβ1 (Plcb 1); prolactin receptor (Prlr); RAS oncogene family member RAB1B (Rab 1B); ras related protein Rap-2a (Rap 2 a); retinoid-related orphan receptor beta (Rorb); sirtuin (Sirt 1) 1 (Sirt mating message regulatory 2 homolog); solute carrier family 12 (potassium/chloride transporter), member 6 (Slc 12a 6); solute carrier family 5 (choline transporter), member 7 (Slc 5a 7); solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 (Slc 6a 4); trans-acting transcription factor 1 (Sp 1); synaptic membrane bubble glycoprotein 2b (Sv 2 b); the synuclein protein gene (encoding nephrin-1) (Syne 1); synaptotagmin I (Syt 1); synaptotagmin II (Syt 2); synaptotagmin III (Syt 3); transforming growth factor beta receptor II (Tgfbr 2); thyroid hormone beta receptor (Thrb); transient receptor potential cation channel, subfamily C, member 6 (Trpc 6); vesicle associated membrane protein 2 (Vamp 2); wingless MMTV integration site family member 3 (Wnt 3); zinc finger protein BED4 antibody (Zbed 4).

Table 7: putative targets of miR-135 associated with mental diseases

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TABLE 7, succession

As used herein, "nucleic acid" generally refers to a molecule (single-or double-stranded oligomer or polymer) comprising RNA of nucleobases or derivatives, mimics or analogs thereof. Nucleobases include, for example, naturally occurring purine or pyrimidine bases found in RNA (e.g., adenine "a", guanine "G", uracil "U", or cytosine "C"). The term "nucleic acid" includes the terms "oligonucleotide" and "polynucleotide", each of which is a subgenera of the term "nucleic acid". The term "nucleic acid" also includes synthetic polynucleotide and/or oligonucleotide molecules derived from compositions of naturally occurring bases, sugars, and covalent internucleoside linkages (e.g., backbones), as well as nucleic acids of synthetic polynucleotides and/or oligonucleotides having non-naturally occurring portions that have similar functions to the respective naturally occurring portions. Such modified or substituted oligonucleotides may be preferred over the natural form because of their desirable properties, e.g., enhanced cellular uptake, enhanced affinity for nucleic acid targets, and enhanced stability in the presence of nucleases, as discussed in further detail below.

The term "mimetic" or "analog" refers to a molecule that may or may not be structurally similar to a naturally occurring molecule, but has a similar function.

The synthetic miR-135 molecules of some embodiments of the invention are double-stranded nucleic acid molecules comprising miR-135b and a complementary strand. Such double stranded nucleic acid molecules are similar in structure to naturally occurring miRNA precursors and can be bound by cellular protein complexes and processed into active mature mirnas.

According to a specific embodiment, miR-135b (also referred to as a "guide strand" or "active strand") comprises a sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence homology or identity to endogenous mature miR-135 b. According to a specific embodiment, miR-135b comprises a sequence that is equivalent (e.g., identical) to endogenous mature miR-135 b.

As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes residues that are identical when aligned in the two sequences. When using percentages of sequence identity in proteins, it is recognized that different residue positions typically differ by conservative amino acid substitutions, where an amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) and therefore does not alter the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upward to correct the conservative nature of the substitution. Sequences that differ by such conservative substitutions are considered to have "sequence similarity" or "similarity". Methods for making such adjustments are well known to those skilled in the art. Typically, this involves scoring conservative substitutions as partial rather than complete mismatches, thereby increasing the percent sequence identity. Thus, for example, when the same amino acid score is 1 and the non-conservative substitution score is zero, the conservative substitution score is between 0 and 1. For example, scores for conservative substitutions are calculated according to algorithms such as Henikoff S and Henikoff JG [ amino acid substitution matrices from protein blocks, proc. Natl. Acad. Sci. U.S.A.), 1992,89 (22): 10915-9 ].

Any homology comparison software may be used, including, for example, blastN software of the National Center for Biotechnology Information (NCBI), for example, by determining identity (e.g., percent homology) using default parameters.

According to some embodiments of the invention, the identity is global identity (global identity), i.e. the identity of the entire nucleic acid sequence of the invention, but not of parts thereof.

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

According to some embodiments of the invention, the homology is global homology (global homology), i.e. the homology of the entire nucleic acid sequence of the invention, but not of parts thereof.

Various known sequence comparison tools may be used to determine the degree of homology or identity between two or more sequences. The following is a non-limiting description of such tools that may be used with some embodiments of the present invention.

When starting from a polynucleotide sequence and comparing to other polynucleotide sequences, the EMBOSS-6.0.1 Nedemann-Wells algorithm (available from Emboss. Sourcefuge. Net/apps/cvs/Emboss/apps/needle. Html) may be used.

According to some embodiments, the determination of the degree of homology also requires the use of a Smith-Waterman algorithm (for protein-protein comparison or nucleotide-nucleotide comparison).

According to some embodiments of the invention, the sequence preselected by overall homology (e.g., 60% identity over 60% sequence length) to the polypeptide or polynucleotide of interest is subjected to overall homology prior to overall homology (e.g., 80% overall homology over the entire sequence) to the polypeptide or polynucleotide of interest. For example, BLAST software is used to select homologous sequences, the first stage using Blastp and tBlastn algorithms as filters, and the second stage using needle (EMBOSS package) or frame+ algorithm alignment. The definition of local identity (Blast alignment) has a very loose cut-off-60% identity over a span of 60% sequence length, since it only acts as a filter for the global alignment stage. In this particular embodiment (when local identity is used), default filtering of Blast packages (by setting parameters "-F F") is not used. In the second stage, homologs are defined based on at least 80% global identity to the core gene polypeptide sequence.

According to a specific embodiment, miR-135b comprises the sequence shown in SEQ ID NO. 37.

According to a specific embodiment, miR-135b comprises a sequence shown in any one of SEQ ID NOs 1, 5, 7, 8, 10, 14, 16, 41, 42, 43, 44, 45 or 46.

According to a specific embodiment, the complementary strand (also referred to herein as the "passenger strand") of the synthetic miR-135 molecule comprises a sequence that is at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to the mature miR-135b sequence.

According to one embodiment, the complementary strand comprises a sequence that is 100% complementary (i.e., perfectly matched) to the guide strand.

According to one embodiment, the complementary strand comprises the sequence shown in SEQ ID NO. 40.

According to one embodiment, the complementary strand comprises the sequence shown in SEQ ID NO. 2, 3, 4, 6, 9, 11, 12, 13, 15 or 47.

According to specific embodiments, the nucleic acid sequence of miR-135b (e.g., as shown in SEQ ID NO: 37) and the nucleic acid sequence of the complementary strand (e.g., as shown in SEQ ID NO: 40) are 100% complementary over the entire nucleic acid sequence (e.g., over the entire lengths of SEQ ID NO:37 and SEQ ID NO: 40).

According to a specific embodiment, there is no single stranded overhang (overs) between the guide strand and the passenger strand of the synthetic miR-135 molecules of some embodiments of this invention.

According to one embodiment, the synthesized miR-135 molecule comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 37.

According to one embodiment, the synthetic miR-135 molecule comprises the complementary strand shown in SEQ ID NO. 40.

The synthetic miR-135 molecules of the invention are optionally 100 nucleotides or less, optionally 90 nucleotides or less, optionally 80 nucleotides or less, optionally 70 nucleotides or less, optionally 60 nucleotides or less, optionally 55 nucleotides or less, or optionally 50 nucleotides or less in length.

According to one embodiment, the synthetic miR-135 molecule comprises 46 to 50 residues.

According to one embodiment, the synthetic miR-135 molecule comprises 46 to 55 residues.

According to one embodiment, the synthetic miR-135 molecule comprises 46 to 60 residues.

According to one embodiment, the synthetic miR-135 molecule comprises 46 to 70 residues.

According to one embodiment, the synthetic miR-135 molecule comprises 46 to 80 residues.

According to a specific embodiment, the synthetic miR-135 molecule comprises 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.

According to specific embodiments, the synthetic miR-135 molecule comprises 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.

According to a specific embodiment, the synthetic miR-135 molecule contains no more than 52 nucleic acids.

According to a specific embodiment, the synthetic miR-135 molecule contains no more than 50 nucleic acids.

According to a specific embodiment, the synthetic miR-135 molecule contains no more than 48 nucleic acids.

According to a specific embodiment, the synthetic miR-135 molecule comprises 52 nucleic acids.

According to a specific embodiment, the synthesized miR-135 molecule comprises 50 nucleic acids.

According to a specific embodiment, the synthetic miR-135 molecule comprises 48 nucleic acids.

According to a specific embodiment, the synthetic miR-135 molecule comprises 46 nucleic acids.

According to one embodiment, the synthesized miR-135 molecule does not contain a junction region. In this case, the miR-135b region and the complementary region (i.e., the guide and passenger strand, respectively) are independent and anneal as a duplex.

According to 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 located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

According to 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 are located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

According to 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.

According to 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.

According to one embodiment, the synthetic miR-135 molecule comprises a junction region between a nucleic acid sequence (e.g., a guide strand) of miR-135b and a nucleic acid sequence of a complementary sequence (e.g., a passenger strand). Such a junction region may create a hairpin loop. Thus, the synthetic miR-135 molecules of some embodiments of the invention are capable of forming hairpin loop structures due to binding between the miR-135b region and the complementary region of the molecule.

According to one embodiment, the linking region comprises 2 to 30 residues.

According to one embodiment, the linking 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.

According to one embodiment, the linking region comprises 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.

According to one embodiment, the synthetic miR-135 molecule can include flanking sequences located 5 'or 3' of the miR-135b (e.g., the guide strand) and/or the complementary sequence (e.g., the passenger strand).

According to one embodiment, there are at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides on one or both sides of the miR-135b (e.g., guide strand) and/or complementary sequence (e.g., passenger strand).

According to one embodiment, miR-135b (e.g., the guide strand) and/or a complementary sequence (e.g., the passenger strand) does not contain a flanking sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule (e.g., miR-135b and/or the complementary strand) comprises one or more modifications.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule (e.g., miR-135b and/or the complementary strand thereof) comprises a single modification in comparison to the endogenous mature miR-135b sequence or its complementary strand (also referred to as the native miR-135b sequence).

According to another embodiment, the nucleic acid sequence of the miR-135 molecule (e.g., the nucleic acid sequence of miR-135b and/or the complementary strand) comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more modifications, as compared to the native miR-135b sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule comprises 1 to 2 modifications compared to the native miR-135b sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule comprises 3 to 4 modifications compared to the native miR-135b sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule comprises 5 to 10 modifications compared to the native miR-135b sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule comprises 10 to 15 modifications compared to the native miR-135b sequence.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule comprises 16 to 26 modifications compared to the native miR-135b sequence.

Thus, the synthetic miR-135 molecules of the invention can be synthesized to include modifications that confer desired characteristics. For example, the modification can improve stability, hybridization thermodynamics with the target nucleic acid, targeting a particular tissue or cell type, or cell permeability, e.g., by endocytosis-dependent or endocytosis-independent mechanisms. Modification can also increase sequence specificity and thus decrease non-site targeting.

According to one embodiment, the synthetic miR-135 molecule comprises a modification selected from the group consisting of an insertion, deletion, substitution, or point mutation of a nucleic acid, provided that the molecule retains at least about 90%, 95%, 99% or 100% of its biological activity (e.g., miR-135 silencing activity).

According to one embodiment, the synthetic miR-135 molecules include 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) as well as the pyrimidine bases thymine (T), cytosine (C) and uracil (U). "modified" bases include, but are not limited to, 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-mercaptocytosine; 5-halouracils and cytosines; 5-propynyluracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine; 5-halogen, in particular 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; 3-deazaguanine and 3-deazaadenine. Additional modified bases include those disclosed in the following documents: U.S. Pat. nos. 3,687,808; kroschwitz, j.i. edit (1990), "encyclopedia of polymer science and engineering," pages 858-859, john wili father-child publishing company (John Wiley & Sons); englisch et al (1991), "application chemistry (Angewandte Chemie)", international version 30,613; and Sanghvi, y.s., "antisense research and applications (Antisense Research and Applications)", chapter 15, pages 289-302, s.t. rooke and b.lebleu editions, 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 nucleic acid duplex stability by 0.6℃to 1.2 ℃ (Sanghvi, Y.S. et al (1993), "antisense research and applications", pages 276-278, CRC Press, bokapton) and are presently preferred base substitutions, especially when combined with 2' -O-methoxyethyl sugar modifications. Other base modifications are described in Deleavey and Damha, chemistry and biology (Chemistry and Biology) (2012) 19:937-954, which are incorporated herein by reference.

According to one embodiment, the modification is a chemical modification.

According to one embodiment, the synthetic miR-135 molecules of the invention can have a chemical modification on a nucleotide in an internal (i.e., non-terminal) region that is not complementary to the target nucleic acid. For example, modified nucleotides may be incorporated into the bulge-forming region of the miRNA. Modification may include, for example, a ligand linked to the miRNA via a linker. Modifications may, for example, improve the pharmacokinetics or stability of the polynucleotide, or improve the hybridization characteristics (e.g., hybridization thermodynamics) of the polynucleotide to the target nucleic acid.

In some embodiments, the direction of the modification or ligand incorporated into or attached to the protruding region of the polynucleotide is oriented to occupy space in the protruding region. For example, the modification may include a modified base or sugar on the nucleic acid strand or a ligand as an intercalator. These are preferably located in the protrusions. The intercalator may be an aromatic compound, such as a polycyclic aromatic compound or a heterocyclic aromatic compound. Polycyclic intercalators may have stacking capability and may include systems having 2, 3 or 4 fused rings. In some embodiments, the direction of the modification or ligand incorporated into or attached to the protruding region of the polynucleotide is oriented to occupy space in the protruding region. Such orientation may contribute to an improvement in hybridization properties or other desired properties of the polynucleotide.

In one embodiment, the synthetic miR-135 molecules can include aminoglycoside ligands, which can confer improved hybridization properties or improved sequence specificity to the polynucleotide. Exemplary aminoglycosides include glycosylated polylysines; galactosylated polylysine; neomycin B; tobramycin; kanamycin A; and aminoglycoside acridine conjugates, such as neo-N-acridine, neo-S-acridine, neo-C-acridine, tobra-N-acridine and Kanaa-N-acridine. The use of acridine analogs can increase sequence specificity. For example, neomycin B has a high affinity for RNA but low sequence specificity compared to DNA. In some embodiments, a guanidine analog of an aminoglycoside (guanidyl glycoside) is linked to a polynucleotide agent. In the guanidyl glycoside, the amine group on the amino acid is substituted with a guanidyl group. Ligation of guanidine analogs can enhance cell permeability of polynucleotides.

To increase nuclease resistance and/or binding affinity to a target, the synthetic miR-135 molecules of the invention can include 2' -O-methyl, 2' -fluoro, 2' -O-methoxyethyl, 2' -O-aminopropyl, 2' -amino, and/or phosphorothioate linkages. Nucleic acid analogs comprising Locked Nucleic Acids (LNA), e.g., nucleic acid analogs comprising a "locked" ribose ring with methylene bridges connecting the 2'-O atom and the 4' -C atom; ethylene Nucleic Acids (ENA), such as 2'-4' -ethylene bridge nucleic acids; and certain nucleobase modifications, such as 2-amino-a, 2-thio (e.g., 2-thio-U) (G-clamp modifications), can also increase binding affinity to a target. The inclusion of pyranose in the oligonucleotide backbone can also reduce endoribonucleolysis.

The miR-135 molecules can be further modified by inclusion of a 3' cationic group or by ligation of inverted terminal nucleosides with 3' -3 '. In another alternative, the 3 '-terminus may be blocked with an aminoalkyl group, such as 3' C5-aminoalkyldT. Other 3' conjugates can inhibit 3' -5' exonuclease cleavage. While not being bound by theory, 3 'conjugates, such as naproxen or ibuprofen, can inhibit exonuclease cleavage by sterically blocking the binding of an exonuclease to the 3' end of an oligonucleotide. Even small alkyl chains, aryl or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5' -exonucleases.

According to one embodiment, the 5 '-end may be blocked with an aminoalkyl group (e.g., a 5' -O-alkylamino substituent). Other 5' conjugates can inhibit 5' -3' exonuclease cleavage. While not being bound by theory, 5 'conjugates, such as naproxen or ibuprofen, can inhibit exonuclease cleavage by sterically blocking the binding of an exonuclease to the 5' end of an oligonucleotide. Even small alkyl chains, aryl or heterocyclic conjugates or modified sugars (D-ribose, deoxyribose, glucose, etc.) can block 3'-5' -exonucleases

In one embodiment, the synthetic miR-135 molecules include modifications that improve targeting. Examples of modifications that target an oligonucleotide reagent to a particular cell type include carbohydrate sugars, such as galactose, N-acetylgalactosamine, mannose; vitamins, such as folate; other ligands, such as RGDS and RGD mimics; and small molecules including naproxen, ibuprofen, or other known protein binding molecules (discussed further below).

According to one embodiment, the modification is selected from sugar modifications, nucleobase modifications and internucleotide linkage modifications, as broadly described below.

Specific examples of synthetic miR-135 molecules useful in accordance with this aspect of the invention include those that contain a modified backbone (e.g., a sugar-phosphate backbone) or a non-natural nucleoside linker. Oligonucleotides or polynucleotides having modified backbones include those that retain phosphorus atoms in the backbone, such as those disclosed in the following documents: US patent US4,739,047; US4,469,863; US4,476,301; US5,023,243; US5,177,196; US5,188,897; US5,264,423; US5,276,019; US5,278,302; US5,286,717; US5,321,131; US5,399,676; US5,405,939; US5,453,496; US5,455,233; US5,466,677; US5,476,925; US5,519,126; US5,536,821; US5,541,306; US5,550,111; US5,563,253; US5,571,799; US5,587,361; US5,625,050 and US8,017,763; and U.S. patent application US20100222413, the entire disclosures of which are incorporated herein by reference.

According to one embodiment, the nucleic acid sequence of the miR-135 molecule (e.g., the nucleic acid sequence of miR-135b and/or the complementary strand) comprises a phosphorus-modified internucleotide linkage at the 5 'or 3' end of the nucleotide sequence.

Exemplary internucleotide linkage modifications include, but are not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, alkylphosphonates (including 3' -alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (including 3' -phosphoramidates), aminoalkylphosphoramidates, thiocarbonylphosphoramidates (thiophosphorylamites), thiocarbonylalkylphosphates, borane phosphates (e.g., having a normal 3' -5' to 5' linkage, 2' -5' linked analogs thereof, and those of opposite polarity, wherein pairs of adjacent nucleoside units link 3' -5' to 5' -3' or 2' -5' to 5' -2 '), borophosphonates, phosphodiesters, phosphonoacetates (PACE), morpholino, peptide Nucleic Acids (PNA), and Threo Nucleic Acids (TNA). Various salts, mixed salts and free acid forms of the above modifications may also be used. Other internucleotide linkage modifications are described in Deleavey and Damha, chemistry and biology (2012) 19:937-954, which are incorporated herein by reference.

According to one embodiment, the internucleotide linkage modification comprises phosphorothioate.

According to one embodiment, the synthetic miR-135 molecule comprises at least one phosphorothioate linkage modification in the nucleic acid sequence or the complementary strand of miR-135 b.

According to one embodiment, the synthetic miR-135 molecule comprises 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 or complementary strand of miR-135 b.

According to one embodiment, the synthetic miR-135 molecule comprises a phosphorothioate at the internucleotide junction at the 5' or 3' end of the nucleotide sequence (e.g., in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence).

According to one embodiment, the synthesized miR-135 molecule comprises a phosphorothioate between the last two nucleotides of the 3' end of the nucleic acid sequence or complementary strand of miR-135 b.

According to one embodiment, the synthesized miR-135 molecule comprises a phosphorothioate between the last two nucleotides of the 5' end of the nucleic acid sequence or complementary strand of miR-135 b.

According to one embodiment, the synthetic miR-135 molecule comprises phosphorothioates between the last two nucleotides of the 3 'end and between the last two nucleotides of the 5' end of the nucleic acid sequence or complementary strand of miR-135 b.

According to one embodiment, the synthesized miR-135 molecule comprises phosphorothioates in both the nucleic acid sequence of miR-135b and the nucleic acid sequence of the complementary strand.

According to one embodiment, the synthetic miR-135 molecule comprises a borophosphonate at the internucleotide junction at the 5' or 3' end of the nucleotide sequence (e.g., in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence).

According to one embodiment, the synthetic miR-135 molecule comprises a methylphosphonate at the internucleotide junction at the 5' or 3' end of the nucleotide sequence (e.g., in the last nucleotide at the 5' end of the miR-135 nucleic acid sequence).

According to one embodiment, the synthetic miR-135 molecule comprises a phosphodiester at the internucleotide junction at the 5' or 3' end of the nucleotide sequence (e.g., in the last nucleotide at the 5' end of the miR-135 nucleic acid sequence).

According to a specific embodiment, the synthetic miR-135 molecule comprises a phosphate at the internucleotide junction at the 5' or 3' end of the nucleotide sequence (e.g., in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence).

Alternatively, the synthetic miR-135 molecular backbone, which does not include a phosphorus atom therein, has a backbone 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 an morpholino linkage (formed in part by the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; methylacetyl and thiomethylacetyl backbones; methylene methylacetylacetyl and thiomethylacetyl backbones; an olefin-containing backbone; a sulfamate backbone; methylene imino and methylene hydrazine backbones; sulfonate and sulfonamide backbones; an amide backbone; n, O, S and CH with mixing ₂ Other components of the component parts, as disclosed in the following documents: US patent US5,289,047; US5,034,506; US5,166,315; US5,185,444; US5,214,134; US5,216,141; US5,235,033; US5,264,562; U.S. Pat. No. 5,264,564; US5,405,938; US5,434,257; US5,466,677; US5,470,967; US5,489,677; US5,541,307; US5,561,225; US5,596,086; US5,602,240; US5,610,289; US5,602,240; US5,608,046; US5,610,289; US5,618,704; US5,623,070; US5,663,312; US5,633,360; US5,677,437 and US5,677,439.

According to one embodiment, the synthetic miR-135 molecule comprises at least one sugar modification (e.g., a ribose modification).

According to one embodiment, the synthetic miR-135 molecule comprises 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., ribose modifications).

According to one embodiment, at least one nucleic acid of the miR-135 molecule (e.g., a nucleic acid of miR-135b and/or the complementary strand) comprises a modification corresponding to position 2 of ribose.

According to one embodiment, the sugar modification is located in the last nucleotide at the 3' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to one embodiment, the sugar modification is located in the last nucleotide at the 5' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to one embodiment, the sugar modifications are located in the last nucleotide of the 3 'and 5' ends of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to a specific embodiment, the sugar modification is located in the first 1 to 2 nucleotides of the 5 'end of the nucleic acid sequence of the miR-135b strand and/or in the last 1 to 3 nucleotides of the 3' end of the nucleic acid sequence of the miR-135b strand.

According to a specific embodiment, the sugar modification is located in the first two nucleotides at the 5 'end of the nucleic acid sequence of the complementary strand and/or in the last nucleotide at the 3' end of the nucleic acid sequence of the complementary strand.

According to one embodiment, the sugar modification is located in both the nucleic acid sequence of miR-135b and the nucleic acid sequence of the complementary strand.

Exemplary sugar modifications include, but are not limited to: 2' -modified nucleotides, for example 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-dimethylaminoethyl-oxyethyl (2 ' -O-DMAEO), 2' -fluoroarabino-oligonucleotides (2 ' -F-ANA), 2' -O-N-methylacetamido (2 ' -O-NMA), 2' -NH ₂ Or Locked Nucleic Acid (LNA). Other sugar modifications are described in Deleavey and Damha, chemistry and biology (2012) 19:937-954, which are incorporated herein by reference.

According to one embodiment, the synthetic miR-135 molecule comprises at least one 2 '-O-methyl (2' -O-Me) modified nucleotide.

According to one embodiment, the 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.

According to one embodiment, all nucleotides of the synthesized miR-135 molecule include a 2 '-O-methyl (2' -O-Me) modification.

According to a specific embodiment, the 2' -O-methyl (2 ' -O-Me) modified nucleotide is located at the 5' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to a specific embodiment, the 2' -O-methyl (2 ' -O-Me) modified nucleotide is located at the 3' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to a specific embodiment, the 2 '-O-methyl (2' -O-Me) modified nucleotide is located at the last 2 nucleotides of the 5 'end of the nucleic acid sequence of the miR-135b strand or the complementary strand and/or at the last nucleotide of the 3' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to one embodiment, the synthetic miR-135 molecule comprises at least one 2 '-O-methoxyethyl (2' -O-MOE) modified nucleotide.

According to one embodiment, the 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.

According to specific embodiments, the 2' -O-methoxyethyl (2 ' -O-MOE) modified nucleotide is located at the 5' end of the nucleic acid sequence of the miR-135b strand or complementary strand.

According to a specific embodiment, the 2' -O-methoxyethyl (2 ' -O-MOE) modified nucleotide is located at the 3' end of the nucleic acid sequence of the miR-135b strand or complementary strand.

According to a specific embodiment, the 2 '-O-methoxyethyl (2' -O-MOE) modified nucleotide is at the last 2 nucleotides of the 3 'end of the nucleic acid sequence of the miR-135b strand or the complementary strand and/or at the last nucleotide of the 5' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to specific embodiments, 2'-O-MOE modifications are effected on adenine (A) nucleotides of the miR-135b strand or the complementary strand (e.g., at the 3' end of the nucleic acid sequence of the miR-135b strand).

According to a specific embodiment, the 2'-O-MOE modification also includes 5' -ribose methylation (designated 2'-O-MOE-5' -Me).

According to particular embodiments, the 2' -O-MOE-2' -Me modification is effected on uracil (U) nucleotides of the miR-135b strand or the complementary strand (e.g., at the 5' end of the nucleic acid sequence of the miR-135b strand).

According to one embodiment, the synthetic miR-135 molecule comprises at least one 2 '-fluoro (2' -F) -modified nucleotide.

According to one embodiment, the synthetic miR-135 molecule comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more 2 '-fluoro (2' -F) -modified nucleotides.

According to specific embodiments, the 2' -fluoro (2 ' -F) -modified nucleotide is located at the 5' end of the nucleic acid sequence of the miR-135b strand or the complementary strand.

According to specific 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 the complementary strand.

According to one embodiment, the synthetic miR-135 molecules comprise modified internucleotide linkages and sugar modifications. Thus, synthetic miR-135 molecules that can be used according to the invention are those molecules that are modified in both sugar and internucleoside linkages, i.e., the backbone of the nucleotide unit is substituted with a new group. The base units are maintained to be complementary to the appropriate polynucleotide target. Examples of such oligonucleotide mimics include Peptide Nucleic Acids (PNAs). PNA oligonucleotides refer to oligonucleotides in which the sugar backbone is replaced by an amide-containing backbone, in particular an aminoethylglycine backbone. The base is retained and bound directly or indirectly to the nitrogen heteroatom of the amide moiety of the backbone. U.S. patents teaching the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082; US5,714,331 and US5,719,262, each of which is incorporated herein by reference. Other backbone modifications useful in the present invention are disclosed in U.S. patent No. 6,303,374.

According to one embodiment, the synthetic miR-135 molecule comprises a phosphorus-modified internucleotide linkage and at least one sugar modification (e.g., a 2' -modified nucleotide).

According to one embodiment, the synthetic miR-135 molecule comprises a phosphorus-modified internucleotide linkage, at least one phosphorothioate-modified internucleotide linkage, and at least one sugar modification (e.g., a 2' -modified nucleotide).

According to a specific embodiment, the synthetic miR-135 molecule comprises a phosphate in the last nucleotide at the 5' -end of the miR-135b nucleic acid sequence, and a 2' -O-Me modification in the last nucleotide at the 3' -end of the miR-135b nucleic acid sequence.

According to a specific embodiment, the synthetic miR-135 molecule comprises a phosphate in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, a 2' -O-Me modification in the last two nucleotides at the 5' end of the miR-135b nucleic acid sequence, and a 2' -O-Me modification in the last nucleotide at the 3' end of the miR-135b nucleic acid sequence.

According to a specific embodiment, the synthetic miR-135 molecule comprises a phosphate in the last nucleotide at the 5 'end of the miR-135b nucleic acid sequence, a 2' -O-MOE-5'-Me modification in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, and a 2'-O-Me modification in the last nucleotide at the 3' end of the miR-135b nucleic acid sequence.

According to a specific embodiment, the synthetic miR-135 molecule comprises a phosphate in the last nucleotide at the 5 'end of the miR-135b nucleic acid sequence, a 2' -O-MOE-5'-Me modification in the last nucleotide at the 5' end of the miR-135b nucleic acid sequence, and a 2'-O-MOE modification in the last two nucleotides at the 3' end of the miR-135b nucleic acid sequence.

According to a specific embodiment, the synthetic miR-135 molecule comprises a 2'-O-Me modification in the last nucleotide at the 3' -end of the complementary strand nucleic acid sequence, and a 2'-O-Me modification in the last two nucleotides at the 5' -end of the complementary strand nucleic acid sequence.

According to one embodiment, at least one 2' -F modified nucleotide is also included in any of the above-synthesized miR-135 molecules.

According to one embodiment, at least one phosphorothioate modified internucleotide linkage is also included in any of the above-synthesized miR-135 molecules.

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.

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

According to a specific embodiment, the synthetic miR-135 molecules of the invention have a nucleic acid sequence of miR-135b as shown in SEQ ID NO. 10.

According to a specific embodiment, the synthetic miR-135 molecules of the invention have a nucleic acid sequence of miR-135b as shown in SEQ ID NO. 16.

According to a specific embodiment, the synthetic miR-135 molecules of the invention have a nucleic acid sequence of miR-135b as shown in SEQ ID NO. 41.

According to a specific embodiment, the synthetic miR-135 molecules of the invention have a nucleic acid sequence of miR-135b as shown in SEQ ID NO. 42.

According to a specific embodiment, the synthetic miR-135 molecules of the invention have the nucleic acid sequence of the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecules of the invention have the nucleic acid sequence of the complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 37 and the complementary strand shown in SEQ ID NO. 40.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 10 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 41 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 42 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 43 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 44 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 45 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 46 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 16 and the complementary strand shown in SEQ ID NO. 13.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 10 and a complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 41 and the complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 42 and a complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 43 and a complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 44 and a complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 45 and a complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 46 and the complementary strand shown in SEQ ID NO. 47.

According to a specific embodiment, the synthetic miR-135 molecule of the invention comprises the nucleic acid sequence of miR-135b shown in SEQ ID NO. 16 and the complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 10 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 41 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 42 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 43 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 44 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 45 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 46 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 16 and a complementary strand shown in SEQ ID NO. 13.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 10 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 41 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 42 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 43 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 44 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 45 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 46 and a complementary strand shown in SEQ ID NO. 47.

According to one aspect of the invention, there is provided a composition of matter comprising a synthetic miR-135 molecule, which synthetic miR-135 molecule comprises a miR-135b nucleic acid sequence shown in SEQ ID NO. 16 and a complementary strand shown in SEQ ID NO. 47.

The synthetic miR-135 molecules of the invention can be constructed using chemical synthesis and/or enzymatic ligation reactions, using methods known in the art (as discussed in detail below). For example, polynucleotides can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biostability of the molecule or to increase the physical stability of the duplex formed between the polynucleotide and the target nucleic acid, such as phosphorothioate derivatives and acridine substituted nucleotides (as discussed in detail above) can be used.

Synthetic miR-135 molecules designed according to the teachings of the invention can be produced according to any oligonucleotide synthesis method known in the art, including enzymatic synthesis and solid phase synthesis. Various mechanisms of oligonucleotide synthesis have been disclosed in the following documents: for example, US patent 4,659,774; US4,816,571; US5,141,813; US5,264,566; US4,959,463; US5,428,148; US5,554,744; US5,574,146; US5,602,244, each of which is incorporated herein by reference.

According to one embodiment, chemical synthesis may be achieved by a diester method, a triester method, a polynucleotide phosphorylase method, and by solid phase chemistry. These methods are discussed in detail below.

Diester process: the diester process is the first to develop into a usable state. The basic step is to ligate two appropriately protected deoxynucleotides to form a dideoxynucleotide containing a phosphodiester linkage.

Triester process: the main difference between the diester and triester processes is the presence of additional protecting groups on the reactant and product phosphate atoms. The phosphate protecting group is typically chlorophenyl, which renders the nucleotide and polynucleotide intermediates soluble in organic solvents. Thus, the purification was performed in chloroform solution. Other improvements to this process include (i) block coupling of trimers and larger oligomers, (ii) the extensive use of high performance liquid chromatography to purify intermediates and final products, and (iii) solid phase synthesis.

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 predominantly a single nucleotide to a short oligonucleotide. Chromatographic purification allows the desired single adduct to be obtained. At least one trimer is required to initiate the process, and the primer must be obtained by some other method. The polynucleotide phosphorylase method is viable, with the advantage that the processes involved are familiar to most biochemists.

Solid phase method: using techniques developed for solid phase synthesis of polypeptides, it is possible to attach the initial nucleotide to a solid support material and continue the stepwise addition of nucleotides. All mixing and washing steps are simplified and the process becomes easily automated. These syntheses are now generally carried out using automated nucleic acid synthesizers. The equipment and reagents for performing the solid phase synthesis are commercially available from, for example Applied Biosystems.

To date, phosphoramidite chemistry has become the most widely used coupling chemistry for the synthesis of oligonucleotides. As is well known to those skilled in the art, phosphoramidite synthesis of oligonucleotides involves activation of nucleoside phosphoramidite monomer precursors by reaction with an activator to form an activated intermediate, which is then added in turn to a growing oligonucleotide chain (typically anchored at one end to a suitable solid support) to form an oligonucleotide product.

The recombination method comprises the following steps: recombinant methods for producing nucleic acids in cells are well known to those of skill in the art and can be performed without the inclusion of chemical modifications to the synthesized miR-135 molecules. These include the use of vectors, plasmids, cosmids, and other vectors to deliver nucleic acids to cells, which may be target cells or simply host cells (to produce a large number of desired RNA molecules). Alternatively, such vectors may be used in the case of cell-free systems, provided that reagents for producing RNA molecules are present. Such methods include those described in Sambrook,2003; methods described in Sambrook,2001 and Sambrook,1989, which are incorporated herein by reference.

Any other method for such synthesis may also be used; the actual synthesis of oligonucleotides is well within the capabilities of the skilled person and can be accomplished by established methods described, for example, in the following documents: sambrook, j. And Russell, d.w. (2001), "molecular cloning: laboratory manual "; ausubel, R.M. et al (1994,1989), "molecular biology experiments (Current Protocols in Molecular Biology)", volumes I-III, john Willi father publishing company, balmo, mali; perbal, b. (1988), "molecular cloning utility guide (A Practical Guide to Molecular Cloning)", john wili parent-child publishing company, new york; and Gait, m.j. Edit (1984), "oligonucleotide synthesis (Oligonucleotide Synthesis)"; using solid phase chemistry methods such as cyanoethyl phosphoramidite, followed by deprotection, desalting, and purification by, for example, an automated trityl process or HPLC.

According to one embodiment, the synthetic miR-135 molecules are linked to ligands (also referred to as moieties) that are selected to improve stability, distribution, cellular uptake, cross the Blood Brain Barrier (BBB), or target the synthetic miR-135 molecules to cells of interest. Thus, the synthetic miR-135 molecules can be modified to include non-nucleotide portions, as discussed in detail below.

According to one aspect of the invention, conjugated miR-135 molecules are provided.

The term "conjugate" as used herein refers to any compound resulting from the covalent attachment of two or more individual compounds. In the present invention, a conjugate refers to a molecule comprising a covalently coupled synthetic miR-135 molecule and a cell targeting moiety.

As used herein, the expression "cell targeting moiety" refers to any substance that binds to a molecule expressed or presented on a target cell of interest, preferably expressed/presented in a specific manner, e.g., not expressed/presented on other cell types, or at a higher level than other cell types. According to a specific embodiment, the molecule is a receptor. This binding specificity allows for delivery of the synthetic miR-135 molecule (which is linked to a cell targeting moiety) to a cell, tissue, or organ that expresses or presents the molecule. In this way, conjugates carrying a cell targeting moiety will be specific for a cell when administered to a subject (e.g., a human) or contacted with a different type of cell population in vitro.

The cell targeting moiety according to the invention may exhibit a Kd of at least about 10 for a target (a molecule expressed or presented on a target cell of interest, e.g., a receptor) ^-4 M, or at least about 10 ^-5 M, or at least about 10 ^-6 M, or at least about 10 ^-7 M, or at least about 10 ^-8 M, or at least about 10 ^-9 M, or at least about 10 ^-10 M, or at least about 10 ^-11 Or at least about 10 ^-12 M or greater.

The term "receptor" refers to a cell-associated protein that binds to a biologically active molecule called a "ligand".

According to one embodiment, the molecule expressed or presented on the target cell of interest (e.g., receptor) is expressed in a cell-specific manner (e.g., on a central nervous system cell, bone cell, muscle cell, cancer cell, gastrointestinal cell, etc.).

Receptors that may be targeted by the cell targeting moiety of the invention include, but are not limited to: 5-hydroxytryptamine receptors (e.g., 5-HT1A, 5-HT1B, 5-HT2A, 5-HT3, 5-HT1D, 5-HT 6); adenosine receptors (e.g., A1, A2A); adrenergic receptors (e.g., alpha 1A-adrenergic receptor, alpha 1B adrenergic receptor, alpha 1D adrenergic receptor); angiotensin receptor (e.g., AT 2); bombesin receptors (e.g., BB1, BB2, BB 3); bradykinin receptors (e.g. B1, B2); calcitonin receptors (e.g., AM1, AMY1, CGRP, CT-R, AM, AMY 3); chemokine receptors (e.g., CXCR 4); cholecystokinin receptors (e.g., CCK 2); corticotropin releasing factor receptors (e.g., CRF1, CRF 2); dopamine receptors (e.g., D1, D2); endothelin receptors (e.g., etα, etβ); ephrin receptors (e.g., ephA1, ephA2, ephA3, ephA4, ephB1, ephB2, ephB 3); formyl peptide receptors (e.g., FPR1, FPR2, FPR 3); frizzled receptors (e.g., FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD 10); galanin receptors (GAL 1, GAL2, GAL 3); growth hormone secretagogue receptors (growth hormone releasing peptides) (e.g., GHS-R1 a); a Kisspeptin (Kisspeptin) receptor; melanocortin receptors (e.g., MC1, MC2, MC3, MC 4); melatonin receptors (e.g., MT1, MT 2); neuropeptide FF/neuropeptide AF receptors (e.g., NPFF1, NPFF 2); neuropeptide S receptors (e.g., NPS); neuropeptide W/neuropeptide B receptor (e.g., NPBW 2); neuropeptide Y receptors (e.g., Y1, Y2, Y4, Y5); neurotensin receptors (e.g., NTS1, NTS 2); opioid receptors (e.g., delta, kappa, mu); orexin receptors (e.g., OX1, OX 2); peptide P518 receptor (e.g., QRFP); prostanoid receptors; SLC6 neurotransmitter transporter family (e.g., DAT, NET, SERT, glyT 1); somatostatin receptors (e.g., sst1, sst2, sst3, sst4, sst 5); tachykinin receptors (e.g., NK1, NK2, NK 3); toll-like receptors (e.g., TLR 7); vasopressin and oxytocin receptors (e.g., OT, V1A, V1B, V2); VEGF receptors (e.g., VEGFR1, VEGFR2, VEGFR 3) and G Protein Coupled Receptors (GPCRs).

According to one embodiment, the molecule expressed or presented on the target cell of interest (e.g., receptor) is on a cell of the Central Nervous System (CNS), including, but not limited to, cells of the hypothalamus, brainstem, cortex, cerebellum, striatum, midbrain, hippocampus, glia, and/or spinal cord.

According to one embodiment, the molecule (e.g., receptor) is expressed or presented on brain cells.

According to one embodiment, the molecule (e.g., receptor) is expressed or presented on a neuronal cell.

According to one embodiment, the molecule (e.g., receptor) is expressed or presented on a glial cell (glial cell) or neuroglia.

According to one embodiment, the molecule expressed or presented on the target cell (e.g., receptor) of interest is a neurotransmitter transporter.

According to one embodiment, the second component of the conjugate according to the invention is a cell targeting moiety that specifically binds to a neurotransmitter transporter.

The term "neurotransmitter transporter" as used herein refers to a protein belonging to a class of membrane transporters that span neuronal cell membranes, whose primary function is to carry neurotransmitters across these membranes and direct their further transport to specific intracellular locations.

Neurotransmitter transporters that may be targeted by cell targeting moieties of some embodiments of the invention include, but are not limited to, the cytoplasm present in neurons and glial cellsUptake carriers in the membrane, which pump neurotransmitters from the extracellular space into cells. The process depends on Na transmembrane ⁺ Gradient, especially Na ⁺ Is described. Two protein families have been identified. One family includes GABA, monoamines (e.g., norepinephrine), dopamine, serotonin, and transporters of 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. The homologous protein family is derived from Na ⁺ And Cl ^- The ion is co-transported with the neurotransmitter into the cell (Na/Cf neurotransmitter transporter) to gain energy. The second family includes transport proteins for excitatory amino acids (e.g., glutamate). Common structural components include putative 6 to 10 transmembrane domains, cytoplasmic N-and C-termini, and glycosylation in the extracellular loop. Excitatory amino acid transporters are independent of Cl ^- And may require intracellular K ⁺ Ion (Na) ⁺ /K ⁺ Neurotransmitter transporters) (Liu, y. Et al (1999) Cell biology progress (Trends Cell biol.) 9:356-363).

Neurotransmitter transporters that may be targeted by the cell targeting moiety of the present invention also include, but are not limited to, uptake vectors present in the plasma membranes of neurons and glial cells, which pump neurotransmitters from the extracellular space into cells. The process depends on Na transmembrane ⁺ Gradient, especially Na ⁺ Is described. Two protein families have been identified. One family includes GABA, monoamines (e.g., norepinephrine), dopamine, serotonin, and transporters of 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. The homologous protein family is derived from Na ⁺ And Cl ^- The ion is co-transported with the neurotransmitter into the cell (Na/Cl neurotransmitter transporter) to gain energy. The second family includes transport proteins for excitatory amino acids (e.g., glutamate). Common structural components include putative 6 to 10 transmembrane domains, cytoplasmic N-and C-termini toAnd glycosylation in the extracellular loop. Excitatory amino acid transporters are independent of Cl ^- And may require intracellular K ⁺ Ion (Na) ⁺ /K ⁺ Neurotransmitter transporters) (Liu, y. Et al (1999) Cell biology progress (Trends Cell biol.) 9:356-363).

Neurotransmitter transporters that may be targeted by the cell targeting moiety of the present invention also include neurotransmitter transporters that are present in intracellular vesicle membranes (typically synaptic vesicles), the main function of which is to concentrate neurotransmitters from the cytoplasm into vesicles prior to exocytosis of the vesicle contents during synaptic transmission. Vesicle transport and utilization H ⁺ -an electrochemical gradient across the vesicle membrane produced by atpase. Two protein families are involved in neurotransmitter transport to vesicles. One family uses mainly proton exchange to drive transport into secretory vesicles, and includes monoamine and acetylcholine transporters. For example, monoamine transporters exchange two luminal protons for each cytoplasmic transmitter molecule. The second family includes GABA transporters, which rely on positive charges within synaptic vesicles. These two classes of vesicle transporters have no sequence similarity to each other and have a structure different from that of the plasma membrane carrier (Schloss, P.et al (1994) Cell biology evolution (Trends Cell biol.) 9:356-363).

According to one embodiment, the types of neurotransmitter transporters that can be targeted with the cell targeting moiety of the present invention include, for example, dopamine transporter (DAT), serotonin transporter (SERT) and norepinephrine transporter (NET).

Dopamine transporter (also known as DAT or SLC6 A3) refers to a molecule which is an integral membrane protein that transports the neurotransmitter dopamine from the synaptic cleft and deposits it into surrounding cells, thereby terminating neurotransmitter signaling.

Serotonin transporter (also known as SERT or SLC6 A4) refers to a polypeptide which is an integral membrane protein that transports the neurotransmitter serotonin from the synaptic cleft into presynaptic neurons.

Norepinephrine transporter (also referred to as NET or SLC6 A2) refers to a transmembrane protein molecule that transduces synaptically released norepinephrine back to presynaptic neurons.

Specific types of neurotransmitter transporters that may be targeted with the cell targeting moiety of the present invention include, but are not limited to: a glutamate/aspartate transporter comprising: excitatory amino acid transporter 1 (EAAT 1), excitatory amino acid transporter 2 (EAAT 2), excitatory amino acid transporter 3 (EAAT 3), excitatory amino acid transporter 4 (EAAT 4), excitatory amino acid transporter 5 (EAAT 5), vesicle glutamate transporter 1 (VGLUT 1), vesicle glutamate transporter 2 (VGLUT 2), and vesicle glutamate transporter 3 (VGLUT 3); GABA transporters, including GABA transporter type 1 (GAT 1), GABA transporter type 2 (GAT 2), GABA transporter type 3 (GAT 3), betaine transporter (BGT 1), and saccular GABA transporter (VGAT); a glycine transporter comprising: glycine transporter 1 (GlyTl), glycine transporter 2 (GlyT 2); a monoamine transporter comprising: dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), vesicle monoamine transporter 1 (VMAT 1), vesicle monoamine transporter 2 (VMAT 2); an adenosine transporter comprising: balanced nucleoside transporter 1 (ENT 1), balanced nucleoside transporter 2 (ENT 2), balanced nucleoside transporter 3 (ENT 3) and balanced nucleoside transporter 4 (ENT 4) and cystic acetylcholine transporter (VAChT).

According to one embodiment, the conjugates of the invention comprise a cell targeting moiety that specifically binds to a tumor associated antigen.

As used herein, the phrase "tumor-associated antigen" refers to a protein that is common to a particular hyperproliferative disease (e.g., cancer) and that is produced by tumor cells.

The types of tumor-associated antigens to which the present invention relates include tumor-specific antigens (TSA) or tumor-associated antigens (TAA). "TSA" refers to a protein or polypeptide antigen that is characteristic of tumor cells and that is not found on other cells in the body. "TAA" refers to a protein or polypeptide antigen expressed by a tumor cell. For example, a TAA may be one or more surface proteins or polypeptides, nucleoproteins or glycoproteins, or fragments thereof, of a tumor cell.

According to one embodiment, the tumor-associated antigen is associated with a solid tumor (e.g., colon cancer, breast cancer, prostate cancer, renal Cell Carcinoma (RCC), lung cancer, sarcoma, or melanoma).

According to one embodiment, the tumor-associated antigen is associated with hematological malignancy.

Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, for example: MART-1/melanA (MART-1), gp100 (Pme 117), tyrosinase, TRP-1, TRP-2 and tumor-specific polyclonal antigens (e.g., MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p 15); 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 translocation, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein Barr virus antigen EBVA and Human Papilloma Virus (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, CA19-9, CA 72-4, CAM 17.1, nuMa, K-ras, beta-Catenin (beta-Catenin), CDK4, mum-1, P15, P16, 43-9F, 5T4, 791Tgp72, alpha fetoprotein, beta-HCG, BCA225, BTA, CA 125, CA 15-3\CA 27.291\BCA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, ga\CAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70-K, NY, CO 1, TAG-16, TAG-90, TAG-12, and related TAG proteins. Other examples of tumor antigens include, but are not limited to: a33, BAGE, bcl-2, beta-catenin, CAl25, CA19-9, CD5, CD19, CD20, CD21, CD22, CD33, CD37, CD45, CD123, CEA, c-Met, CS-1, cyclin B1 monoclonal antibody (cyclin B1), DAGE, EBNA, EGFR, ephrinB2, 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. Other tumor antigens are provided in Van der Bruggen P, stroobant V, vigneron N, van den Eynde b. Peptide database: t cell-defined tumor antigen, tumor immunity (Cancer Immun) (2013), www, cancer Immunity. Org/peptide, incorporated herein by reference.

According to one embodiment, the conjugates of the invention comprise a cell targeting moiety that specifically binds to a molecule (e.g., receptor) expressed or present on bone cells.

According to one embodiment, the cell targeting moiety targets the skeletal system.

According to one embodiment, the cell targeting moiety is directed against a specific bone cell type (e.g., osteoblast, osteocyte, osteoclast, bone cell progenitor, osteoclast progenitor or bone lining cell).

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

According to one embodiment, the conjugates of the invention comprise a cell targeting moiety that specifically binds to a molecule (e.g., receptor) expressed or presented on a muscle cell.

Exemplary muscle cell molecules that can be targeted by the cell targeting moiety of the invention include, but are not limited to: m Cadherin antibody (M-Cadherin)/Cadherin 15 (Cadherin-15), nidogen, ABCG2 and myogenin.

According to one embodiment, the conjugates of the invention comprise a cell targeting moiety that specifically binds to a molecule (e.g., receptor) expressed or presented on gastrointestinal cells.

Exemplary gastrointestinal cell molecules that may be targeted by the cell targeting moiety of the present invention include, but are not limited to, 5-hydroxytryptamine transporter or 5-hydroxytryptamine receptor 1-7.

The choice of the cell targeting moiety of the invention will depend on the particular type of disease to be treated (e.g., mental disease, cancer, bone disease, etc.).

According to one embodiment, the cell targeting moiety is a small molecule.

According to one embodiment, the cell targeting moiety is a small molecule drug. Exemplary small molecule drugs that can be used to target, for example, 5-HT neurons and postsynaptic neurons include, but are not limited to: ligands for the 5-HT1A receptor [11C ] DASB, [11C ] WAY100635, or [18F ] MPPF.

According to one embodiment, the cell targeting moiety is a synthetic component.

According to one embodiment, the cell targeting moiety is a nanoparticle capable of binding to an antigen, receptor or other protein or non-protein membrane compound of the target cell.

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

It should be noted that affinity can be quantified using known methods, such as Surface Plasmon Resonance (SPR) (described in Scarano S, masini M, turner AP, minnni M, for surface plasmon resonance imaging of affinity-based biosensors, biosensors and bioelectronics (Biosens Bioelectron) 2010, 25:957-66) using, for example, captured or immobilized monoclonal antibodies (MAb) formats to minimize the contribution of affinity, and can be calculated using, for example, dissociation constants Kd such that lower Kd reflects higher affinity.

The affinity binding moiety typically has a binding affinity (K) _D ) (i.e., so long as the binding is specific, i.e., there is no background binding).

According to a specific embodiment, the affinity binding moiety is an aptamer or lectin.

According to a specific embodiment, the affinity binding moiety is an antibody or antibody fragment.

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, and multispecific antibodies formed from antibody fragments capable of binding an antigen. These functional antibody fragments are defined as follows: (1) Fab, fragments containing monovalent antigen binding fragments of an antibody molecule, can be produced by digesting an intact antibody with papain to produce an intact light chain and a portion of a heavy chain; (2) Fab', fragments of antibody molecules, which can be obtained by treating an intact antibody with pepsin, followed by reduction to produce intact light and heavy chains; obtaining two Fab' fragments per antibody molecule; (3) F (ab') 2, an antibody fragment obtainable by treating an intact antibody with pepsin without subsequent reduction; f (ab ') 2 is a dimer of two Fab' fragments bound together by two disulfide bonds; (4) Fv, defined as a genetically engineered fragment containing a light chain variable region and a heavy chain variable region, expressed as two chains; (5) A single chain antibody ("SCA"), a genetically engineered molecule comprising a light chain variable region and a heavy chain variable region, linked by a suitable polypeptide linker as a genetically fused single chain molecule; (6) CDR peptides are peptides encoding a single Complementarity Determining Region (CDR); and (6) a single domain antibody (also referred to as nanobody), which is a genetically engineered single monomer variable antibody domain that selectively binds a specific antigen. Nanobody molecular weights are only 12kDa to 15kDa, much smaller than common antibodies (150 kDa to 160 kDa).

According to one embodiment, the cell targeting moiety binds to a neurotransmitter transporter.

According to one embodiment, the cell targeting moiety that specifically binds to a neurotransmitter transporter is selected from the group consisting of: serotonin Reuptake Inhibitor (SRI), selective Serotonin Reuptake Inhibitor (SSRI), serotonin-adrenoceptor reuptake inhibitor (SNRI), noradrenergic and specific serotonergic antidepressant (NASSA), norepinephrine Reuptake Inhibitor (NRI), dopamine Reuptake Inhibitor (DRI), endogenous cannabinoid reuptake inhibitor (eCBRI), adenosine reuptake inhibitor (AdoRI), excitatory Amino Acid Reuptake Inhibitor (EAARI), glutamate reuptake inhibitor (GluRI), GABA Reuptake Inhibitor (GRI), glycine reuptake inhibitor (GlyRI), norepinephrine-dopamine reuptake inhibitor (NDRI), triple reuptake inhibitor, norepinephrine dopamine double reuptake inhibitor, serotonin single reuptake inhibitor, norepinephrine single reuptake inhibitor, and dopamine single reuptake inhibitor.

The term "serotonin reuptake inhibitor" or "SRI" refers to molecules capable of blocking serotonin uptake, including Serotonin Selective Reuptake Inhibitors (SSRIs) which specifically block serotonin uptake without substantially affecting other neurotransmitters, as well as non-selective serotonin reuptake inhibitors, such as serotonin-norepinephrine reuptake inhibitors (SNRI) and serotonin-norepinephrine-dopamine reuptake inhibitors (SNDRI).

The term "serotonin selective reuptake inhibitor" or "SSRI" refers to a selective inhibitor of serotonin reuptake without substantially affecting other neurotransmitter reuptake or transport systems. These compounds act primarily on presynaptic serotonergic cells, resulting in an increase in the extracellular level of the neurotransmitter serotonin, thereby increasing the level of serotonin available for binding to postsynaptic receptors and reversing the activity deficit of the monoamine neurotransmitter system in the brain. Illustrative, 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), indacene (CAS 63758-79-2), ji Meiding (CAS 56775-88-3), citalopram (CAS 59729-33-8), and escitalopram (CAS 219861-08-2). Any method known in the art can determine whether a given compound acts as an SSRI, including but not limited to determining the ability to reduce serotonin uptake ex vivo and antagonize serotonin-depleting effects of amphetamine without affecting the rat heart's intravenous injection [ ³ H]Norepinephrine uptake, as described in the following documents: koe et al (J.Pharmacol. Exp. Ther.) 1983, 226:686-700).

In one embodiment, the SSRI is sertraline or a structural analog thereof having the structure (formula I)

Wherein R is ₁ 、R ₂ 、R ₃ 、R ₄ 、R ₅ And R is ₆ Independently hydrogen or optionally substituted C ₁ -C ₆ An alkyl group; x and Y are each selected from hydrogen, fluorine, chlorine, bromine, trifluoromethyl, C ₁ -C ₃ Alkoxy and cyano; w is selected from the group consisting of: hydrogen, fluorine, chlorine, bromine, trifluoromethyl, nitro and C ₁ -C ₃ An alkoxy group. In some embodiments, the sertraline analog is in the cis-isomer configuration. The term "cis isomer" refers to NR on the cyclohexene ring ₁ R ₂ And the relative orientation of the phenyl moieties (i.e., they are all on the same side of the ring). Since both the 1-and 4-carbons are asymmetrically substituted, each cis-compound has two optically active enantiomeric forms, denoted (reference 1-carbon) cis- (1R) and cis- (1S) enantiomers.

Some useful sertraline analogs are the following compounds, in (1S) -enantiomer or (1S) (1R) racemic form, and pharmaceutically acceptable salts thereof:

-cis-N-methyl-4- (3, 4-dichlorophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N-methyl-4- (4-bromophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N-methyl-4- (4-chlorophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N-methyl-4- (3-trifluoromethyl-phenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N-methyl-4- (3-trifluoromethyl-4-chlorophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N, N-dimethyl-4- (4-chlorophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine;

-cis-N, N-dimethyl-4- (3-trifluoromethyl-phenyl) -1,2,3, 4-tetrahydro-1-naphthylamine; and

-cis-N-methyl-4- (4-chlorophenyl) -7-chloro-1, 2,3, 4-tetrahydro-1-naphthylamine.

Also of interest are the (1R) -enantiomers of cis-N-methyl-4- (3, 4-dichlorophenyl) -1,2,3, 4-tetrahydro-1-naphthylamine.

Sertraline analogs are also described in U.S. Pat. No. 4,536,518 (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, sertralineTriptyline (reverse) methanesulfonamide, 1R,4R Sertraline enantiomer, N-dimethyl Sertraline, nitroSertraline, sertraline aniline, sertraline iodide, sertraline Qu Linhuang amide NH ₂ Sertraline Qu Linhuang amide ethanol, sertraline Qu Linjing, sertraline-CME, dimethyl Sertraline reverse sulfonamide, sertraline reverse sulfonamide (CH 2 Ilinker), sertraline B ring o-methoxy, sertraline A ring methyl ester, sertraline A ring ethanol, sertraline N, N-dimethyl sulfonamide, sertraline A ring carboxylic acid, sertraline B ring p-phenoxy, sertraline B ring p-trifluoromethane, N-dimethyl Sertraline B ring and p-trifluoromethane, and UK-416244. The structures of these analogs are shown below.

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The term "serotonin-adrenaline reuptake inhibitor" or "SNRI" refers to a family of compounds capable of inhibiting serotonin reuptake by blocking serotonin transporter and noradrenaline reuptake by blocking noradrenaline transporter. This family includes, for example and without limitation, the following compounds: 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). Any method known in the art can determine whether a given compound functions as an SNRI, including but not limited to determining the ability to reduce serotonin and norepinephrine uptake by brain synaptosomes, substantially as described by Bolden-Watson C, richelson e. (Life sciences (sci.) 1993;52 (12): 1023-9).

In one embodiment, the SNRIs are tricyclic antidepressants, which are SNRIs having a general molecular structure comprising three rings. Among the tricyclic antidepressants are the linear tricyclic drugs such as imipramine, desipramine, amitriptyline, nortriptyline, protiline, doxepin, cotinamine, mianserin, dactyloxapine, amoxapine, dibenzepine, melitracin, maprotiline, flupenthixol, azathioprine, thiamphenicol and related compounds exhibiting similar activities. The tricyclics include indriline, chlorodanone, nomifensine and related compounds. A variety of other structurally different antidepressants, such as iprindole, bupropion (wellbatrin), nicotinamide, milnacipran, phenelzine and tranylcypromine have been shown to produce similar activities. They are functionally equivalent to tricyclic antidepressants and are therefore included within the scope of the present invention. Thus, the inventors intend to cover the broad class of antidepressants described above as well as related compounds having common properties, i.e. they all have antidepressant activity, and include, but are not limited to, the following compounds: such as amitriptyline, amitriptyline (amitriptyline oxide), carbamazepine, butyline, clomipramine, dimepin, desipramine, dibenzepine, dimetaline, dulciton (dosupein/dothiepin), doxepin, imipramine, oxamipramine, isoindole, roflumin, melitracin, metapamine, nifedipine, nortriptyline, praeparatum, propiverine, quinipamine and trimipramine.

The term "norepinephrine reuptake inhibitor", "NRI", "NERI", "adrenergic reuptake inhibitor" or "ARI" refers to a family of compounds capable of blocking norepinephrine and epinephrine reuptake by blocking the action of a norepinephrine transporter (NET). This family of compounds includes selective NRIs that specifically block NET without affecting other monoamine transporters as well as non-selective NRIs, such as snrs blocking norepinephrine and serotonin transporters (see above), norepinephrine-dopamine reuptake inhibitors (NDRI) blocking norepinephrine and dopamine transporters (see above), tricyclic antidepressants, and tetracyclic antidepressants (see above). Suitable selective NRIs for use in the present invention include, but are not limited to: tomoxetine (Atomoxetine/Tomoxetine)Or CAS 83015-26-3), mazindol (, A)> Or CAS 22232-71-9), reboxetineOr CAS 98819-76-2) and viloxazine (-/-)>Or CAS 46817-91-8).

The term "dopamine reuptake inhibitor" or "DRI" 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 extracellular dopamine concentration and thus dopaminergic neurotransmission. Suitable DRIs include, but are not limited to, the following: medicaments such as al Mi Geng acid, benztropine (Benzatropine/Benztropine), bupropion, dextromethorphan, esketamine, ethylbenzene tropine (etyben zatropine/ethyl), ethambutol (Ponalide), phencanamine, phenformin, ketamine, liflutamine, meloxamine, methoxamine, methylphenidate, nefopam, nomifensine, benzyl alcohol, pra Luo Lintan, pyrrolidones, teletamine and Qu Bi natal; research chemicals such as o Qu Ping (altropane), amforine, phencyclidine (benocyclidine), balafaxine, briman, DBL-583, dichloroalkane (dichlorpane), diazocine, dieticyclidine, difluoropine, garlicidine, GBR-12,935, indatrex, flupan (ioflupane), iodobenzene (iometope), ma Nifa octyl (manifaxine), radafaxine, tametrexine, tesofoxine, mopalide and valonosetril. Suitable DRIs can be identified using any method known to those skilled in the art, for example using the method disclosed by Kula et al (Life Sciences) 34:2567-2575,1984) et al, performed as described, the putative DRl ability to inhibit high affinity uptake of dopamine by a synaptosome preparation prepared from the rat striatum was determined.

As used herein, the term "endogenous cannabinoid reuptake inhibitor" or "eCBRI" refers to any compound that acts as a reuptake inhibitor of endogenous cannabinoids by blocking the action of endogenous cannabinoid transporters. Compounds with this activity can be identified based on the putative ability of endogenous cannabinoid reuptake inhibitors to block the uptake of cannabinoids by rat neurons and astrocytes using the methods described in Beltramo, m. Et al (Science) 1997,277:1094-1097, including but not limited to AM404, arvanil and ovanii.

The term "adenosine reuptake inhibitor" or "AdoRI" refers to a compound that acts as a reuptake inhibitor of purine nucleosides and neurotransmitter adenosines by blocking the action of one or more balanced nucleoside transporters (ENTs). This in turn leads to an increase in extracellular adenosine concentration, thus increasing adenosine energy neurotransmission. Compounds with AdoRI activity can be identified using in vitro assays based on the putative ability of AdoRI to inhibit the uptake of adenosine by erythrocytes, and in vivo assays based on the putative ability of AdoRI to inhibit the vasodilatory effect of adenosine, as well as the ability to prevent adenosine-mediated collateral vessel growth, all of which can be performed substantially as described in US patent 6,984,642 (incorporated herein by reference). Suitable AdoRI include, but are not limited to: acarditin, acetate, barbiturate, benzodiazepines, calcium channel blockers, carbamazepine, carisoprodol, cilostazol, cyclobenzaprine, diltiazem, dipyridamole, estradiol, ethanol (alcohol), flumazenil, hexidine, hydroxyzine, indomethacin, inosine, KF24345, methamphetamine, nitrobenzylthioguanosine, nitrothioprine, papaverine, pentoxifylline, phenothiazines, phenytoin, progesterone, pra Luo Panfei, propofol, puromycin, R75231, re102BS, cable Lu Huangqin (solaflazine), tomycin, qu Ka and tricyclic antidepressants.

The term "excitatory amino acid reuptake inhibitor" or "EAARI" refers to a compound that inhibits excitatory amino acid reuptake by blocking excitatory amino acid transporters or EEATs. Many compounds are known to bind to EEATs and inhibit transporter function. Inhibitors of EEATs fall into two broad categories, which differ in their mode of action: non-transportable blockers and competitive substrates. Suitable EAARIs include, but are not limited to: DL-threo-beta-Benzyloxyaspartate, kainite, dihydrocarbamate, 2S4R4MG, threo-P-hydroxyaspartate, L-trans-pyrrolidine-2, 4-dicarboxylic acid (t-2, 4-PDC). Suitable EEARIs can be identified based on the putative ability of EEARI to inhibit uptake of radiolabeled glutamate by Cos-1 cells expressing human excitatory amino acid transporter-1 (EAAT 1) or human excitatory amino acid transporter-2 (EEAT 2) using an assay described, for example, by Shimamoto et al (molecular Pharmacol (Molecular Pharmacology) 1998, 53:195-201).

The term "glutamate reuptake inhibitor" or "GluRI" refers to a compound that acts as a reuptake inhibitor of glutamate by blocking the action of one or more glutamate transporters. Suitable glutamate reuptake inhibitors include any of those known in the art, including, illustratively: threo-3-hydroxy-DL-aspartic acid (THA), (2S) -trans-pyrrolidine-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 based on the putative ability of GluRI to inhibit uptake of radiolabeled glutamic acid into Cos-1 cells expressing human excitatory amino acid transporter-1 (EAAT 1) or human excitatory amino acid transporter-2 (EAAT 2) using an assay described, for example, by Shimamoto et al (molecular Pharmacol (Molecular Pharmacology) 1998, 53:195-201).

The term "GABA reuptake inhibitor" or "GRI" refers to a compound that acts as a reuptake inhibitor of the neurotransmitter gamma-aminobutyric acid (GABA) by blocking the action of gamma-aminobutyric acid transporter (GAT). This in turn results in an increase in extracellular GABA concentration, thereby increasing GABAergic neurotransmission. Suitable GABA reuptake inhibitors include, but are not limited to: gaertn (found in Hypericum perforatum (St. John's grass)), CI-966, deramciclane (EGIS-3886), norarecoline (C10149), hyperforin (found in Hypericum perforatum (St. John's grass)), pipecolic acid, NNC 05-2090, NNC-71, SKF-89976A, SNAP-5114, stave and tiagabine (Gabitril), described in Borden LA, european journal of pharmacology (Eur J Pharmacol.) 1994, 269:219-224. Methods for detecting whether a given compound is a GABA reuptake inhibitor are known in the art and are described in the following documents: for example US6,906,177; US6,225,115; US4,383,999 and Ali, f.e. et al (journal of pharmaceutical chemistry (j. Med. Chem.) 1985,28,653-660). These methods generally involve contacting the cell with radiolabeled GABA and detecting uptake of GABA in the presence or absence of the candidate compound.

The term "glycine reuptake inhibitor" or "GlyRI" refers to compounds that act as reuptake inhibitors of the neurotransmitter glycine by blocking the action of glycine transporter (GlyTs), including compounds that block the glycine transporter (type 1) GlyT1 as well as GlyT2, glyT1 being involved in the removal of glycine from the synaptic cleft, glyT2 being necessary for glycine reuptake and reloading into synaptic vesicles (Gomeza et al, (2003), recent views of drug discovery and development (Curr Opin Drug Discov Devel) 6 (5): 675-82). Suitable glycine reuptake inhibitors for use in the present invention include: glyT 1-specific inhibitors such as, but not limited to, N-methyl-N- [ [ (1R 2S) -1,2,3, 4-tetrahydro-6-methoxy-1-phenyl-2-naphthyl ] methylglycine (free base of MTHMPNM glycine), 4- [ 3-fluoro-4-propoxyphenyl ] -spiro [ 2H-1-benzopyran-2, 4 '-piperidine ] -1' acetic acid (free base of FPPSBPAA), are described in PCT publications WO/0007978 and WO/0136423; ALX 5407; sarcosine; 5, 5-diaryl-2-amino-4-pentenoate; or a compound described in PCT publication WO/0208216; and GlyT 2-specific inhibitors, such as those described in PCT publication WO/05044810A, the entire contents of which are incorporated herein by reference. Methods for detecting GlyT 1-specific or GlyT 2-specific reuptake inhibitors are known in the art and include, for example, the methods described in PCT publication WO/05018676A or WO/05044810, wherein cells expressing a relevant receptor (GlyT 1 or GlyT 2) are contacted with radiolabeled glycine in the presence of a compound to be tested for reuptake inhibiting activity and the amount of glycine found in the cells after a given time is determined.

As used herein, the term "norepinephrine-dopamine reuptake inhibitor" or "NDRI" refers to a compound that acts as a reuptake inhibitor of the neurotransmitters norepinephrine and dopamine by blocking the action of the norepinephrine transporter (NET) and the dopamine transporter (DAT), respectively. This in turn results in an increase in extracellular norepinephrine and dopamine concentrations, thereby increasing adrenergic and dopaminergic neurotransmission. NDRIs suitable for use in the conjugates of the invention include, but are not limited to: a Mi Geng acidBupropion->Dextro methylphenidate (Focalin), fencanfamin ++> FenkamingRifexostat->Methylphenidate->Nomifenin->Benzyl alcohol->Pr Luo Lintan->Pyrrolidon->Nefopam->Hyperforin (found in Hypericum perforatum (St. John's grass)), cocaine, dexamethasone (2-DPMP), diphenylprolol (D2 PM), methylenedioxypyrrolidon (MDPV), cilobatin, ma Nifa octyl (GW-320,659), radar-fascian (GW-353,162), and tametrine (CP-24,441).

According to one embodiment, the conjugates of the invention comprise a cell targeting moiety that specifically binds to a neurotransmitter transporter, which is a Selective Serotonin Reuptake Inhibitor (SSRI).

According to a specific embodiment, the conjugate of the invention comprises an SSRI selected from the group consisting of sertraline, sertraline structural analogues, fluoxetine, fluvoxamine, paroxetine, indacene, zimeldine, citalopram, dapoxetine, escitalopram and mixtures thereof.

According to a specific embodiment, the conjugate of the invention comprises SSRI sertraline or a structural analogue thereof as defined above.

According to one embodiment, when the cell targeting moiety targets bone cells, the targeting moiety may include a synthetic component, such as tetracycline or Bisphosphonate (BP).

The synthetic miR-135 molecules and cell-targeting moieties can be coupled directly or indirectly via one or more intermediate moieties (e.g., a linker, bridge, or one or more spacer moieties).

According to one embodiment, the synthetic miR-135 molecules and cell targeting moieties can be directly coupled. Alternatively, according to another embodiment, the two moieties may be linked via a linking group.

The terms "linking group", "linker", and grammatical equivalents thereof are used herein to refer to an organic moiety that connects two moieties of a compound. The cell targeting moiety can be attached to any nucleotide in the sense (e.g., guide) or antisense (e.g., passenger) strand within the miR-135 molecule, but it can be preferably coupled via a 3 'terminal nucleotide and/or a 5' terminal nucleotide. The internal conjugate may be attached directly or indirectly to the 2' position of the ribose group, or to a nucleotide at another suitable position, via a linker.

As described above, the synthetic miR-135 molecules of the invention are preferably double-stranded nucleic acid molecules, and thus the conjugate moiety (i.e., the cell-targeting moiety) can be linked to a 3 'nucleotide of the guide sequence, a 5' nucleotide of the guide sequence, a 3 'nucleotide of the passenger sequence (passenger sequence), and/or a 5' nucleotide of the passenger sequence.

Although not wishing to be limited by definition or convention, in this application, the length of a linker is described by counting the number of atoms representing the shortest distance between the atom connecting 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 connected to the miR-135 oligonucleotide. Where the linker comprises one or more ring structures, it is preferred to count the atoms around the ring that represent the shortest path.

Linking groups suitable for use in the present invention include, but are not limited to: modified or unmodified nucleotides; a nucleoside; a polymer; sugar; a carbohydrate; polyvinyl alcohols such as polyethylene glycol and polypropylene glycol; a polyol; polypropylene; a mixture of ethylene glycol and propylene glycol; a polyalkylamine; polyamines such as polylysine and spermidine; polyesters such as poly (ethyl acrylate); polyphosphoric acid diesters (polyphosphoric acid diesters); aliphatic compounds and alkyl olefins (alkyl olefins). Furthermore, linker chemistries based on omega-amino-1, 3-diol, omega-amino-1, 2-diol, hydroxyproline, omega-amino-alkanol, diethanolamine, omega-hydroxy-1, 3-diol, omega-hydroxy-1, 2-diol, omega-thio-1, 3-diol, omega-thio-1, 2-diol, omega-carboxy-1, 3-diol, omega-carboxy-1, 2-diol, co-hydroxy-alkanol, omega-thio-alkanol, omega-carboxy-alkanol, functionalized oligoethylene glycol, allylamine, acrylic acid, allyl alcohol, propargylamine, propargyl alcohol, and the like may be used herein to produce linkers of suitable length.

The linker can also confer other desirable properties, such as improved water solubility, optimal spacing distance between the conjugate moiety (i.e., cell targeting moiety) and the miR-135 molecule, flexibility (or lack thereof), specific orientation, branching, and the like.

According to one embodiment, the linking group has the following structure:

wherein the method comprises the steps of

m, n and p are selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13,

wherein the sum m+n+p is an integer selected from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18,

where k is 0 or 1.

According to one embodiment, p is 5, n is 2, k is 1 and m is 6, resulting in a linker having the structure:

according to one embodiment, p is 5, n and k are 0, and m is 6, resulting in a linker having the structure:

according to one embodiment, the linker comprises more than one coupling for a cell targeting moiety. In a preferred embodiment, the linker is a bivalent or trivalent linker, i.e. 2 or 3 molecules of the agent can be coupled, respectively.

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

According to one embodiment, the divalent or trivalent linker has the formula:

or (b)

m, m ', m ", n ', n", p ', p ", r ', r", s ', s ", t and u are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13;

k. k', k "and v are independently selected from 0 and 1; and

X ¹ 、X ² and X ³ Independently selected from CH ₂ O, S, NH, CO, C (O) O and C (O) NH.

Depending on the values of the above groups, the branched linker may be symmetrical or asymmetrical.

In one embodiment, the linker is a divalent linker as shown above, wherein p and p 'are both 5, n and n' are both 2, k and k 'are both 1, and m' are both 6. In one embodiment, the linker is a divalent linker, wherein p and p 'are both 5, n', k and k 'are both 0, and m' are both 6.

In a specific embodimentIn an embodiment, the linker is a bivalent linker as shown above, wherein r and r 'are both 4, s and s' are both 1, t and v are both 0, and X ¹ And X ² Represents C (O) NH. In another embodiment, the linker is a divalent linker wherein r is 2, r 'is 0, s is 1, s' is 0, t and v are both 0, and X ¹ And X ² Represents CH ₂ 。

In one embodiment, the linker is a divalent linker wherein p and p 'are both 5, n and n' are both 2, k and k 'are both 1, m and m' are both 6, r and r 'are both 4, s and s' are both 1, t and v are both 0, and X ¹ And X ² Represents C (O) NH.

In another embodiment, the linker is a divalent linker wherein p and p 'are both 5, n and n' are both 2, k and k 'are both 1, m and m' are both 6, r is 2, r 'is 0, s is 1, s' is 0, t and v are both 0, and X ¹ And X ² Represents CH ₂ 。

In another embodiment, the linker is a divalent linker wherein p and p 'are both 5, n', k and k 'are both 0 and m' are both 6, r and r 'are both 4, s and s' are both 1, t and v are both 0, and X ¹ And X ² Represents C (O) NH.

In another embodiment, the linker is a divalent linker wherein p and p 'are both 5, n', k and k 'are both 0 and m' are both 6, r is 2, r 'is 0, s is 1, s' is 0, t and v are both 0, and X ¹ And X ² Represents CH ₂ 。

In one embodiment, the linker is a trivalent linker as shown above, wherein p, p ', and p "are all 5, n', and n" are all 2, k ', and k "are all 1, and m, m', and m" are all 6. In one embodiment, the linker is a trivalent linker, wherein p, p ', and p "are all 5, n', n", k ', and k "are all 0, and m, m', and m" are all 6.

In one embodiment, the linker is a trivalent linker as shown above, wherein r, r 'and r "are all 3, s' and s" are all 1, t is 1, v is 0, and X ¹ 、X ² And X ³ And represents O. In another embodiment, the linker is a trivalent linker, wherein rR 'and r' are all 3, s 'and s' are all 1, t is 1, u is 3, v is 1, and X ¹ 、X ² And X ³ And represents O.

In one embodiment, the linker is a trivalent linker, wherein p, p 'and p' are all 5, n 'and n' are all 2, k 'and k' are all 1, m m 'and m' are all 6, r 'and r' are all 3, s 'and s' are all 1, t is 1, v is 0, and X is ¹ 、X ² And X ³ And represents O.

In another embodiment, the linker is a trivalent linker, wherein p, p 'and p' are all 5, n 'and n' are all 2, k 'and k' are all 1, m m 'and m' are all 6, r 'and r' are all 3, s 'and s' are all 1, t is 1, u is 3, v is 1, and X is ¹ 、X ² And X ³ And represents O.

In another embodiment, the linker is a trivalent linker, wherein p, p 'and p' are all 5, n 'and n', k 'and k' are all 0, m m 'and m' are all 6, r 'and r' are all 3, s 'and s' are all 1, t is 1, v is 0, and X is ¹ 、X ² And X ³ And represents O.

In another embodiment, the linker is a trivalent linker, wherein p, p ' and p ' are all 5, n ', k ' and k ' are all 0, m m ' and m ' are all 6, r ' and r ' are all 3, s ' and s ' are all 1, t is 1, u is 3, v is 1, and X is ¹ 、X ² And X ³ And represents O.

According to one embodiment, the linking compound is selected from the group consisting of phosphodiester, phosphorothioate, carbamate, methylphosphonate, guanidine, sulfamate, sulfonamide, methylal, thiomethylal, sulfone, amide and mixtures thereof.

According to one embodiment, the linking compound is C ₁₀ N-hydroxysuccinimide ester linker (i.e., C10 linker).

According to one embodiment, the conjugate of the invention has the following structure:

according to one embodiment, the conjugate of the invention has the following structure:

according to one embodiment, the conjugate of the invention has the following structure:

/>

according to one embodiment, the conjugate of the invention has the following structure:

according to one embodiment, the conjugate of the invention has the following structure:

according to one embodiment, the conjugate of the invention has the following structure:

the conjugates of the invention may be prepared using techniques known to those skilled in the art. The synthesis of conjugates may involve selective protection and deprotection of functional groups. Suitable protecting groups are well known to those skilled in the art. For example, wuts, p.g.m. and Greene t.w. in protecting groups in organic synthesis (Protecting Groups in Organic Synthesis) (4 th edition, U.S. wili publishing company) and Kocienski p.j. in protecting groups (Protecting Groups) (3 rd edition, georg Thieme Verlag publishing company) are reviewed for protecting groups in organic chemistry.

For the purposes of the present invention, a "protecting group" is understood to mean a chemical modification that has been introduced at either end of a miR-135 oligonucleotide. Non-limiting examples of 5' -caps include: reverse abasic residues (moieties), 4',5' -methylene nucleotides; 1- (beta-D-erythrofuranosyl) nucleotides, 4' -thio nucleotides, carbocyclic nucleotides; 1, 5-anhydrohexitol nucleotides; l-nucleotides; an alpha-nucleotide; modified base nucleotides; an inter dithiophosphate linkage; threo-pentofuranosyl nucleotides; acyclic 3',4' -Zhong Hegan acid; acyclic 3, 4-dihydroxybutyl nucleotides; an acyclic 3, 5-dihydroxyamyl nucleotide, a 3'-3' -inverted nucleotide moiety; a 3'-3' -inverted abasic moiety; a 3'-2' -inverted nucleotide moiety; a 3'-2' -inverted abasic moiety; 1, 4-butanediol phosphate; 3' -phosphoramidates; hexyl phosphate; amino hexyl phosphate; 3' -phosphate; 3' -phosphorothioate; dithiophosphate esters; or a bridged or unbridged methylphosphonate moiety. Details are described in WO 97/26170, incorporated herein by reference. The 3' -cap includes, for example, 4',5' -methylene nucleotides; 1- (β -D-erythrofuranosyl) nucleotide; 4' -thio-nucleotides, carbocyclic nucleotides; 5' -amino-alkyl phosphate; 1, 3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1, 2-aminododecyl phosphate; hydroxypropyl phosphate; hydroxypropyl phosphate; l-nucleotides; an alpha-nucleotide; modified base nucleotides; dithiophosphate esters; threo-pentofuranosyl nucleotides; acyclic 3',4' -Zhong Hegan acid; 3, 4-dihydroxybutyl nucleotide; 3, 5-dihydroxyamyl nucleotide, 5'-5' -inverted nucleotide moiety; a 5'-5' -inverted abasic moiety; 5' -phosphoramidate; 5' -phosphorothioate; 1, 4-butanediol phosphate; a 5' -amino group; bridged and/or unbridged 5 '-phosphoramidates, phosphorothioates and/or phosphorodithioates, bridged or unbridged methylphosphonates and 5' -mercapto moieties. See also Beaucage and Iyer,1993, tetrahedron (Tetrahedron) 49,1925; the contents of which are incorporated herein by reference.

According to one embodiment, the synthetic miR-135 molecules or conjugates of this invention can be further modified by chemically linking one or more moieties or conjugates to a miR-135 nucleic acid or protective group, in order to enhance activity, cell distribution or cell uptake of the miR-135 nucleic acid.

According to one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the invention further comprises at least one cell penetrating moiety. Such parts include, but are not limited to: 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; cholic acid (Manoharan et al, bioorganic chemistry and medicinal chemistry communication (Biorg. Med. Chem. Let.)) 1994 4 1053-1060; thioethers, for example, beryllium-S-tritylthiol (Manoharan et al, new York academy of sciences (Ann. N.Y. Acad. Sci.) 1992,660,306-309; manoharan et al, bioorganic chemistry and medicinal chemistry communication (Biorg. Med. Chem. Let.) 1993,3,2765-2770); mercaptocholesterol (Oberhauser et al, nucleic acids research (nucleic acids Res.) 1992,20,533-538); aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al, J. European molecular biology (EMBO J) 1991,10, 11-1118; kabanov et al, european society of Biochemical Association (FEBS Lett.) 1990,259,327-330; svinarchhuk et al, biochemistry (Biochimie) 1993,75,49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethyl-ammonium 1, 2-di-O-hexadecyl-rac-glycerol-3-H phosphonate (Manoharan et al, tetrahedron letters) 1995,36,3651-3654; shea et al, nucleic acids research (nucleic acids res.) 1990,18,3777-3783); polyamine or polyethylene glycol chains (Manoharan et al, nucleosides and nucleotides (Nucleosides and Nucleotides) 1995,14,969-973) or adamantane acetic acid (Manoharan et al, tetrahedron Lett.) 1995,36,3651-3654; palm-based fraction (Mishra et al, biochem & biophysics, acta) 1995,1264,229-237; or octadecylamine or hexylamino-carbonyloxy cholesterol moiety (Crooke et al, J.Pharmacol.exp.Ther.) 1996,277,923-937.

Other lipid moieties that may be used according to the present invention include, but are not limited to: a fatty acid; fat; oils; a wax; cholesterol; sterols; fat-soluble vitamins such as vitamins A, D, E and K; monoglycerides; diglycerides and phospholipids. According to one embodiment, fatty acids include, for example, lauric acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), behenic acid (C22), and mixtures of lithocholic acid and oleylamine (lithocholic amine, C43).

According to a specific embodiment, the lipid moiety is palmitoyl.

According to a specific embodiment, the lipid moiety is a cholesteryl group.

According to one embodiment, the lipid moiety is a C18-C18 (i.e., a C18-phosphodiester-C18), for example wherein C18 is provided in the form of a phosphoramidite, added to the 5' end of the oligonucleotide by a coupling reaction, and then the second C18 is attached to the previous C18 after the same coupling reaction.

According to one embodiment, the cell penetrating moiety is a peptide or protein.

According to a specific embodiment, the cell penetrating moiety is an oxytocin peptide or a derivative compound thereof (e.g., recombinant or synthetic oxytocin).

According to a specific embodiment, the cell penetrating moiety is human serum albumin or other plasma protein or a partial peptide thereof.

According to one embodiment, the cell penetrating moiety is a peptide-shuttle (peptide-shuttle). Exemplary peptide shuttles include, but are not limited to, angiopep-2, RGV29, and THR.

According to one embodiment, the cell penetrating moiety is a small molecule drug. Exemplary small molecule drugs that may be used to target, for example, 5-HT neurons and postsynaptic neurons include, but are not limited to, ligands for the 5-HT1A receptor [11C ] DASB, [11C ] WAY100635, or [18F ] MPPF.

Alternatively, the moiety capable of enhancing cell distribution may be a low molecular weight compound or polypeptide capable of being specifically transported across the biological barrier by receptor-mediated endocytosis using specific transport proteins present in the biological barrier. A variety of uptake receptors and carriers, as well as larger amounts of receptor-specific ligands, are known in the art. Preferred ligands for receptors that mediate endocytosis and/or endocytosis for use according to the present invention include ligands that specifically bind to or are associated with: for example, thiamine transporter, folate receptor, vitamin B12 receptor, asialoglycoprotein receptor, alpha (2, 3) -sialoglycoprotein receptor (e.g., FC5 and FC44 nanobodies composed of camelid single domain antibodies (sdAbs) as receptor-specific ligands), transferrin-1 and-2 receptor, scavenger receptor (type a or B, type I, II or III, or CD36 or CD 163), low Density Lipoprotein (LDL) receptor, LDL-related protein 1 receptor (LRP 1, type B), LRP2 receptor (also known as macroglobulin or glycoprotein 330), diphtheria toxin receptor (DTR, which is a membrane-bound precursor of heparin-binding epidermal growth factor-like growth 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.

Exemplary ligands for use in accordance with the invention that bind to these receptors include, for example, ligands selected from the group consisting of: lipoprotein lipase (LPL), α2-macroglobulin (α2m), receptor-related protein (RAP), lactoferrin, desmopressin, tissue and urokinase type plasminogen activator (tPA/uPA), plasminogen activator inhibitor (PAI-I), tPA/uPA); PAI-l complex, melanotransferrin (or P97), thrombospondin 1 and 2, liver lipase, factor Vlla/Tissue Factor Pathway Inhibitor (TFPI), factor VIIIa, factor IXa, Aβ1-40, amyloid- β precursor protein (APP), C1 inhibitor, complement C3, apolipoprotein E (apoE), pseudomonas exotoxin A, CRM66, HIV-1Tat protein, rhinovirus, matrix metalloproteinase 9 (MMP-9), MMP-13 (collagenase-3), sphingolipid Activating Protein (SAP), gestagen protein, antithrombin III, heparin cofactor II alpha 1-antitrypsin, heat shock protein 96 (HSP-96), platelet Derived Growth Factor (PDGF), apolipoprotein J (apoJ or clusterin), ABETA binding to apoJ and apoE, aprotinin, vascular peptide, very Low Density Lipoprotein (VLDL), transferrin, insulin, leptin, insulin-like growth factor, epidermal growth factor, lectin, receptor specific peptidomimetics and/or humanized monoclonal antibodies or peptides, haemoglobin, non-toxic portions of the diphtheria toxin polypeptide chain, diphtheria toxin B chain (including DTB-His (e.g., spilsberg et al, 2005, toxin (Toxicon), 46 (8): 900-6)), all or a portion of a nontoxic mutant of diphtheria toxin CRM197, apolipoprotein B, apolipoprotein E (e.g., after binding to a polysorbate-80 coating on nanoparticles), vitamin D binding protein, vitamin a/retinol binding protein, vitamin B12/cobalamin plasma carrier protein, glutathione and cobalamin-B12.

According to a specific embodiment, the conjugates of the invention further comprise a group that facilitates transport of the conjugate across a biological membrane. According to one embodiment, the group is amphiphilic. Exemplary agents include, but are not limited to: penetration protein (penetratin), tat protein fragment comprising amino acids 48-60, signal sequence based peptide, PVEC, transporter, amphiphile peptide, arg9, bacterial cell wall penetrating peptide, LL-37, cecropin P1, alpha-defensin, beta-defensin, bacitracin (bacteriocin), PR-39 and antibacterial peptide (endolicidin). If the agent is a peptide, it may be modified, including peptidomimetics, converted proteins, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helicant is preferably an alpha-helicant, which preferably has a lipophilic phase and a lipophobic phase.

According to one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the invention further comprises at least one moiety for transport across the BBB.

According to one embodiment, the moiety for transport across the BBB is a peptide or protein.

According to one embodiment, the moiety for transport across the BBB is a neurophilic peptide or neurotoxin derived peptide or variant thereof.

Exemplary peptides include, but are not limited to: EPO fusion proteins (e.g., fused to a peptidomimetic antibody having affinity for human insulin receptor, an anti-transferrin receptor (TfR) monoclonal antibody, rabies virus glycoprotein (e.g., chimeric RVG fragment peptide)), as discussed in Razzak et al, journal of molecular science (int.J.mol.Sci.) (2019), 20:3108, are incorporated herein by reference.

According to one embodiment, the moiety for transport across the BBB is a BBB shuttle (also known as trojan horseAn antibody). Exemplary BBB-shuttles include, but are not limited to: angiopep-2, DAngiopep-2, apoB, apoE, THR, THRre, RVG, TGN, ^D CDX, melittin, TGN and TAT (47-57). Other BBB-shuttles are discussed in the following documents: oller-Salvia et al, review of the society of chemistry (chem. Soc. Rev.) (Royal society of chemistry, UK) 2016, incorporated herein by reference.

In another embodiment of the invention, the conjugates of the invention may further comprise an endosomolytic ligand (endosomolytic ligand). Endosomolytic ligands facilitate the lysis of endosomes and/or the transport of the composition or components of the invention from the endosomes to the cytoplasm of the cells. The endosomal cleavage ligand may be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusion. In certain embodiments, the endosomal lytic ligand exhibits its active conformation at endosomal pH. Exemplary endosomolytic ligands include: for example, GAL4 peptide (Subbarao et al, biochemistry (Biochemistry) 1987, 26:2964-2972), EALA peptide (Vogel et al, american society of chemistry (J.am. Chem. Soc) 1996, 118:1581-1586), and derivatives thereof (Turk et al, biochemistry and biophysics (biochemita) 2002, 1559:56-68), INF-7 peptide, infHA-2 peptide, diINF-7 peptide, diINF3 peptide, GLF peptide, GALA-INF3 peptide and INF-5 peptide.

The skilled artisan can select any of the above ligands or moieties (e.g., cell penetrating moieties or moieties for transport across the BBB) in view of the target tissue, target cell, route of administration, route of intended follow by the oligonucleotide, etc.

According to one embodiment, when the ligand or moiety (e.g., a cell penetrating moiety or a moiety for transport across the BBB) is a peptide or peptide product, it can be modified in vitro (e.g., pegylated, lipid modified, etc.) to confer stability (e.g., anti-protease activity) and/or solubility (e.g., in biological fluids such as blood, digestive fluids, etc.) to the amino acid sequence of the peptide while retaining its biological activity and extending its half-life.

The conjugates of the invention are typically synthesized using standard procedures in organic synthesis. The skilled artisan will appreciate that the exact step of synthesis will depend on the exact structure of the conjugate to be synthesized. For example, if the conjugate comprises a single nucleic acid strand conjugated to a cell targeting moiety through its 5' end, synthesis is typically performed by contacting an amino activated oligonucleotide with a reactively activated cell targeting moiety.

According to one embodiment, when the conjugate comprises a double-stranded miR-135 nucleic acid, then the sense (e.g., guide) strand and the antisense (e.g., passenger) strand are synthesized, respectively, and annealed in vitro using standard molecular biology methods (as discussed in detail above). In a typical conjugate, a first nucleic acid strand carries a cell targeting moiety and a second nucleic acid strand carries a protecting group.

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 may also be at the 3' end of the nucleic acid strand.

In one embodiment, the cell targeting moiety is coupled to the 5 'end of the passenger strand and/or the protecting group is attached to the 5' end of the guide strand.

It is to be appreciated that when the cell targeting moiety is coupled to the 5 'or 3' end of the sense (e.g., guide) or antisense (e.g., passenger) strand of the synthetic miR-135 molecule, the cell-penetrating moiety and/or the moiety for transport across the BBB can be coupled to any remaining end (5 'or 3') of the double-stranded molecule.

According to one embodiment, the cell penetrating moiety and/or the moiety for transport across the BBB may be coupled to a cell targeting moiety.

According to one embodiment, the lipid moiety (e.g., cholesterol) is coupled to a cell targeting moiety (e.g., SSRI, such as sertraline).

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

According to one embodiment, the synthetic miR-135 molecule or conjugate of some embodiments of the invention and the cell-penetrating moiety and/or the moiety for transport across the BBB can be directly coupled. Alternatively, according to another embodiment, the two moieties may be linked by a linking group.

Suitable linking groups for use in the present invention are discussed above.

According to a specific embodiment, the linker is a palmitoyl modified linker (wherein ASO is an oligonucleotide) having the structure:

according to one embodiment, the linker is a phosphodiester unit (e.g., a Phosphodiester (PO) -trinucleotide linkage and a hexylamino spacer) to conjugate palmitate to the oligonucleotide.

According to one embodiment, the linker is a cholesterol linker. Exemplary linkers include triethylene glycol [ TEG ] linkers and 2-aminobutyl-1-3-propanediol [ C7] linkers.

According to one embodiment, the conjugates of the invention are synthesized using the following steps:

(i) Activating a cell penetrating moiety (e.g., which specifically binds to a neurotransmitter transporter). According to one embodiment, the activating group in the agent is a succinimide or an amino group.

(ii) Activating the passenger strand (or guide strand) at the 5' end thereof. According to a specific embodiment, the activating group in the oligonucleotide is an amino group (wherein the agent has been activated by a succinimide group) or a carboxyl group (wherein the agent has been activated by an amine group).

(iii) The activated cell-penetrating moiety is contacted with an activated passenger strand (or an activated guide strand) under conditions sufficient to allow a reaction between the two activating groups.

According to one embodiment, the following steps may also be performed:

(iv) The protecting group is added to the guide chain (or passenger chain). This step can be performed using oligonucleotides whose reactive groups are blocked by acetylation or benzylation (furanose groups), 2-cyanoethylation (phosphodiester bonds) and FMOC (exocyclic amino groups).

According to one embodiment, the following steps may be further performed:

(v) The guide strand and the passenger strand are annealed.

According to one embodiment, as described above, the synthetic miR-135 molecules can be administered to target cells (e.g., brain cells, such as glial cells, oligodendrocytes, choroidal Plexus (CP) cells, stem cells, or differentiated stem cells) as part of an expression construct, without comprising chemical modification. In this case, the synthetic miR-135 molecules are linked in a nucleic acid construct under the control of cis-acting regulatory elements (e.g., promoters) capable of directing expression of micrornas in target cells (e.g., brain cells, such as glial cells, oligodendrocytes, CP cells, stem cells, or differentiated stem cells) in a constitutive or inducible manner.

The expression constructs of the invention may also include other sequences (e.g., shuttle vectors) that render them suitable for 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 invention may further include enhancers, which may be adjacent to or remote from the promoter sequence, and may play a role in up-regulating its transcription. Polyadenylation sequences may also be added to the expression constructs of the present invention to increase expression efficiency.

In addition to the embodiments already described, the expression constructs of the invention may generally contain other specific elements aimed at increasing the expression level of cloned nucleic acids or facilitating the identification of cells carrying recombinant DNA. The expression constructs of the invention may or may not include eukaryotic replicons.

The nucleic acid construct can be introduced into a target cell (e.g., brain cell, such as glial cell) of the invention using an appropriate gene delivery vector/method (transfection, transduction, etc.) and an appropriate expression system. Such methods are generally described in the following documents: sambrook et al, molecular cloning: laboratory Manual (Molecular Cloning: A laboratory Manual), new York (1989, 1992); ausubel et al, molecular biology experiments (Current Protocols in Molecular Biology), johnwei international publication (John Wiley and Sons, baltimore, md.), barmor, maryland (1989); chang et al, somatic gene therapy (Somatic Gene Therapy), CRC Press, anaba, michigan (1995); vega et al, gene Targeting (Gene Targeting), CRC Press, anabag, michigan (1995); and (3) a carrier: a review of molecular cloning vectors and their uses, butterworth, boston ma (1988); and Gilboa et al [ biotechnology (Biotechniques) 4 (6): 504-512,1986], and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

To bypass the blood brain barrier, constructs of the invention may be administered directly into the brain (via ventricle), olfactory bulb (via intranasal administration) through the spinal cord (e.g., via epidural catheter) or through expression in the choroid plexus, as further detailed herein. Other modes of administration will be discussed in detail below.

Additionally or alternatively, lipid-based systems can be used to deliver constructs or conjugated miR-135 molecules into target cells (e.g., brain cells, such as glial cells) of the invention.

Liposomes include any synthetic (i.e., non-naturally occurring) structure consisting of a lipid bilayer that encloses a volume. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes can be prepared by any method known in the art [ Monkkonen, J. Et al, 1994, journal of targeted drugs (J. Drug Target) 2:299-308; monkkonen et al, 1993, journal of International calcification research (Calcif. Tissue int.) 53:139-145; lasic d., liposome technologies company (Liposomes Technology inc.), elmins, 1993,63-105 (chapter 3); winterhalter M, lasic D, lipid chemistry and Physics (Chem Phys Lipids) 1993, month 9, 64 (1-3): 35-43]. Any method known in the art may be used to incorporate micrornas (e.g., synthetic miR-135 molecules) into liposomes. For example, microrna polynucleotide agents (e.g., synthetic miR-135 molecules) can be encapsulated within liposomes. Alternatively, it may be adsorbed on the surface of the liposome. Other methods that may be used to incorporate agents into liposomes of the invention are those described in Alfonso et al: [ science and practice of pharmacy, mack publication, iston, pa., 19 th edition, (1995) ] and methods described in Kulkarni et al [ J.Microencapulous article ] 1995,12 (3) 229-46].

Liposomes used in the methods of the invention can cross the blood barrier. Thus, according to one embodiment, the liposomes of the invention do not comprise a blood barrier targeting polysaccharide (e.g., mannose) in their membrane portion. To determine liposomes particularly suitable for the present invention, screening assays may be performed, such as those described in the following documents: US20040266734 and US20040266734; and Danenberg et al, J cardiovascular pharmacology (Journal of cardiovascular pharmacology), 2003,42:671-9; circulation (2002, 106:599-605); circulation 2003,108:2798-804.

According to one embodiment, dual targeting liposomes are used. Exemplary dual targeting liposomes include, but are not limited to: angiopep-2-oligo-arginine, T7-TAT, THR-transporter, tf-TAT, tf-transmembrane (Tf-penetratin) or Tf-yellow bee toxin (Tf-mastoparan).

Additionally or alternatively, non-lipid based vesicles may be used according to this aspect of the invention, including exosomes, e.g., modified exosomes such as EVOX. Such exosomes may cross the BBB and release therapeutic compositions into the brain, such as cerebral cerebrospinal fluid (CSF).

Other non-lipid based vesicles that may be used according to this aspect of the invention include, but are not limited to, polylysine, dendrimers, and Gagomers.

Regardless of the method or construct employed, an isolated cell is provided that comprises a nucleic acid construct encoding a synthetic miR-135 molecule as described above, or comprises a synthetic miR-135 molecule or a conjugated form thereof.

According to a specific embodiment, the cell is a glial cell (i.e., a neuron or glial cell, such as an oligodendrocyte or astrocyte).

According to one embodiment, the glial cell is a neuron, e.g., a serotonergic neuron.

According to a specific embodiment, the cell is a cancer cell.

According to a specific embodiment, the cells are bone cells.

According to a specific embodiment, the cell is a muscle cell.

According to a specific embodiment, the cell is a gastrointestinal cell.

The synthetic miR-135 molecules of the invention will be provided to cells, i.e., target cells (e.g., glial cells) of the invention, either in vivo (i.e., within an organism or subject) or ex vivo (e.g., in tissue culture). In the case of ex vivo treatment of cells, the method preferably comprises the step of administering such cells back to the individual (ex vivo cell therapy).

For ex vivo treatment, it is preferred to treat cells (e.g., brain cells, such as glial cells, e.g., oligodendrocytes, CP cells, stem cells, or differentiated stem cells) with a composition of the invention (e.g., a synthetic miR-135 molecule or conjugated form thereof), and then administer it to a subject in need thereof.

Administration of the ex vivo treated cells of the invention may be accomplished using any suitable route of introduction, such as intravenous, intraperitoneal, intrarenal, gastrointestinal, subcutaneous, transdermal, intramuscular, intradermal, intrathecal, epidural, and intrarectal (as discussed further below). According to a presently preferred embodiment, the ex vivo treated cells of the present invention may be introduced into an individual using intravenous, intrarenal, intragastrointestinal and/or intraperitoneal administration.

The cells of the invention (e.g., glial cells such as oligodendrocytes, CP cells, stem cells, differentiated stem cells and/or cardiomyocytes) may be derived from autologous or allogeneic sources, such as human cadavers or donors. Since non-autologous cells may induce an immune response when administered to the body, several methods have been developed to reduce the likelihood of rejection of non-autologous cells. These include inhibition of the recipient immune system or encapsulation of non-autologous cells in an immunoisolatory semipermeable membrane prior to transplantation.

Encapsulation techniques are generally classified into microencapsulation, which involves small spherical carriers, and macroencapsulation, which involves larger flat and hollow fiber membranes (Uludag, h. Et al, (2000) mammalian cell encapsulation techniques, advanced drug delivery reviews (Adv Drug Deliv Rev) 42,29-64).

Methods of preparing microcapsules are known in the art and include those disclosed in the following documents: for example Lu, m.z. et al (2000), cell encapsulation with alginate and α -phenoxycinnamylidene-acetylated poly (allylamine), biotechnology and bioengineering (Biotechnol Bioeng) 70,479-483; chang, T.M. and Prakash, S. (2001), methods for microencapsulation of enzymes, cells and genetically engineered microorganisms, molecular biotechnology (Mol Biotechnol) 17,249-260; and Lu, m.z. et al (2000), a novel cell encapsulation method using photosensitive poly (allylamine α -cyanocinnamylidene acetate), journal of microencapsulation (J microencapsulation) 17,245-521.

For example, microcapsules were prepared using modified collagen composited with a terpolymer shell of 2-hydroxyethyl methacrylate (HEMA), methacrylic acid (MAA) and Methyl Methacrylate (MMA), resulting in a capsule thickness of 2 μm to 5 μm. Such microcapsules may be further encapsulated with other 2 μm to 5 μm terpolymer shells to impart a negatively charged smooth surface and minimize plasma protein absorption (Chia, s.m. et al, (2002) multilayer microcapsules for cell encapsulation, biomaterials (Biomaterials) 23, 849-856).

Other microcapsules are based on alginate, a marine polysaccharide (sambianis, a. (2003) microencapsulated islets in diabetes treatment, diabetes technology and treatment (Diabetes Thechnol Ther) 5, 665-668) or derivatives thereof. For example, microcapsules may be prepared by polyelectrolyte complexation between polyanionic sodium alginate and sodium cellulose sulfate and polycationic poly (methylene-co-guanidine).

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, for example, when the capsule size is reduced from 1mm to 400 μm, the quality control, mechanical stability, diffusion properties and in vitro activity of the encapsulated cells are improved (Canaple, L. Et al (2002), improved cell encapsulation by size control, journal of biological materials science: polymer edition (J Biomater Sci Polym Ed) 13,783-96). In addition, nanoporous biocapsules with well controlled pore sizes as small as 7nm, tailored surface chemistry and precise microstructure were found to successfully immunoisolate the microenvironment of the cells (see: williams, d., (1999), small i.e. microparticles and nanoparticles technology in medical devices, medical science and technology (Med Device Technol) 10,6-9, and Desai, t.a. (2002) micromachining technology of pancreatic cell encapsulation, biotherapeutic expert view (Expert Opin Biol Ther) 2, 633-646).

Examples of immunosuppressants that may be used in conjunction with ex vivo treatment include, but are not limited to: methotrexate, cyclophosphamide, cyclosporine a, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazine (sulphasalazopyrine)), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximabExamples of etanercept, tumor necrosis factor antagonists (TNF.alpha.blocking, biologicals for inflammatory cytokines, and non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs include, but are not limited to, acetylsalicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salicylic acid, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, cox-2 inhibitors, and tramadol.

For in vivo treatment, the composition (e.g., a synthetic miR-135 molecule or conjugated form thereof) is administered to a subject, either as such or as part of a pharmaceutical composition.

As used herein, "pharmaceutical composition" refers to a formulation of one or more of the active ingredients described herein with other chemical ingredients, such as physiologically suitable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to an organism.

Herein, the term "active ingredient" refers to a molecule responsible for a biological effect (e.g., a synthetic miR-135 molecule or conjugated form thereof).

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably to refer to a carrier or diluent that does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound. Excipients are included in these phrases.

Herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starches, cellulose derivatives, gelatin, vegetable oils, and polymers such as polyethylene glycol.

The formulation and administration techniques of drugs can be found in the following documents: "Remington' sPharmaceutical Sciences," Mitsui publishing company, iston, pa., the latest edition, incorporated herein by reference.

Suitable routes of administration may include, for example, oral, rectal, transmucosal, especially nasal, intranasal, ocular, enteral or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal or intraocular injections.

Alternatively, the pharmaceutical composition may be administered in a local manner rather than a systemic manner, for example, by injecting the pharmaceutical composition directly into a tissue region of a patient.

According to a specific embodiment, the composition is for oral administration.

Conventional methods for delivering drugs to the Central Nervous System (CNS) include: neurosurgical strategies (e.g., intra-brain injection or intra-ventricular infusion); molecular manipulation of agents (e.g., production of synthetic miR-135 molecules linked to cell targeting moieties having affinity for brain cell surface molecules, as discussed above), attempts to exploit one of the BBB's endogenous transport pathways; designing a pharmacological strategy for increasing the lipid solubility of the agent (e.g., conjugation of a synthetic miR-135 molecule to a lipid or cholesterol carrier); and temporary disruption of BBB integrity by hypertonic disruption (caused by infusion of mannitol solution into carotid artery or use of bioactive agents such as angiotensin peptides).

Drug delivery methods behind the BBB include intracerebral implantation (e.g., with needles) and convection enhanced distribution. Mannitol can be used to bypass the BBB. Likewise, mucosal (e.g., nasal) administration can be used to bypass the BBB.

According to a specific embodiment, the composition is for intranasal administration.

Intranasal administration may be used to deliver therapeutic agents to the Central Nervous System (CNS). Delivery is by olfactory epithelial cells located in the posterior upper part of the nasal cavity. Neurons of the olfactory epithelium project into the olfactory bulb in the brain, thus enabling a direct connection between the brain and the external environment. The transfer of the drug into the brain is thought to occur through slow transport of the olfactory nerve cells or through faster transfer into the cerebrospinal fluid in the brain along the perinerve spaces surrounding the olfactory nerve cells. It is considered to be a non-invasive mode of administration, allowing macromolecules that cannot cross the blood brain barrier to enter the central nervous system. This route of administration reduces systemic exposure and thus reduces unwanted systemic side effects. Delivery from the nose to the CNS typically occurs within minutes and does not require drug binding to any receptor or axonal transport.

According to a specific embodiment, the composition is for Intrathecal (IC), intraventricular (ICV), ocular or Intravenous (IV) administration, wherein the composition will allow passage through the Blood Brain Barrier (BBB).

According to one embodiment, the pharmaceutical composition is administered intrathecally, i.e. into the spinal canal or subarachnoid space, thereby allowing it to reach the cerebrospinal fluid (CSF).

According to one embodiment, the pharmaceutical composition is administered by ocular administration.

According to one embodiment, the pharmaceutical composition is administered by Intraventricular (ICV) administration, i.e. by direct injection into the cerebrospinal fluid in the ventricle.

According to one embodiment, the pharmaceutical composition is administered by Intravenous (IV) administration.

The pharmaceutical compositions of the invention may be prepared by methods well known in the art, for example by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping (entering), or lyophilizing processes.

Thus, the pharmaceutical compositions for use according to the invention may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active ingredients into preparations which can be used pharmaceutically. The appropriate formulation depends on the route of administration selected.

For injection, the active ingredient of the pharmaceutical composition may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, ringer's solution or physiological salt 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.

For oral administration, the pharmaceutical compositions may be readily formulated by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient. Pharmaceutical formulations for oral use may be prepared using solid excipients, optionally grinding the resulting mixture, and, if desired, processing the mixture of granules after adding suitable adjuvants to obtain tablets or dragee cores. Suitable excipients are in particular fillers, for example sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and/or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents can be added, for example crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores have suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbomer gels, polyethylene glycol, titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to the tablets or dragee coatings for identifying or characterizing different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules (push-fit capsules) made of gelatin and sealed soft capsules made of gelatin and a plasticizer (e.g., glycerol or sorbitol). Push-in capsules may contain the active ingredient in admixture with fillers (e.g., lactose), binders (e.g., starches), lubricants (e.g., talc or magnesium stearate) and, optionally, stabilizers. In soft capsules, the modified DNase proteins may be dissolved or suspended in a suitable liquid, such as a fatty oil, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers may be added. The dosages of all formulations for oral administration should be appropriate for the chosen route of administration.

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

For administration by nasal inhalation, the active ingredient used according to the invention is conveniently delivered in the form of an aerosol spray from a pressurized pack or nebulizer, and suitable propellants may be used, for example dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin may be formulated containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

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

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agents in water-soluble form. Alternatively, suspensions of the active ingredients may be prepared as appropriate oil-or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils (e.g. sesame oil), or synthetic fatty acid esters (e.g. ethyl oleate), triglycerides or liposomes. The aqueous injection suspension may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredient, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water, before use.

The pharmaceutical compositions of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., using conventional suppository bases such as cocoa butter or other glycerides.

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

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

Determination of a therapeutically effective amount is well within the ability of those skilled in the art, particularly in light of the detailed disclosure provided herein.

For any formulation used in the methods of the invention, a therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, the dosage may be formulated in an animal model to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in the human body.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell culture or in experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration employed. The exact formulation, route of administration and dosage may be selected by the individual physician in view of the patient's condition. (see, e.g., fingl et al, 1975, "therapeutic pharmacological foundation (The Pharmacological Basis of Therapeutics)", ch.1p.1).

The dose and interval can be adjusted individually to provide sufficient plasma active ingredient levels to induce or inhibit biological effects (minimum effective concentration (MEC). MEC will vary for each formulation but can be estimated from in vitro data.

Depending on the severity and responsiveness of the condition to be treated, the administration may be a single administration, with the course of treatment lasting from days to weeks, or until cure, or a reduction in the disease state is achieved.

Of course, the amount of the composition to be administered will depend on the subject being treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, and the like. The dosage and time of administration will vary according to careful and continuous monitoring of individual condition changes.

It is understood that there is an animal model by which the synthetic miR-135 molecules of the invention can be tested prior to human treatment. For example, animal models of depression, stress, anxiety, such as a learning unassisted model (LH), a Chronic Mild Stress (CMS) model, a social frustration stress (SDS) model, and a maternal loving deprivation model, and a sleep deprivation model, may be used. For example, animal models of bipolar disorder that may be used include, for example, transgenic mice with neuronal specific expression of mutant Polg (D181A) [ as taught by Kato et al, neurological and biological behavior reviews (Neuroscience and Biobehavioral Reviews) (2007) 6 (31): 832842, incorporated herein by reference ], and established amphetamine-induced manic rat models [ e.g., as taught in U.S. Pat. No. 3, 6,555,585 ] and ketamine-induced hyperactivity [ e.g., as taught by Ghedim et al, (Journal of Psychiatric Research), (2012) 46:1569-1575], incorporated herein by reference.

The compositions of the invention may be presented in a package or dispenser device, such as a U.S. FDA (U.S. food and drug administration approved kit, which may contain one or more unit dosage forms containing the active ingredient, for example, the package may comprise a metal or plastic foil, such as a blister pack (blister pack), which may be accompanied by instructions for administration, the package or dispenser may also be accompanied by a notice associated with a container in a form prescribed by a government agency regulating the manufacture, use or sale of the pharmaceutical product, the notice reflecting approval by the agency of the form of the composition for use in humans or veterinarian, such notice may be, for example, a prescription drug label approved by the U.S. food and drug administration, or approved product instructions.

It is to be understood that in addition to the synthetic miR-135 molecules or conjugated forms thereof, therapeutic compositions of the invention can also comprise other known drugs for treating CNS-related disorders (e.g., psychotic disorders such as depression, stress, anxiety, sleep deprivation, etc.), such as, but not limited to: selective Serotonin Reuptake Inhibitors (SSRI), serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NaSSAs), norepinephrine Reuptake Inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors, selective serotonin reuptake enhancers, norepinephrine-dopamine inhibitors, tricyclic antidepressants (e.g. imipramine), monoamine oxidase inhibitors (MAOIs). These medicaments may be contained in the article in a single package or in individual packages.

According to one embodiment, in addition to the synthetic miR-135 molecule or conjugated form thereof, the therapeutic composition of the invention comprises a drug or any combination of drugs, including, but not limited to: lithium (e.g., lithium carbonate, lithium citrate, lithium sulfate), antipsychotics (e.g., typical antipsychotics and atypical antipsychotics, as described in detail below), mood stabilizer drugs (e.g., valproic acid (VPA, valproate), minerals, anticonvulsants, antipsychotics), and antidepressants.

Examples of typical antipsychotics that may be used according to the present invention include, but are not limited to, low-potency drugs: chlorpromazineTalbond->Thioridazine->Mesopyridazine and levo-promethazine; the medium-effect medicament comprises the following components: rosapine->Morin->Perphenazine->And thiothioxanthone->High-efficiency medicine: haloperidol-> FluphenazineNorfloxacin, zuclopidol +.>Flupentixol->Prochlorlazine and trifluoperazine +.>Furthermore, prochlorperazine +.>And pimozide->

Exemplary atypical antipsychotics (also referred to as second-generation antipsychotics) that may be used in accordance with the present teachings include, but are not limited to: amisulprideAripiprazole->Asenapine->Blonanserin->Bitolperistin (RG 1678), epipiprazole (OPC-34712), carbimidazine +.>Chlorapamin->Clozapine->Carilazine (RGH-188), iloperidoneLurasidone->LY2140023, meipigron->Mo Shapa MingOlanzapine->Paliperidone->Perropine->Pimuvanserin->Quinidine->Remomobili->Risperidone->Serndole->Shu Bi li->Vabicaserin/>ZiprasidoneZotepine->And Ji Luona (Lu 31-130).

Exemplary embodiments that may be used in accordance with the present teachingsMood stabilizers include, but are not limited to, minerals (e.g., lithium); anticonvulsant mood stabilizer comprising valproic acid Divalproex sodium->And sodium valproateLamotrigine->Carbamazepine->OxcarbazepineTopiramate->Riluzole->And gabapentin +.>Antipsychotics (as described above); and food supplements (e.g., omega-3 fatty acids).

Exemplary antidepressants that may be used in accordance with the present invention include, but are not limited to: selective serotonin reuptake inhibitors (SSRIs, such as citalopram, escitalopram, fluoxetine, fluvoxamine, paroxetine and sertraline); serotonin-norepinephrine reuptake inhibitors (SNRIs, such as desmethylvenlafaxine, duloxetine, milnacipran, and venlafaxine); noradrenergic and specific serotonergic antidepressants (e.g., mianserin and mirtazapine); norepinephrine reuptake inhibitors (NRIs, such as tomoxetine, mazindol, reboxetine, and viloxazine); norepinephrine-dopamine reuptake inhibitors (e.g., bupropion); selective serotonin reuptake enhancers (e.g. tenecteplatine); norepinephrine-dopamine inhibitors (NDDIs, such as agomelatine); tricyclic antidepressants (including tertiary amine tricyclic antidepressants and secondary amine tricyclic antidepressants); monoamine oxidase inhibitors (MAOIs).

According to one embodiment, the antidepressant includes a Selective Serotonin Reuptake Inhibitor (SSRI), a tricyclic antidepressant, and a Norepinephrine Reuptake Inhibitor (NRI).

According to one embodiment, the antidepressant includes a Selective Serotonin Reuptake Inhibitor (SSRI).

It should be appreciated that additional non-drug treatment strategies may be used in connection with the present teachings, including but not limited to: clinical psychology, electroconvulsive therapy, involuntary supervision, phototherapy, psychotherapy, transcranial magnetic stimulation and cognitive behavioral therapy.

According to another aspect of the present invention there is provided a method of treating a CNS-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the present invention, thereby treating the CNS-related disorder.

According to another aspect of the present invention there is provided a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the present invention for use in treating a CNS-related disorder in a subject in need thereof.

The term "treating" refers to inhibiting or preventing the development of a disease, disorder or condition and/or causing the alleviation, alleviation or regression of a disease, disorder or condition, or preventing the occurrence of a disease, disorder or medical condition in a subject who may be at risk of a disease, disorder or condition but has not yet been diagnosed as having a disease, disorder or condition (i.e., prophylaxis). Those of skill in the art will appreciate that various methods and assays may be used to assess the progression of a disease, disorder or condition, and similarly, various methods and assays may be used to assess the reduction, alleviation or regression of a disease, disorder or condition.

As used herein, the term "subject" or "subject in need thereof" includes mammals of any age, such as humans, men or women, at risk of suffering from or developing a pathology.

As used herein, the phrase "CNS-related disorder" or "central nervous system-related disorder" includes psychotic disorders (e.g., panic syndrome or panic attacks, anxiety disorders such as generalized anxiety disorder, all types of phobia syndrome, e.g., social phobia, mania, manic depression, e.g., manic depression, hypomania, all forms and/or types of depression, e.g., unipolar depression, stress disorders, PTSD, somatoform disorders, personality disorders, compulsive behavior, psychosis, and schizophrenia); addiction or substance related disorders such as drug dependence [ e.g. alcohol, psychostimulants (e.g. kukka, cocaine, fast pills, iceps), opioids and nicotine ], stress, fatigue, epilepsy, headache, acute pain, chronic pain, neuropathy, cerebral ischemia, dementia (e.g. alzheimer's and multi-infarct dementia), memory loss, cognitive dysfunction (e.g. impaired cognitive function), sleep disorders (e.g. insomnia, wakefulness and/or hypersomnia), eating disorders (e.g. bulimia, anorexia, body-image deformation, binge eating disorders), autism spectrum disorders, tourette, childhood disorders, movement disorders, multiple sclerosis, growth disorders, reproduction disorders, accommodation disorders, delirium. Other patients are described, for example, in the diagnostic and statistical handbook (DSM) for mental disorders, fifth edition (DSM-5). Typically, such conditions have complex genetic, biochemical and/or environmental components.

According to a specific embodiment, the CNS related disorder is a psychotic disorder.

According to a specific embodiment, the mental disorder is an emotional disorder.

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 medical condition, and many other conditions. See, e.g., DSM-5 (described above).

According to a specific embodiment, the mental disorder is depression.

Non-limiting examples of depression include, but are not limited to: major Depressive Disorder (MDD), atypical depression, melancholic depression, major psychotic depression or psychotic depression, stress depression, postpartum depression, seasonal Affective Disorder (SAD), acute depression, chronic depression (dysthymia), bipolar depression, unspecified depression, depressive Personality Disorder (DPD), recurrent transient depressive disorder (RBD), mild depression (mild depression), premenstrual syndrome, premenstrual anxiety disorder, depression caused by chronic medical conditions (e.g., cancer, chronic pain, chemotherapy, chronic stress), and combinations thereof. Various subtypes of depression are described, for example, in DSM-5.

According to a specific embodiment, the mental disorder is bipolar disorder.

Non-limiting examples of bipolar disorders include, but are not limited to: mania, acute mania, severe mania, hypomania, depression, moderate depression, dysthymia, major depression, episodes of mania and/or depression, psychotic/psychotic symptoms (e.g. hallucinations, delusions), mixed bipolar-type states, bipolar I disorder (mania with or without major depression), bipolar II disorder (hypomania with major depression), rapid cycling bipolar disorder, cycling bipolar disorder and/or unspecified bipolar disorder (BD-NOS). See, e.g., DSM-5 (described above). Bipolar disorder is also known as manic depression.

According to a specific embodiment, the psychotic disorder is schizophrenia.

According to one embodiment, schizophrenia refers to a mental disorder involving the individual's departure from reality. Symptoms include two or more of the following symptoms in at least one month: delusions, hallucinations, unstructured speech, severe unstructured or stressful behavior, or negative symptoms (i.e., affective flaccidity, unconsciousness, or self-dialation). Schizophrenia includes mental diseases such as schizoaffective disorders. Diagnosis of schizophrenia is described, for example, in DSM-5. Types of schizophrenia include, but are not limited to, paranoid, catatonic, undifferentiated, and residual. See, e.g., DSM-5 (described above).

According to a specific embodiment, the CNS related disorder is an autism spectrum disorder.

According to one embodiment, autism spectrum disorder refers to a neurological disorder characterized by impaired social interactions and communication, accompanied by repetitive and clatter behaviors. Autism includes a series of impaired social interactions and communications; however, the disorder can be broadly classified as "high-function autism" or "low-function autism" according to the extent of impaired social interaction and communication. Individuals diagnosed with "hyperfunctional autism" have minimal but identifiable impairment of social interactions and communication (e.g., asperger syndrome). Other information on the presence of autism spectrum disorders can be found in the following: such as DSM-5; sicile-Kira and Grandin, "autism spectrum disorder: a complete guideline for understanding autism, albert syndrome, pervasive developmental disorders, and other autism spectrum disorders ", as well as other ASD,2004,Perigee Trade publishers; duncan et al, autism spectrum disorder [ two volumes ]: parent and professional manuals, 2007, prasugrel press.

According to a specific embodiment, the CNS-related disorder is a neuroimmune-based psychotic disorder. Non-limiting examples of neurologic based psychiatric disorders include, but are not limited to: mood disorders such as depression (e.g., major depressive disorder) and bipolar disorder, schizophrenia, autism spectrum disorder, pediatric acute episodic neuropsychiatric syndrome (PANS), and childhood autoimmune neuropsychiatric disorder (PANDAS).

According to another aspect of the present invention there is provided a method of treating a depression-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the present invention, thereby treating the depression-related disorder.

According to another aspect of the present invention there is provided a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the present invention for use in treating a depression-related disorder in a subject in need thereof.

According to one embodiment, the depression-related disorder is selected from major depressive disorder, obsessive-compulsive disorder (OCD), pervasive Developmental Disorder (PDD), post-traumatic stress disorder (PTSD), anxiety disorder, bipolar disorder, eating disorders, and chronic pain.

According to one embodiment, treating a CNS-related disorder (e.g., a psychotic disorder) or a depression-related disorder may further be achieved by administering to the subject an additional drug (or any combination of drugs) for treating the CNS-related disorder (e.g., a psychotic disorder) or depression-related disorder.

Exemplary drugs that may be used in accordance with the present teachings for treating mental disorders or depression-related disorders include, but are not limited to: selective Serotonin Reuptake Inhibitors (SSRIs), serotonin-norepinephrine reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NaSSAs), norepinephrine Reuptake Inhibitors (NRIs), norepinephrine-dopamine reuptake inhibitors, selective serotonin reuptake enhancers, norepinephrine-dopamine inhibitors, tricyclic antidepressants (e.g. imipramine), monoamine oxidase inhibitors (MAOIs).

According to one embodiment, treating a psychotic disorder or a depression-related disorder may further be achieved by administering to the subject an additional agent (or any combination of agents) for treating a psychotic disorder or depression-related disorder, including but not limited to: lithium (e.g., lithium carbonate, lithium citrate, lithium sulfate), antipsychotics (e.g., typical antipsychotics and atypical antipsychotics, as detailed above), mood stabilizer drugs (e.g., valproic acid (VPA, valproate), minerals, anticonvulsants, antipsychotics), and antidepressants. Other drugs that may be used in accordance with the present teachings are described in detail above.

According to one embodiment, treatment is determined to be effective (e.g., psychotic disorder treatment, e.g., antidepressant/mood disorder treatment) when a significantly higher miR-135 expression level is obtained after treatment compared to the miR-135 expression level prior to treatment.

The expression level of miR-135 in the post-treatment subject can be about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% greater than the pre-treatment subject.

Monitoring treatment may also be accomplished by assessing the health of a patient, and additionally or alternatively, by subjecting the subject to behavioral testing, MRI, or any other method known to those of skill in the art.

According to another aspect of the present invention there is provided a method of treating a cancer disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the invention, thereby treating the cancer disease.

According to another aspect of the present invention there is provided a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the present invention for use in treating a cancer disease in a subject in need thereof.

Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Specific examples of cancer diseases include, but are not limited to: myeloid leukemia, such as chronic myelogenous leukemia; acute myelogenous leukemia with maturation, acute promyelocytic leukemia, acute non-lymphocytic leukemia with basophilic granulocytosis, acute monocytic leukemia, acute myelomonocytic leukemia with eosinophilic granulocytosis; malignant lymphomas, such as Burkitt's Non-Hodgkin's lymphomas; lymphocytic leukemia, such as acute lymphoblastic leukemia, chronic lymphoblastic leukemia; myeloproliferative diseases, such as solid tumors, benign meningiomas, salivary gland mixed tumors, colon adenomas; adenocarcinomas, such as small cell lung cancer, kidney, uterus, prostate, bladder, ovary, colon, sarcoma, liposarcoma, myxoid tumor, synovial sarcoma, rhabdomyosarcoma (alveoli), extraosseous mucoid chondrosarcoma, ewing's tumor; others include testicular and ovarian vegetative cell tumors, retinoblastomas, wilms' cell tumors, neuroblastomas, malignant melanoma, mesothelioma, breast, skin, prostate and ovary.

According to a specific embodiment, the cancer comprises ovarian, colorectal or prostate cancer.

According to one embodiment, the treatment of the cancer disease may be further achieved by administering to the subject an additional drug (or any combination of drugs) for treating the cancer disease.

Such drugs may include, but are not limited to, radiation therapy, chemotherapy, biological therapy (e.g., immunotherapy or bone marrow transplantation).

According to one embodiment, the anticancer therapy is determined to be effective when the tumor mass is reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more, or when tumor growth ceases, as compared to a subject not treated with a composition of the invention, or as compared to the same subject receiving the treatment but prior to the treatment.

Those skilled in the art will appreciate that various methods and assays may be used to assess the efficacy of cancer treatment, such as CT scan, MRI, X-ray, ultrasound, blood testing, and the like.

The composition of matter of some embodiments of the invention may further be used to enhance muscle cell differentiation and bone regeneration.

Accordingly, a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the invention can be used to treat a bone-related disease or disorder or a muscle-related disease or disorder in a subject in need thereof.

According to one embodiment, there is provided a method of promoting bone regeneration or muscle cell differentiation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition of matter or conjugate of some embodiments of the invention, thereby promoting bone regeneration or muscle cell differentiation.

Exemplary bone-related diseases or conditions that may be treated according to the present teachings include, but are not limited to: injury involving bone injury; fractures, such as closed, open, and non-union fractures; a growth defect; osteolytic bone diseases, such as cancer; periodontal disease and defects, as well as other dental restorative processes; skeletal disorders such as age-related osteoporosis, postmenopausal osteoporosis, glucocorticoid-induced osteoporosis or disuse osteoporosis and arthritis, osteoarthritis, or any disorder that benefits from stimulation of bone formation. The compositions of the invention may also be used for the treatment of congenital, trauma-induced, or surgically resected bone (e.g. for cancer treatment), as well as for cosmetic surgery.

Exemplary muscle-related diseases or disorders that may be treated in accordance with the present teachings include, but are not limited to: muscle degeneration diseases, neuromuscular diseases, spinal muscular atrophy, inflammatory muscle diseases, and metabolic muscle diseases. Exemplary muscle diseases include, but are not limited to: muscular dystrophy, such as Duchenne Muscular Dystrophy (DMD), becker muscular dystrophy, facial shoulder brachial muscular dystrophy, and myotonic muscular dystrophy; amyotrophic Lateral Sclerosis (ALS), myasthenia gravis, lambert-eaton syndrome, botulism, and cerebrovascular accidents.

According to one embodiment, the anti-cancer treatment is determined to be effective when bone regeneration (e.g., in a bone cell mass) or muscle cell differentiation (e.g., in a bone cell mass) is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% or more compared to a subject not treated with a composition of the invention, or compared to the same subject receiving the treatment but prior to the treatment.

Those skilled in the art will appreciate that various methods and assays may be used to assess bone regeneration and promotion of myocyte differentiation, such as CT scan, MRI, X-ray, ultrasound, blood testing, and the like.

It is expected that during the life of a patent from the beginning of this application many relevant miRNA modifications will be developed and the scope of the term modification is intended to include all such new techniques a priori.

As used herein, the term "about" refers to 10%.

The terms "include (comprises, comprising, includes, including)", "having (has)" and its cognate words (conjugates) mean "including but not limited to.

The term "consisting of … …" is intended to be "inclusive of and limited to".

The term "consisting essentially of … … (consisting essentially of)" means that a composition, method, or structure can include additional ingredients, steps, and/or portions, provided that the additional ingredients, steps, and/or portions do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound (at least one compound)" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of the invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be interpreted as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as individual values within the range. For example, a description of a range such as 1 to 6 should be considered to have specifically disclosed sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within the range, e.g., 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, is applicable.

Whenever numerical ranges are indicated herein, it is intended to include any reference number (fractional or integer) within the indicated range. The expressions "range between the first indicator number and the second indicator number" and "range from the first indicator number to the second indicator number" are used interchangeably herein and are meant to include the first indicator number and the second indicator number and all fractions and integers therebetween.

As used herein, the term "method" refers to means, techniques, and procedures for accomplishing a given task including, but not limited to, those means, techniques, and procedures known to, or readily developed from, practitioners of the chemical, pharmacological, biological, biochemical, and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered as essential features of such embodiments unless the embodiment is not functional without such elements.

Various embodiments and aspects of the invention as described above and as claimed in the claims section below are experimentally supported in the following examples.

It is to be understood that any sequence identifier (SEQ ID NO) disclosed in the present application may refer to a DNA sequence or an RNA sequence, depending on the context in which the SEQ ID NO is mentioned, even if the SEQ ID NO is expressed only in the form of a DNA sequence or in the form of an RNA sequence. For example, SEQ ID NO 10 is expressed as an RNA sequence (e.g., uracil is denoted by U), but it may refer to an RNA sequence corresponding to a miR-135b nucleic acid sequence, or a DNA sequence of a miR-135b molecule nucleic acid sequence. Furthermore, although some sequences are expressed in the form of RNA sequences (e.g., uracil is denoted by U), it may refer to the sequence of an RNA molecule comprising dsRNA, or the sequence of a DNA molecule corresponding to the illustrated RNA sequence, depending on the actual type of molecule described. In any event, DNA and RNA molecules having the disclosed sequences are contemplated, as are any alternatives.

Examples

Reference is now made to the following examples, which together with the above description illustrate the invention in a non-limiting manner.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbial, and recombinant DNA techniques. These techniques are explained in detail in the literature. See, for example: "molecular cloning: laboratory Manual (Molecular Cloning: A laboratory Manual) "Sambrook et al, (1989); "molecular biology experiments (Current Protocols in Molecular Biology)", volumes I-III, ausubel, R.M. editions (1994); ausubel et al, "molecular biology experiments (Current Protocols in Molecular Biology)", johnwei international publication company (John Wiley and Sons, baltimore, md.), barmor, maryland (1989); perbal, "molecular cloning Utility guide (A Practical Guide to Molecular Cloning)", john wei liqueur, new york (1988); watson et al, "recombinant DNA (Recombinant DNA)", science American book (Scientific American Books), new York; birren et al (edit), "genome analysis: a series of laboratory manuals (Genome Analysis: A Laboratory Manual Series) ", volumes 1-4, cold spring harbor laboratory Press (Cold Spring Harbor Press), new York (1998); the method as described in the following U.S. patents: US4,666,828, US4,683,202, US4,801,531, US5,192,659 and US5,272,057; "cell biology: laboratory Manual (Cell Biology: A Laboratory Handbook) ", volumes I-III, cellis, J.E. editions (1994); "Current immunological protocols" volume I-III, coligan J.E. edit (1994); stites et al, edited, "basic and clinical immunology (Basic and Clinical Immunology)" (eighth edition), appleton & Lange Press, norwalk, CT (1994); mishell and Shiigi editions, "selected methods in cell immunology (Selected Methods in Cellular Immunology)", W.H. Mannheim publishing company, new York (1980); useful immunoassays are widely described in the patent and scientific literature, see, for example: U.S. Pat. nos. 3,791,932, US3,839,153, US3,850,752, US3,850,578, US3,853,987, US3,867,517, US3,879,262, US3,901,654, US3,935,074, US3,984,533, US3,996,345, US4,034,074, US4,098,876, US4,879,219, US5,011,771 and US5,281,521; "oligonucleotide Synthesis (Oligonucleotide Synthesis)", gait, M.J. edit (1984); "nucleic acid hybridization (Nucleic Acid Hybridization)", hames, B.D. and Higgins S.J. editions (1985); "transcription and translation (Transcription and Translation)", hames, b.d., and Higgins s.j. Edit (1984); "animal cell culture (Animal Cell Culture)", fresnel, r.i. edit (1986); "immobilized cells and enzymes (Immobilized Cells and Enzymes)", IRL Press (1986); "molecular cloning Utility guidelines (A Practical Guide to Molecular Cloning)", perbal, B. (1984) and "methods in enzymology", volumes 1-317, american academy of publishing; "PCR protocol: method and application guidelines (A Guide To Methods And Applications) ", academic press, san diego, CA (1990); marshak et al, "protein purification and characterization strategy-laboratory curriculum handbook (Strategies for Protein Purification and Characterization-A Laboratory Course Manual)", CSHL publishing (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are also provided herein. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

General materials and Experimental procedure

Animal and arrangement

Adult C57BL/6 male mice of 9-11 weeks of age were used. Mice were kept in a temperature controlled room (22.+ -. 1 ℃) for 12 hours light/dark cycle. Food and water are available at will. All experimental protocols were performed on male mice and were approved by the institutional animal care and use committee of the institute of the science, weizmann.

Microdissection and RNA preparation

The brain was removed immediately after the decapitation and placed on a 1mm metal substrate (cat #51380; wu Dedai mol. Ston, illinois). The brain was sectioned into 1mm or 2mm sections using standard blades (GEM, 62-0165) and then flash frozen on dry ice. Brain regions were extracted from sections removed from the matrix using blunt syringes of different diameters and then stored at-80 ℃. Using Nucleospin ^TM RNA XS kit (Macherey-Nagel) ^TM Du Ren, germany) for RNA extraction. RNA was reverse transcribed into cDNA using a high capacity RNA to cDNA kit (applied biosystems, inc., forst Calif.). The cDNA was then analyzed by quantitative real-time PCR (RT-PCR).

MicroRNA purification and quantitative real-time PCR expression analysis

According to the manufacturer's instructions, use Mini kit (Qiagen)) Isolation of mRNAs including microRNAs and use +.>The reverse transcription kit is processed to produce cDNA. Then use SYBR ^TM Green PCR kit (Qiagen) according to manufacturer's guidelines at Applied Biosystems ^TM The samples were analyzed in a 7500 thermocycler (applied biosystems, inc.). Specific primers for each miR were used with commercial universal primers, while U6 snRNA was used as an internal control. For mRNA quantification, specific primers were designed for each transcript using software Primer Express 2 (applied biosystems, usa) and expression was tested using real-time PCR.

Cloning of target transcript 3' UTR into psiCHEK-2 luciferase expression plasmid

The 3' UTR sequences of Slc6a4 and Htr1a were PCR amplified from mouse genomic DNA. Ligation of 3'UTRs PCR fragments according to manufacturer's guidelinesIn the easy vector (Promega), and further subcloned into a single NotI site 3' of luciferase in the psiCHECK-2 reporter plasmid (Promega). Cloning direction was confirmed by diagnostic cleavage and sequencing.

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: a psiCHECK-2 plasmid containing a wild-type or mutated 3' utr and an over-expression vector for a specific miRNA, or an air-over-expression plasmid. miR over-expression plasmids were taken from miR-Vec libraries. 24 hours after transfection, cells were lysed and luciferase reporter activity was determined as described previously [ Kuperman Y. Et al, molecular Endocrinol (2011) 25:157-169]. Renilla luciferase values were normalized to control firefly luciferase levels, transcribed from the same vector but unaffected by the 3' UTR, then tested under each condition and repeated six times on average.

Table 2: oligonucleotide primers for cloning

Table 3: oligonucleotide primers for real-time PCR of microRNAs

Table 4: oligonucleotide primers for real-time PCR of mRNA

Synthesis of miR-135 Single-stranded oligonucleotides (optionally conjugated with Sertraline)

The oligonucleotide sequences were assembled using standard procedures using standard 2' -deoxy, 2' -O-Me or 2' -MOE phosphoramidite blocks, for example:

the synthesis was performed using a typical experimental method of solid phase synthesis on CPG (controlled pore glass) supports. In short, a typical oligonucleotide synthesis is performed by a series of cycles consisting of four steps (deprotection, coupling, capping, and oxidation) which are repeated until the 5' nucleotide is attached (as shown in FIG. 1).

(a) Deprotection/detritylation

Cleaving the acid labile 5' -dimethoxytrityl protecting group from the base anchored to the solid support (the starting nucleoside or the subsequently grown oligomeric chain); thereby obtaining free reactive hydroxyl functionality. Dichloro or trichloroacetic acid in methylene chloride was used as cleavage reagent.

(b) Coupling of

The free 5' -OH group is now able to react with the added phosphoramidite. As a result, the two nucleosides are linked by a phosphite bridge. The phosphoramidite must first be activated using a weak acid (e.g., 1H-tetrazole).

(c) End capping

In order to prevent the remaining free OH-groups (about 1%) from reacting in the synthesis cycle, thereby generating non-specific sequences, all free reactive groups are blocked using acetylation in the capping step, thereby eliminating them as reaction partners during further synthesis.

(d) Oxidation

The internucleotide phosphate groups produced in the coupling step are oxidized to their phosphate salts using an iodine solution.

The new synthesis cycle is repeated again from step (a). These reactions are repeated until the desired oligonucleotide sequence is produced. The loop can be terminated to give a solid support-bound oligonucleotide with free 5'-OH groups or an oligonucleotide with protected 5' -OH groups (DMT).

Incorporation of phosphorothioate linkages:

the difference between synthesizing a normal oligomer with a fully phosphodiester backbone and an oligomer with a partially or fully phosphorothioate backbone is based on the choice of oxidizing agent used in the oxidation of step (d).

The phosphodiester bond was created by adding a fourth oxygen to the phosphate using iodine and water. Phosphorothioate linkages were created by adding sulfur to the phosphate esters using Beaucage reagent. Once the sulfur or oxygen is attached to the phosphate, the bond is stabilized from subsequent chemical cycling. By switching back and forth between the two oxidants, a chimeric backbone can be constructed.

Sertraline conjugated miR-135 synthesis

Guide RNA strands and passenger RNA strands were synthesized on an automated synthesizer using phosphoramidite chemistry. Use of rA ^Bz ；rC ^Ac ；rG ^iBu The method comprises the steps of carrying out a first treatment on the surface of the rU (r U) _2'Ome A ^Bz ； _2'OMe C ^Ac ； _2'OMe U phosphoramidite monomer extension sequences. After strand extension, the RNA strand is immobilized therefromThe support was cut off and deprotected using a mixture of ammonium hydroxide and methylamine. The 2' OH position is deprotected using a solution containing fluoride ions. The RNA strand was then purified by anion exchange HPLC.

Sertraline modified passenger RNA strands were synthesized on an automated synthesizer using phosphoramidite chemistry. Use of rA ^Bz ；rC ^Ac ；rG ^iBu The method comprises the steps of carrying out a first treatment on the surface of the rU (r U) _2'Ome A ^Bz ； _2'OMe C ^Ac ； _2'OMe U phosphoramidite monomer extension sequences. The C10N-hydroxysuccinimide ester linker was grafted to the 5' end of the passenger RNA sequence that was still attached to the solid support prior to the deprotection step. Sertraline-C ₆ Acy-NH ₂ Condensation of the ligand with the C10N-hydroxysuccinimide ester linker was performed in dimethylformamide containing 3% N, N-diisopropylethylamine at room temperature for 48 hours (FIG. 2). Following conjugation, the sertraline modified passenger RNA strand is cleaved from its solid support and deprotected using a mixture of ammonium hydroxide and methylamine. The 2' OH position is deprotected using a solution containing fluoride ions. The conjugate RNA strand was then purified by reverse phase HPLC.

The guide and passenger RNA strands were quantified by UV spectroscopy using extinction coefficients calculated based on nearest neighbor method. The duplex incorporates equimolar amounts of guide and passenger strands. Annealing is performed in water. The duplex was not further purified.

Oligonucleotide primers, RNA strands and sertraline conjugated RNA strands and RNA duplex were prepared by the company columbach (axolarabs, gmbH) germany according to the instructions and designs discussed herein.

The structure is as follows:

naked miR-135 duplex 1 (duplex 11 according to Table 1 below and duplex 1 according to Table 6 below)

Sertraline conjugated miR-135 duplex 1 (miCure-135-1 according to Table 6 below)

Sertraline-acyl C ₆ -NHCO-C ₉ -5'miR-135 sequence 3' -OH

Naked control (duplex 16 according to Table 1 below, and duplex 6 according to Table 6 below)Duplex 8 of (d)

Sertraline conjugated controls (miCure-135-8 according to Table 6 below)

Naked miR-135 duplex 2 (according to Table 6 below)

Guide chain-5' -P/UmUAUGGCUUUUUUUUCCUUAUGUGA (SEQ ID NO: 41)

Passenger chain-5' -ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13)

Sertraline conjugated miR-135 duplex 2 (miCure-135-2 according to Table 6 below, see

Naked miR-135 duplex 3 (according to Table 6 below)

Guide strand-5' -P/UmUAUGGCUUUUUUUUCCUUGUAAmsAm (SEQ ID NO: 42)

Passenger chain-5' -ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13)

Sertraline conjugated miR-135 duplex 3 (miCure-135-3 according to Table 6 below)

Naked miR-135 duplex 9 (according to Table 6 below)

Guide chain-5' -P/UAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 10)

Passenger strand-5' -uscacauaggaaugaaaagccasusa (SEQ ID NO: 47)

Sertraline conjugated miR-135 duplex 9 (miCure-135-9 according to Table 6 below)

Naked miR-135 duplex 10 (according to Table 6 below)

Guide chain-5' -P/UmUAUGGCUUUUUUUUCCUUAUGUGA (SEQ ID NO: 41)

Passenger strand-5' -uscacauaggaaugaaaagccasusa (SEQ ID NO: 47)

Sertraline conjugated miR-135 duplex 10 (miCure-135-10 according to Table 6 below)

Naked miR-135 duplex 10 (according to Table 6 below)

Guide strand-5' -P/UmUAUGGCUUUUUUUUCCUUGUAAmsAm (SEQ ID NO: 42)

Passenger strand-5' -uscacauaggaaugaaaagccasusa (SEQ ID NO: 47)

Sertraline conjugated miR-135 duplex 11 (miCure-135-11 according to Table 6 below)

For all duplex:

capital letters (e.g., N, A, U, C, G): RNA (ribonucleic acid)

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

Lowercase letter "s": phosphorothioates. The absence of indication means normal phosphodiester linkages.

P: phosphoric acid esters

And (3) underlined: 2 '-fluoro, i.e. 2' -F

(C10) The method comprises the following steps Carboxyl modifier C ₁₀

(Grignard study: 10-1935)

Intranasal administration

Mice were lightly anesthetized by inhalation of 2% isoflurane and placed in the supine position. Mu.l of conjugated control or conjugated miR-135 drops were applied alternately to each nostril once daily. A total of 10 μl of the 166 μg containing solution was delivered.

Intra-cerebral injection

For stereotactic surgery and compound delivery, a computer-guided stereotactic instrument and motorized nanosyringe (Angle Two ^TM Stereotaxic Instrument stereotactic, myNeurolab Inc.). The mice were placed on a stereotactic apparatus in the general anesthesia state and at 20 ° tilt, lentiviral preparations were delivered to coordinates as determined by Franklin and Paxinos profiles to DR ML 1mm; AP-4.5mm; DV-4.2mm. Injection was performed at a rate of 0.2. Mu.l/1 min.

Chronic administration in brain chamber

Cerebral perfusion kit 3 (ALZET, DURECT Corporation, cooperation, calif. Cooperation from bregma: posterior, -0.7mm, -1.5mm lateral, -2.0 mm) was used. The perfusion cannula was connected to a mini osmotic pump (ALZET; pump model 1007D) which was implanted subcutaneously in the back of the animal just behind the scapula. The osmotic pump was perfused for about 8 hours prior to implantation and filled with duplex/control (0.5 nmol/day). Once implanted, the pump was continuously perfused with compound or negative control at a rate of 0.5 μl/h for 7 days.

Intra-cerebral microdialysis

Extracellular 5-HT concentrations were measured by in vivo microdialysis. Briefly, one concentric dialysis probe (copper spun, 1 mm-long) was implanted into mPFC (AP, 2.2; ml, -0.2; dv, -3.4) of pentobarbital anesthetized mice. Experiments were performed 48-72 hours after surgery. 1mM citalopram (SSRI; lundbeck A/S, copenhagen Warewratio) was added to the artificial cerebrospinal fluid. At 6. Mu.l min ^-1 (WPI model sp220 i) was pumped to artificial cerebrospinal fluid and 6 min samples were collected. Tail suspension experiments were performed when fraction 8 was collected. The 5-HT concentration was analyzed by high performance liquid chromatography-amperometric detection (Hewlett-packard 1049; p0.6V, palo alto, calif., U.S.A.), with a limit of detection of 1.5fmol sample. Baseline 5-HT levels were calculated as the average of four pre-dose samples. Tar violet staining (cresyl-violet staining) was used to verify correct probe placement.

Behavior assessment

All behavioral assessments were performed in the dark period 2 hours after adapting the test chamber prior to each test. The experimenter performing the test had no knowledge of the group of mice.

Dim Light Transfer (DLT) test: the DLT laboratory apparatus consisted of a polyvinyl chloride box divided into a black darkroom (14X 27X 26 cm) and an associated white 1200lux illumination room (30X 27X 26 cm). During the experiment of 5 minutes, the time spent in the light room, the distance travelled in the light area and the number of bright-dark transitions were quantified with a video tracking system (VideoMot 2; TSE system, bard honburg, germany).

Tail suspension experiment: the mice were hung 30cm above the bench with tape placed about 1cm from the tail tip. Mice were monitored and recorded using a camera system (Smart, panlab company) and were kept stationary for 6 minutes.

8-hydroxy-DPAT induced hypothermia

Body temperature was measured intrarectally using a lubricating probe inserted at B2 cm and a digital thermometer (AZ 9882, panlab, barcelona, spain). Mice were individually kept in clean cages for 20 minutes prior to measurement, and then two baseline temperature measurements were made. After 10 minutes, the animals were intraperitoneally injected with 8-OH-DPAT 1mg/kg, and body temperature was recorded every 15 minutes for 120 minutes. Data are expressed as changes from the average baseline measurement.

In experiments to detect the effects of different miR-135 mimics (as described in the "results" section below), body temperature was measured using a microchip. The mice were subcutaneously implanted with programmable subcutaneous microchip transponders (IPTT-300 extended precision calibration; biomedical systems, inc., schiff, DE) according to the manufacturer's instructions. Briefly, the process involves rapidly inserting a large gauge needle delivery device containing a microchip and depressing a plunger on the device to expel the microchip from the delivery device. Temperature measurements from microchip transponders were obtained using compatible readers (catalog number WRS6007, model IPTT300, biomedical systems inc.). The reader was placed 5cm to 6cm from the back of the mouse according to the manufacturer's instructions. Hearing a beep (after 1 to 3 seconds) indicates that the reading is complete and the temperature displayed is recorded. The repeat temperature is obtained by taking two readings in succession.

Tissue preparation for in situ hybridization and receptor autoradiography

Mice were sacrificed with excess pentobarbital and brains were removed quickly and stored at-20 ℃. Tissue sections 14mm thick were cut using a frozen microtome (HM 500 OM, meikang, waldov, germany), thawed and mounted onto 3-amino-propyltriethoxysilane coated slides (Sigma-Aldrich, spanish Madeli) and stored at-20℃until use.

Receptor autoradiography

Autoradiographic binding assays for 5-HT1A and 5-HT1B receptors and serotonin transporter (SERT) were performed using the following radioligands, respectively: (a) [ solution to the problem ] ³ H]-8-OH-DPAT(233Cimmol ^-1 )，(b)[ ¹²⁵ I]Cyanopindolol (2200 Cimmol) ^-1 ) And (c) [ ³ H]Citalopram (70 Cimmol) ^-1 ) (Amersham-GE Healthcare, basil, and Perkin-Elmer, spanish Madeli). 8-OH-DPAT, isoproterenol, pargyline and 5-HT were from Sigma-Aldrich, and fluoxetine from Tocris. The experimental conditions are summarized in table 5 below.

Table 5: summary of conditions for labeling serotonin receptors and transporters

Exposing tissue toMR film (Kodak Co.) ³ H-micro standard (Amersham-GE Health-care Co.). All experimental and control brains in one group were treated in duplicate and exposed to film in batches.

The films were analyzed by microscopic densitometry using a computer-aided image analyzer (AIS, imaging study, san josep, onta, ca). The 5-HT1AR mRNA and 5-HT1B binding sites in selected brain regions were measured in respective autoradiograms to obtain relative optical densities. For 5-HT1AR and SERT binding, the system is used ³ H-microscale calibration to obtain fmol mg from relative optical density data ^-1 Protein equivalent. The AlS system is also used to acquire false color images. Black and white photographs were taken from the autoradiogram using a Wild 420 microscope (comes, halberd, germany) equipped with a Nikon DXM 1200F digital camera and ACT-1Nikon software (Soft Imaging System Gmbh company, minster, germany). The image was processed with Photoshop (Adobe Systems, mountain view city) by using the same contrast and brightness values.

Peripheral Blood Mononuclear Cell (PBMCs) study

This study was performed by Axolabs www.axolabs.com. Briefly, human PBMCs were isolated from buffy coats (obtained from Blood Bank Suhl, germany, institute of transfusion medicine (Institutefor Transfusion Medicine)) of healthy donors. The buffy coat was used in a volume of about 28ml to 32ml and aseptically delivered in a sealed infusion bag about 19 hours after donation. Human PBMCs (huPBMCs) was isolated using Ficoll gradient centrifugation.

huPMBCs were treated with different mimics (as described in the "results" section below) by transfection or direct incubation (for 24 hours at three concentrations).Transfection was performed using Lipofectamine 2000. Multiplex assays run on Meso Scale Discovery (MSD) platformsTo analyze the supernatant for the presence of cytokines. The whole process is completed by Axolabs (www.axolabs.com).

Corticosterone-chronic restraint stress model

For the chronic restraint stress regimen, mice were isolated and treated 3 days before the start of the experiment. During the 28 day period, mice were gently placed into 50mL Falcon tubes with holes, fixed in plastic trays, and left in a darkened, quiet room for 2 hours, once a day. Control mice were not isolated and were treated for only 1 minute per day.

Corticosterone (Cortic, sigma-Aldrich, spanish Madrid) was dissolved in commercially available mineral water and adjusted to pH 7.0-7.4 with HCl. In a chronic restraint stress regimen over 28 days, isolated mice were given a corticosterone solution, 30 μg ml ^-1 For 15 days, followed by 15. Mu.g ml ^-1 For 3 days, 7.5. Mu.g ml ^-1 For 10 days. The corticosterone solution was kept in opaque bottles for no more than 3 days to avoid irradiation. Control mice only had mineral water.

Chronic social frustrating stress

C57BL/6J mice at 9 weeks of age were subjected to the chronic social stress-frustrating (CSDS) protocol as described previously [ Krishnan V. Et al, cell (Cell) (2007) 131:391-404]. Briefly, mice were placed in feeder cages of challenged ICR (CD 1) inbred mice (Harlan) and allowed to physically interact for 5 minutes. During this time, ICR mice challenged the intruder mice, which exhibited compliant attitudes. A perforated plexiglas spacer was then placed between the animals and the mice were kept in the same cage for 24 hours, allowing for sensory contact. This process was then repeated daily with unfamiliar ICR mice for 10 consecutive days. Control mice were kept in the same room as the social frustrating mice, but were removed from the room during 5 minutes of interaction with ICR mice. Control mice were treated daily, two mice were kept per cage, with perforated transparenciesThe partition plates are spaced apart.

Immunohistochemistry

Mice were anesthetized with pentobarbital and transcardiac perfused with 4% Paraformaldehyde (PFA) in sodium phosphate buffer (pH 7.4). Brains were extracted, post-fixed in PFA at 4 ℃ for 24 hours, and left to stand in a 10-30% gradient sucrose solution at 4 ℃ for 3 days. After cryopreservation, 30 μm thick sections were cut continuously to obtain prefrontal cortex (PFC), caudal putamen (CPu), hippocampus (HPC) and dorsal central slit nucleus (DRN). Brain sections were washed and incubated in 1 XPBS/Triton 0.2% solution containing normal serum from the secondary antibody host. Primary antibodies (primary antibodies) against the following were used: neuN (anti-NeuN 1:1000; ref: MAB377, millipore Inc.), iba1 (anti-Iba 11:1000; ref:019-197741, wako Inc.), and TPH (anti-TPH 11:2500; ref: AB1541, inc.). Briefly, primary antibodies were incubated overnight at 4℃and then incubated with the corresponding biotinylated anti-mouse IgG1 (1:200; ref: A-10519,Life Technologies company) (for anti-NeuN), biotinylated anti-rabbit (1:200; ref: BA-1000,Vector Laboratories company) (for anti-Iba 1), and biotinylated anti-sheep (1:200; ref BA-6000,Vector Laboratories company) (for anti-TPH 1). The color reaction was carried out by incubation with diaminobenzidine tetrahydrochloride (DAB) (ref: 18865.02, quimigen Co.). Sections were mounted and embedded in Entellan (Electron Microscopy Sciences). The number of NeuN positive cells, iba1 positive cells and TPH positive cells in the DRN was evaluated using imageJ software (v 1.51s, NIH, bethesda, maryland, USA) and these sections corresponded to different levels of front and back from bregma of 4.24mm to-4.84 mm. In three consecutive DRN sections, all labeled cells whose nuclei are in the counting frame were counted.

Immunofluorescence

Mice were anesthetized with pentobarbital and transcardiac perfused with 4% Paraformaldehyde (PFA) in sodium phosphate buffer (pH 7.4). Brains were extracted, post-fixed in PFA at 4 ℃ for 24 hours, and left to stand in a 10-30% gradient sucrose solution at 4 ℃ for 3 days. After cryopreservation, 30 μm thick sections were serially cut to obtain Olfactory Bulb (OB), CPu, HPC and DRN. Brain sections were washed and incubated in 1 XPBS/Triton 0.2% solution containing normal serum from the secondary antibody host. Primary antibodies Alexa488 (anti-Alexa 488:1000; ref.: A11094, invitrogen) and TPH (anti-TPH 1:1541; ref.: ab1541, abcam Inc.) were used. Briefly, primary antibodies were incubated overnight at 4 ℃, rinsed, and treated with secondary antibodies A555-anti-sheep (1:500; ref.: A-21436,Life Technologies company) and Alexa 488-anti-rabbit (1:500; ref.: A-21206,Life Technologies company) for 120 minutes. Nuclei were stained with Hoechst (1:10.000, ref.: H3570, life Technologies).

TPH was observed using an inverted Nikon Eclipse Ti2-E microscope (Nikon Instruments company) attached to a rotating disk unit Andor Dragonfly (Oxford Instruments company) ⁺ Intracellular localization and imaging of Alexa 488-conjugated miR-135 in neurons. For all experiments, oil immersion objectives (Plan Apochromat Lambda blue60 ×, numerical Aperture (NA), 1.4, oil) were used. The samples were excited with 405nm, 488nm and 561nm laser diodes. The beam is coupled into a multimode fiber that passes through Andor Borealis unit, reshaping the beam from a gaussian profile to a uniform flat top. From there it passes through a 40 μm pinhole disc. Tissue sections were imaged on a high resolution scientific complementary metal oxide semiconductor (sCMOS) camera (Zyla 4.2,2.0Andor,Oxford Instruments company). Acquisition was performed using Fusion software (Andor, oxford Instruments company) and image processing was performed using ImageJ/Fiji (1.51 s, open source software).

Western blot

Tissue samples of OB, PFC, CPu, HPC, DRN and cerebellum (Cb) were excised from brain sections and homogenized in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 5mM EDTA, 0.1% SDS, 50mM Tris, pH 8.0) containing protease and phosphatase inhibitors. Using Pierce ^TM The BCA protein assay kit (ThermoFisher Scientific company) quantitates proteins. Protein lysates (10-15 g) were separated using 4-15% SDS-PAGE and electrotransferred to nitrocellulose membranes. Using Western blots were probed for primary antibodies against SERT (1:1000, ref: ab130130, abcam Inc.) and 5HT1AR (1:1000, ref: ab85165, abcam Inc.), as well as against β -actin (1:50000, ref: A3854, sigma-Aldrich Inc.) as a loading control, followed by incubation with the corresponding HRP conjugated anti-goat (1:20000, ref: P0449, dako Inc.) for SERT and HRP conjugated anti-rabbit IgG (1:10000, ref: NA934, GE Healthcare Life Inc.) for 5HT1 AR. Using SuperSignal ^TM Chemiluminescent ECL substrate kit (Thermo Fisher Scientific company) was tested by chemiluminescence and using ChemiDoc ^TM Imaging system (Bio-Rad) was photographed. Using ImageLab ^TM The images were analyzed by software (Bio-Rad).

Statistical analysis

Data are expressed as mean ± standard deviation. Data analysis used student t-test, one-way or two-way analysis of variance (as the case may be), followed by post-hoc test (Newman-Keuls). Significance level was set to P <0.05 (double tail).

Example 1

In vitro design, synthesis and validation of miR-135 mimetic oligonucleotides

miR-135 mimics (oligonucleotides) were designed to mimic the antidepressant effects demonstrated by overexpression and viral manipulation of endogenous miR-135 (in mouse 5HT neurons or DRNs, respectively) in transgenic mouse models. Oligonucleotides (miR-135 mimics) are designed based on endogenous miR-135 with only minor modifications aimed at improving stability and cell penetration. The modified oligonucleotides were synthesized by a company of oligonucleotide manufacture named BioSpring (www.biospring.de) using conventional chemistry, each of which served as a duplex to improve stability. 17 different mimics were designed, synthesized and screened in vitro using luciferase assays (see table 1) to verify their effect on Htr1a and Slc6a4 target transcripts. For luciferase assays, the human cell line HEK293T and transfection reagent, and lipofectamine 2000 (Thermo Fisher Scientific company) were used.

Duplex 11 was found to be the most potent miR-135 mimetic oligonucleotide, significantly affecting 5HTR1A and SLC6a4 levels (using 3' utr luciferase construct; fig. 3A-B).

The design of the duplex 11 mimetic of miR-135 is as follows:

guide chain-5 Ph/UAUGGCUUUUCAUUCCUAUGUGa (SEQ ID NO: 10)

Passenger chain-ucACAUAGGAAUGAAAAGCCAUa (SEQ ID NO: 13)

Lowercase letters (e.g., a, u, c, g): 2' -O-Me modification

Ph=phosphate, 5 indicates that this is the 5' end of the sequence

Mimics identified as consistently reducing target transcripts (Htr 1a and Slc6a 4) in various formulations were further used for in vivo experiments.

Table 1: miR-135 mimics (oligonucleotide) of test

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Example 2

Influence of miR-135 mimics on serotonergic function in vivo

To examine the effect of miR-135 mimics on the serotonergic system in vivo, stereotactic surgery was used to deliver miR-135 mimics (duplex 11) directly into the dorsal central nucleus (DRN) of wild-type mice. Naked (i.e., unconjugated) mimics (100 μg) were administered acutely and the physiological consequences of 5HT1AR autoreceptor (HTR 1 a) silencing were detected using a hypothermia response induced by a selective 5-HT1AR agonist, 8-OH-DPAT, which effect was mediated only by presynaptic 5-HT1AR in mice. mice treated with the miR-135 mimetic (duplex 11) did not exhibit 8-OH-DPAT-induced hypothermia, whereas the two control groups: mice treated with artificial cerebrospinal fluid (aCSF) or mice treated with control miR (i.e., a naked control) exhibited the expected low temperature response (fig. 4A-D). Baseline temperatures were not different between groups (fig. 4E). The kinetics of this effect was demonstrated at 4 different time points (24, 48, 72 and 96 hours) after acute dosing (fig. 4A-D).

Example 3

In vivo effects of sertraline conjugated miR-135 mimics on serotonergic function

After verifying miR-135 mimetic efficiency in vitro and in vivo, the oligonucleotides were delivered non-invasively to the brain using sertraline conjugated intranasal administration using the previously reported method (Ferres-Coy et al, molecular psychiatry (mol. Psychiatr.) (2016) 21 (3): 328-38). Sertrales-Coy et al (2016, supra) have previously reported that intranasal administration of sertraline conjugated small interfering RNAs (siRNAs) silences SERT expression/function in mice. After crossing the permeable olfactory epithelium, the sertraline conjugated siRNA is internalized and transported to the serotonin cell body through deep Rab-7 related intimal vesicles.

Modified miR-135 mimics and control oligonucleotides were conjugated to nonfunctional sertraline (purchased from NEDKEN SOLUTIONS, S.L. company, barcelona, spain) and acutely administered to the dorsal mid-slit nuclei (DRN) of naive mice. Mice treated with a 100 μg dose of sertraline-conjugated miR-135 mimetic Duplex 11 (designated miCure-135-1, as shown in SEQ ID Nos. 10 and 13) showed No hypothermia response after 8-OH-DPAT administration. This effect persisted for up to 7 days after acute administration (fig. 5A-D).

Example 4

Acute intracerebral administration of sertraline conjugated miR-135 mimics (30 μg) silence 5HT1a and SERT and elicit antidepressant-like responses

To further explore the efficacy of sertraline conjugated miR-135 in downregulating gene expression in the serotonergic system, which gene expression was previously demonstrated to be directly regulated by miR-135, lower doses of miCure-135-1 (30 μg) were acutely administered to DRN in adult mice. First, the physiological consequences of 5-HT1A-auto receptor (HTR 1A) silencing were detected using a hypothermia response induced by 8-OH-DPAT, as performed with higher doses (100. Mu.g). The results indicate that a single 30 μg dose of miCure-135-1 was sufficient to eliminate hypothermia induced by the selective HTR1A agonist, without changing the baseline temperature for 7 days of treatment (fig. 6A-E).

Next, microdialysis probes were immobilized into the inner prefrontal cortex (mPFC) of mice, enabling them to collect CSF in real time, and subsequently measuring 5-HT levels using liquid chromatography. Mice were housed in a single cage and 15 fractions were collected 6 minutes each. Part 8 was collected during the tail-suspension experiment (test for assessing mouse depression-like behavior). The results showed a significant decrease in immobility time in mice treated with miCure-135-1 compared to the control group (FIG. 6G), indicating that sertraline-conjugated miR-135 has antidepressant-like effects. Notably, consistent with behavioral results, the mPFC 5-HT levels of the treated mice were significantly higher (fig. 6F), indicating that sertraline-conjugated miR-135 treated mice had a better response mechanism. Next, the inventors evaluated whether sertraline-conjugated miR-135 could silence SERT genes. Histological examination of 1-4 days post-dose showed a significant decrease in SERT binding density of the dorsal nucleus of the middle suture 24 hours and 96 hours post-injection (75% of control; fig. 6H).

Example 5

Acute intranasal administration of sertraline conjugated miR-135 mimics (166 μg) silence 5HT1a and elicit an antidepressant/anxiolytic-like response

Intranasal delivery (where anesthetized mice received 5 μl of miCure-135-1 in each nostril, 166 μg total) was used to examine the potential of miCure-135-1 to act as an antidepressant following a non-invasive delivery method. 8-OH-DPAT induced hypothermia was detected 5 days after treatment, and the group of miCure-135-1 treated mice was found to exhibit lower hypothermia response (FIG. 7A, which is the physiological result of HTR1a silencing. In addition, the immobility time in the tail-suspension experiment was reduced in the miCure-135-1 treated mice compared to the control group (FIG. 7B), and the treated mice were longer to wait in the bright room of the dark/light transfer assay compared to the control group, showing anxiolytic effects (FIG. 7C).

Example 6

In vitro design, synthesis and validation of 16 advanced chemical modification miR-135 mimic oligonucleotides

As described above, other miR-135 mimics (oligonucleotides) were designed based on endogenous miR-135 (see Table 1 above), but include advanced chemical modifications aimed at improving stability and reducing innate immune toxicity without resulting in reduced efficacy.

16 different mimics were designed and synthesized using conventional chemistry (as described in the "general materials and Experimental procedures" section above) (see FIG. 8 and Table 6 below) and each oligonucleotide was used as a duplex to improve stability. 16 different miR-135 mimics were screened in vitro using a luciferase assay to determine their effect on validating Htr1a and Slc6a4 target transcripts. For luciferase assays, the human cell line HEK293T and transfection reagent, and lipofectamine 2000 (Thermo Fisher Scientific company) were used.

MiCure-135-1, miCure-135-2, miCure-135-3, miCure-135-9, miCure-135-10, and MiCure-135-11 were found to be the most potent miR-135 mimic oligonucleotides, significantly affecting 5HTR1A and SLC6a4 levels (using the 3' UTR luciferase construct; FIGS. 9A-B).

Mimics identified as consistently reducing target transcripts (Htr 1a and Slc6a 4) in various formulations were further used in other experiments.

Table 6: other miR-135 mimics (oligonucleotides) of the test

Example 7

Advanced chemical modification of miR-135 oligonucleotides alters cytokine secretion patterns of PBMC in vitro

Oligonucleotides that the body does not recognize as self typically induce an immune response by releasing pro-inflammatory cytokines. Chemical modification of oligonucleotides is intended to increase efficacy and potentially reduce innate immune activation. To test the immune activation pattern induced by the new modifications, human Peripheral Blood Mononuclear Cells (PBMCs) were isolated by Ficoll density gradient centrifugation, starting from fresh buffy coats (obtained from a blood donation center, as described in the "general materials and experimental methods" section above) of healthy volunteers. PBMCs were treated with different mimics for 24 hours and supernatants were analyzed for the presence of cytokines using multiplex assays run on a Meso Scalediscovery (MSD) platform.

The results of this assay showed that 2 of the new duplex, miCure-135-2 and miCure-135-3, did not induce secretion of the test cytokines (FIGS. 10A-E and 11A-F, respectively), miR-135-1 showed modest activation of TNF- α (FIG. 10A) and IFN- α -2a (FIG. 10B), miR-135-9 induced high secretion of IFN- α -2a (FIG. 11A) and low secretion of IFN- α (FIG. 11B), miR-135-11 induced high secretion of IFN- α -2a (FIG. 11A), and low secretion of TNF- α (FIG. 11B) and IFN- γ (FIG. 11C). The results of MiCure-135-10 revealed very high secretion of TNF- α and IFN- α -2a (FIGS. 10A and 10B, respectively). The duplex identified as inducing the lowest level of secretion (i.e., miCure-135-2 and miCure-135-3) were further tested in vivo. The relatively high cytokine secretion induced by the miscure-135-9, miscure-135-10 and miscure-135-11 may be related to a shared passenger strand consisting of two phosphorothioate linkages that replace the normal phosphodiester at the 5 'and 3' ends, respectively, which were tested.

Example 8

Acute DRN administration of sertraline conjugated miR-135 mimics affect serotonergic function and cause antidepressant-like reversal

Should be

To further examine the effect of miR-135 mimics on the serotonergic system in vivo, three miR-135 mimics conjugated with nonfunctional 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 to the mid-slit dorsal nucleus (DRN) using stereotactic surgery.

Acute (30 μg) dosing of both miCure-135-2 and miCure-135-9 and detection of physiological consequences of 5HT1AR autoreceptor (HTR 1 a) silencing using hypothermia induced by selective 5-HT1AR agonists, 8-OH-DPAT, was only mediated by presynaptic 5-HT1AR in mice. As shown in FIGS. 12A-D, sertraline conjugated miR-135 treated mice (mimics miCure-135-2 and miCure-135-9) did not show hypothermia, whereas the two control groups, i.e., mice treated with artificial cerebrospinal fluid (aCSF), and mice treated with control miR exhibited the expected hypothermia response. Baseline temperatures were not different between groups (fig. 12E). The kinetics of this effect was demonstrated at 3 different time points after acute administration.

Next, the miCure-135-3 and sertraline conjugated control acutely (100 μg) was administered to the mouse DRN and the functional consequences of serotonin transporter (SERT) silencing were examined. To this end, microdialysis probes were immobilized into the inner prefrontal cortex (mPFC) of mice, which was able to collect CSF in real time and subsequently measure 5-HT levels using liquid chromatography. Mice were housed in a single cage and 12 fractions were collected 6 minutes each. From part 7, infusion of a locally selective serotonin reuptake inhibitor (citalopram 10 μm) by reverse dialysis resulted in an increase in extracellular 5-hydroxytryptamine (5-HT). The increase in 5-HT levels in the mocure-135-3 treated mice was significantly lower compared to the levels of the control group (FIG. 12F), indicating that mocure-135-3 resulted in a decrease in available serotonin transporter. The same experimental group (i.e., mice injected once with 100 μg of miCure-135-3 or conjugated control to DRN) was tested in a tail-suspension experiment. The results demonstrate that mice treated with miCure-135-3 exhibited significantly reduced immobility time compared to the control group (FIG. 12G), which suggests that sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) have antidepressant-like effects.

Example 9

Intranasal and intraventricular administration of sertraline conjugated miR-135 mimics silence 5HT1a and SERT

The use of non-invasive methods of administration (i.e., intranasal administration) demonstrated the potential of sertraline-conjugated miR-135 mimics as antidepressants and employed clinically acceptable methods of administration. Mice were treated acutely with miCure-135-3 and tested for 8-OH-DPAT-induced hypothermia 48 hours after dosing. Administration of 200 μg (FIG. 13C) and 100 μg (FIG. 13B) of miCure-135-3 significantly reduced the hypothermia response in the treated mice compared to the control treated mice. Acute intranasal administration of 50 μg of miCure-135-3 showed a clear trend of hypothermia response (p=0.054) (fig. 13A). A similar response was shown 7 days after intranasal administration of miCure-135-2 (200 μg/day) (FIG. 13D).

The intra-cerebral chamber (ICV) delivery method is intended to mimic intrathecal administration, as in both cases the drug is administered directly into the CSF. In this experiment, a subcutaneous osmotic minipump was connected to a cannula placed in the ventricle of the mouse. The miCure-135-3 was delivered to the second ventricle continuously at a rate of 200 μg/day for 7 days. Compared to the control group, a smaller magnitude of hypothermia was observed in the mocure-1352 treated group (fig. 13E). Taken together, the hypothermic results (FIGS. 13A-E) indicate that sertraline conjugated miR-135 mimics successfully reduce serotonergic autoreceptors (HTR 1 a).

Next, to examine the effect of sertraline-conjugated miR-135 mimics on serotonin transporter (SERT) levels, microdialysis probes were immobilized into the inner prefrontal cortex (mPFC) of mice, enabling them to collect CSF in real-time and subsequently measure 5-HT levels using liquid chromatography. Mice were housed in a single cage and 18 fractions were collected 20 minutes each. From part 7, infusion of a locally selective serotonin reuptake inhibitor (citalopram 10 μm) by reverse dialysis resulted 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 compared to the levels in the control group (FIG. 13F), indicating that intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) reduced 5-hydroxytryptamine transporter in the dorsal aspect of the central slit.

Example 10

Acute intranasal administration of miCure-135-3 affects serotonergic function and causes antidepressant-like responses

To further explore the effect of sertraline conjugated miR-135 mimics, which were administered in a non-invasive manner, on serotonergic function, the intranasal route of administration was used to deliver MICure-135-3 to wild-type mice. To assess whether sertraline conjugated miR-135 could reduce SERT and 5-HT 1A-autoreceptor (HTR 1A) protein levels, mice were acutely treated with miCure-135-3, and samples taken 3 days post-dose were subjected to immunoblot analysis, which showed significant reduction in SERT and HTR1Ar protein levels in the dorsal suture nuclei at 72 hours post-injection (75% for both proteins in the control group; FIGS. 14A-D).

Next, to examine the effect of sertraline-conjugated miR-135 mimics on serotonin transporter (SERT) levels, microdialysis probes were immobilized into the inner prefrontal cortex (mPFC) of mice, enabling them to collect CSF in real-time and subsequently measure 5-HT levels using liquid chromatography. The mice were housed in a single cage 48 hours after dosing and 12 fractions were collected for 20 minutes each. From part 7, infusion of a locally selective serotonin reuptake inhibitor (citalopram 10 μm) by reverse dialysis resulted 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 compared to the levels in the control group (FIG. 14F), indicating that intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) reduced 5-hydroxytryptamine transporter in the dorsal aspect of the central slit.

Additional microdialysis experiments were performed on the same group of mice using the same probe immobilized in mPFC, aimed at detecting the effect of acute intranasal administration of miCure-135-3 on HTR1AR levels. CSF was collected in real time 72 hours after dosing in order to measure 5-HT levels using liquid chromatography. Mice were housed in a single cage and 12 fractions were collected for 20 minutes each. On section 6, a selective HTR1a agonist (8-OH-DPAT 1mg kg ^-1 Intraperitoneal injection) resulted in a decrease in extracellular 5-hydroxytryptamine (5-HT). The reduction in 5-HT levels in the miCure-135-3 treated group was significantly shorter than in the control group (FIG. 14E), indicating that intranasal administration of sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) reduced 5-HT 1A-autoreceptors in the dorsal aspect of the central slit. The same experimental group (i.e., mice given 2500 μg of miCure-135-3 or conjugated control intranasally) was tested in a tail-suspension experiment. The results demonstrate that mice treated with miCure-135-3 exhibited significantly reduced immobility times compared to the control group (FIG. 14G), which suggests that sertraline-conjugated miR-135 mimics (e.g., miCure-135-3) have antidepressant-like effects.

Example 11

Acute intranasal administration of sertraline conjugated miR-135 mimics reduced depressive-like behavior in mice previously receiving a depressive-like induction regimen

The potential of sertraline-conjugated miR-135 mimics as antidepressants was demonstrated using a depression-like mouse model. Mice were treated with corticosterone in their drinking water and subjected to 2 hours of restrictive stress daily for 28 days. Mice experiencing this pattern exhibited more depression-like behavior. According to this protocol, mice were treated with a dose of miCure-135-3 (2500. Mu.g). Mice treated with mocure-1353 showed antidepressant-like effects 3 days after treatment by reducing immobility time in tail-suspension experiments compared to control group (fig. 15).

Example 12

Selective accumulation of miR-135 mimics in the dorsal nucleus of the central slit after intranasal administration

To examine brain distribution of miR-135 mimics following intranasal administration, alexa 488-labeled miCure-135-3 was synthesized. Labeled molecules were delivered to mice using intranasal administration, and brain tissue was collected 6 hours after administration. Confocal fluorescence microscopy showed that alexa 488-labeled miCure-135-3 was detected intracellularly in TPH 2-positive midbrain 5-HT neurons following intranasal administration (FIGS. 16A-C). Confocal analysis showed that alexa 488-labeled miCure-135-3 was absent from cells near the site of application (olfactory bulb) or near brain regions of the ventricles (hippocampus and striatum) (fig. 16D), supporting that surface SERT expression was necessary for oligonucleotide uptake and internalization, and that miCure-135-3 selectively accumulated in DRN.

Example 13

miR-135 mimic has no effect on cell viability in brain

To further test for safety of miR-135 mimics, mice received a single direct injection, directly into the dorsal nucleus of the midsuture of a conjugate control, miCure-135-3, or a previously reported positive control, which had no effect on cell viability (as taught in Ferres-Coy et al, molecular psychiatry (mol. Psychiatr.) (2016) 21 (3): 328-38, conjugated sertraline-siRNA against SERT). The middle brain midstitch nuclear sections were stained with a neuronal (NeuN-positive), serotonergic neuronal (TPH-positive) or microglial cell (Iba-1-positive) marker. Immunohistochemical analysis showed that mocure-135-3 did not induce neuronal degeneration (FIG. 17A), serotonergic neuronal degeneration (FIG. 17C) and immune response (FIG. 17B). These can also be seen in representative images of midbrain midstitch nuclei stained with NeuN, iba1 and TPH under different treatments (fig. 17D). These data support the specificity and safety of the miCure-135-3.

Example 14

Assessing the effect of acute intranasal administration of sertraline conjugated miR-135 in a depressive-like regimen of chronic social frustration stress

The effect of sertraline-conjugated miR-135 mimics as antidepressants was demonstrated by using chronic social frustrating stress protocols in challenged ICR mice [ previously described in: krishnan V.et al, cells (Cell) (2007) 131:391-404]. Mice experiencing this pattern exhibited more depression-like behavior. Mice were treated with a dose of miCure-135-3 (2500. Mu.g) and tested for antidepressant-like effects by measuring the immobility time in the tail suspension test of the miCure-135-3 treated mice compared to the control group 2 days and 3 days after treatment.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that 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.

It is intended that all publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. As for the chapter titles used, they should not be construed as necessarily limiting. Furthermore, the entire contents of any one or more priority files of the present invention are incorporated herein by reference in their entirety.

Sequence listing

<110> Yeda research and development Co., ltd (Yeda Research and Development Co. Ltd.)

Miku Rate therapy Co., ltd (miCure Therapeutics Ltd.)

A Longchen (CHEN, alon)

Saran Ma Naxi Rov (MANSHIROV, sharon)

Montefilte Luo Anderius, babolo (MONTEFELTRO, andres Pablo)

<120> MODIFIED MIR-135, CONJUGATED FORMs AND USES THEREOF (MODIFIED MIR-135, CONJUGATED FORM therEOF, AND USES OF SAME)

<130> 89938

<150> 63/138,555

<151> 2021-01-18

<150> 63/272,329

<151> 2021-10-27

<160> 47

<170> PatentIn version 3.5

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<223> miR-135 mimetic oligos in-vitro

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<213> Artificial sequence

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<223> miR-135 mimetic oligos

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<221> misc_feature

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<222> (4)..(4)

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<222> (23)..(23)

<223> 2-OMe modification

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<223> 2-OMe modification

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

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<220>

<221> misc_feature

<222> (23)..(23)

<223> 2-OMe modification

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(2)

<223> 2-OMe modification

<220>

<221> misc_feature

<222> (22)..(22)

<223> 2-OMe modification

<400> 11

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(2)

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<210> 13

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(2)

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<221> misc_feature

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<221> misc_feature

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

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<220>

<221> misc_feature

<222> (11)..(13)

<223> 2-OMe modification

<220>

<221> misc_feature

<222> (23)..(23)

<223> 2-OMe modification

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<212> RNA

<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(2)

<223> 2-OMe modification

<220>

<221> misc_feature

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<223> 2-OMe modification

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<213> Artificial sequence

<220>

<223> miR-135 mimetic oligos

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<221> misc_feature

<222> (1)..(1)

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<221> misc_feature

<222> (1)..(2)

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<221> misc_feature

<222> (4)..(4)

<223> 2-OMe modification

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<221> misc_feature

<222> (23)..(23)

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<212> DNA

<213> Artificial sequence

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<400> 20

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<212> DNA

<213> Artificial sequence

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<212> DNA

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<212> DNA

<213> Artificial sequence

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<223> Single strand DNA oligonucleotide

<400> 23

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<212> DNA

<213> Artificial sequence

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<212> DNA

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<212> DNA

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<212> DNA

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<220>

<223> Single strand DNA oligonucleotide

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<212> DNA

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<212> DNA

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<212> DNA

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<212> DNA

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<212> DNA

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<212> DNA

<213> Artificial sequence

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<223> mature miR-135b

<400> 37

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<212> RNA

<213> Artificial sequence

<220>

<223> mature miR-135b*

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<212> RNA

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<212> RNA

<213> Artificial sequence

<220>

<223> RNA oligonucleotide complementary to miR-135b

<400> 40

ucacauagga augaaaagcc aua 23

<210> 41

<211> 24

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

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<220>

<221> misc_feature

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<223> 2-O-MOE-5-Me

<220>

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<223> 2-OMe modification

<400> 41

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<210> 42

<211> 26

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5' phosphorylated

<220>

<221> misc_feature

<222> (1)..(1)

<223> 2-O-MOE-5-Me

<220>

<221> misc_feature

<222> (24)..(24)

<223> 2-OMe modification

<220>

<221> misc_feature

<222> (25)..(26)

<223> 2-OMe modification

<220>

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<223> phosphorothioate bond

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<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5' phosphorylated

<220>

<221> misc_feature

<222> (1)..(1)

<223> 2-O-MOE-5-Me

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (3)..(3)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (4)..(4)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (5)..(5)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (6)..(6)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (7)..(7)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (8)..(8)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (9)..(9)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (10)..(10)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (11)..(11)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (12)..(12)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (13)..(13)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (14)..(14)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (15)..(15)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (16)..(16)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (17)..(17)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (18)..(18)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (19)..(19)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (20)..(20)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (21)..(21)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (22)..(22)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (23)..(24)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (25)..(26)

<223> 2-OMe modification

<220>

<221> misc_feature

<222> (25)..(26)

<223> phosphorothioate bond

<400> 43

uuauggcuuu ucauuccuau gugaaa 26

<210> 44

<211> 26

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5' phosphorylated

<220>

<221> misc_feature

<222> (1)..(1)

<223> 2-O-MOE-5-Me

<220>

<221> misc_feature

<222> (1)..(3)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (3)..(3)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (4)..(4)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (4)..(5)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (5)..(5)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (6)..(6)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (6)..(7)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (7)..(7)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (8)..(8)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (8)..(9)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (9)..(9)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (10)..(10)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (10)..(11)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (11)..(11)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (12)..(12)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (12)..(13)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (13)..(13)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (14)..(14)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (14)..(26)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (15)..(15)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (16)..(16)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (17)..(17)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (18)..(18)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (19)..(19)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (20)..(20)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (21)..(21)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (22)..(22)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (23)..(24)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (25)..(26)

<223> 2-OMe modification

<400> 44

uuauggcuuu ucauuccuau gugaaa 26

<210> 45

<211> 26

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5' phosphorylated

<220>

<221> misc_feature

<222> (1)..(1)

<223> 2-O-MOE-5-Me

<220>

<221> misc_feature

<222> (1)..(3)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (4)..(5)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (6)..(7)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (8)..(9)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (10)..(11)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (10)..(10)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (11)..(11)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (12)..(13)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (14)..(26)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (15)..(15)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (16)..(16)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (17)..(17)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (18)..(18)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (19)..(19)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (20)..(20)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (21)..(21)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (22)..(22)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (23)..(24)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (25)..(26)

<223> 2-OMe modification

<400> 45

uuauggcuuu ucauuccuau gugaaa 26

<210> 46

<211> 26

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(1)

<223> 5' phosphorylated

<220>

<221> misc_feature

<222> (1)..(1)

<223> 2-O-MOE-5-Me

<220>

<221> misc_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (2)..(2)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (10)..(11)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (10)..(10)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (11)..(11)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (15)..(26)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (15)..(15)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (16)..(16)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (17)..(17)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (18)..(18)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (19)..(19)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (20)..(20)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (21)..(21)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (22)..(22)

<223> 2'-fluoro

<220>

<221> misc_feature

<222> (23)..(24)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (25)..(26)

<223> 2-OMe modification

<400> 46

uuauggcuuu ucauuccuau gugaaa 26

<210> 47

<211> 23

<212> RNA

<213> Artificial Sequence

<220>

<223> miR-135 mimetic oligos

<220>

<221> misc_feature

<222> (1)..(2)

<223> 2'-O-Me

<220>

<221> misc_feature

<222> (1)..(1)

<223> Could be conjugated at 5 prime to sertraline or other cell

targeting moiety

<220>

<221> misc_feature

<222> (1)..(2)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (22)..(23)

<223> phosphorothioate bond

<220>

<221> misc_feature

<222> (23)..(23)

<223> 2'-O-Me

<400> 47

ucacauagga augaaaagcc aua 23

Claims (53)

1. A composition of matter comprising a synthetic miR-135 molecule comprising the nucleic acid sequence of miR-135b as set forth in SEQ ID No. 37 and a complementary strand as set forth in SEQ ID No. 40.

2. The composition of matter of claim 1, wherein said miR-135 molecule comprises no more than 50 nucleic acids.

3. The composition of matter of claim 1 or 2, wherein 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 are located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

4. The composition of matter of claim 1 or 2, wherein said nucleic acid sequence of miR-135b as set forth in SEQ ID No. 37 and said complementary strand as set forth in SEQ ID No. 40 form a hairpin loop structure.

5. The composition of matter of claim 1 or 2, wherein said nucleic acid sequence of miR-135b as set forth in SEQ ID No. 37 and said complementary strand as set forth in SEQ ID No. 40 are 100% complementary over the entire lengths of SEQ ID No. 37 and SEQ ID No. 40.

6. The composition of matter of any one of claims 1 to 5, wherein said nucleic acid sequence of said miR-135b and/or said complementary strand comprises one or more modifications selected from the group consisting of sugar modifications, nucleobase modifications, and internucleotide-linkage modifications.

7. The composition of matter of claim 6, wherein said 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' -Fluoroarabinooligonucleotide (FANA).

8. The composition of matter of claim 6 or 7, wherein said sugar in said miR-135b is modified in at least one nucleotide at the 3' end of said nucleic acid sequence.

9. The composition of matter of any one of claims 6 to 8, wherein said sugar in said miR-135b is modified in at least one nucleotide 5' of said nucleic acid sequence.

10. The composition of matter of any one of claims 6 to 9, wherein said sugar modification in said complementary strand comprises a modification in the last nucleotide of said 3' end of said nucleic acid sequence.

11. The composition of matter of any one of claims 6 to 10, wherein said sugar modification in said complementary strand comprises a modification in the first two nucleotides of said 5' end of said nucleic acid sequence.

12. The composition of matter of any one of claims 6 to 11, wherein the sugar modification is a 2 '-O-methyl (2' -O-Me), 2 '-O-methoxyethyl (2' -O-MOE) and/or 2 '-fluoro (2' -F) modification.

13. The composition of matter of any one of claims 6 to 12, wherein said sugar modifications are 2' -O-methoxyethyl (2 ' -O-MOE) and 5' -ribomethylation (2 ' -O-MOE-5' -Me).

14. The composition of matter of any one of claims 6 to 13, wherein said internucleotide linkage modification is selected from the group consisting of: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates, alkylphosphonates, chiral phosphonates, phosphinates, phosphoramidates, aminoalkyl phosphoramidates, thiocarbonyl phosphoramidates, thionalkyl phosphates, thionalkyl phosphotriesters, borane phosphate, phosphodiester, phosphonoacetate (PACE) and Peptide Nucleic Acids (PNA).

15. The composition of matter of any one of claims 6 to 14, wherein said internucleotide linkage modification in said miR-135b is in the last nucleotide at the 5' end of said nucleic acid sequence.

16. The composition of matter of any one of claims 6 to 15, wherein said internucleotide linkage modification comprises a phosphate or phosphorothioate.

17. The composition of matter of claim 16, wherein said phosphorothioate modification is located between the last two nucleotides of the 3' end of said nucleic acid sequence of said miR-135b or said complementary strand.

18. The composition of matter of claim 16 or 17, wherein said phosphorothioate modification is located between the last two nucleotides of the 5' end of said nucleic acid sequence of said miR-135b or said complementary strand.

19. The composition of matter of any one of claims 6 to 18, wherein said nucleic acid sequence comprising said modified miR-135b is as set forth in any one of SEQ ID NOs 10, 16 or 41-46.

20. The composition of matter of any one of claims 6 to 19, wherein said nucleic acid sequence comprising said modified complementary strand is as set forth in any one of SEQ ID NOs 13 or 47.

21. A composition of matter 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.

22. The composition of matter of claim 21, wherein said nucleic acid sequence of said miR-135b is set forth in SEQ ID No. 10 and said complementary strand is set forth in SEQ ID No. 13.

23. The composition of matter of claim 21, wherein said nucleic acid sequence of said miR-135b is set forth in SEQ ID No. 41 and said complementary strand is set forth in SEQ ID No. 13.

24. The composition of matter of claim 21, wherein said nucleic acid sequence of said miR-135b is set forth in SEQ ID No. 42 and said complementary strand is set forth in SEQ ID No. 13.

25. The composition of matter of claim 21, wherein said nucleic acid sequence of said miR-135b is set forth in SEQ ID No. 10 and said complementary strand is set forth in SEQ ID No. 47.

26. The composition of matter of any one of claims 21 to 25, wherein 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 are located on separate nucleic acid sequence molecules forming a double-stranded synthetic miR-135 molecule.

27. The composition of matter of any one of claims 21 to 25, wherein 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 form a hairpin loop structure.

28. The composition of matter of any one of claims 21 to 27, wherein 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 are 100% complementary.

29. A composition of matter comprising the nucleic acid construct of the synthetic miR-135 molecule of any one of claims 1 to 28.

30. A conjugate, comprising:

(i) A composition of matter comprising the synthetic miR-135 molecule of any one of claims 1 to 28; and

(b) A cell targeting moiety.

31. The conjugate of claim 30, wherein the cell targeting moiety is conjugated to the 5' end of the nucleic acid sequence of the complementary strand.

32. The conjugate of claim 30, wherein the cell targeting moiety is conjugated to the 5' end of the nucleic acid sequence of miR-135 b.

33. The conjugate of any one of claims 30 to 32, wherein the synthetic miR-135 molecule and the cell-targeting moiety are linked via a linking group.

34. The conjugate of any one of claims 30 to 33, wherein the cell targeting moiety specifically binds to a molecule expressed or presented on brain cells.

35. The conjugate of any one of claims 30 to 34, wherein the cell targeting moiety specifically binds to a neurotransmitter transporter.

36. The conjugate of claim 35, wherein the cell targeting moiety is selected from the group consisting of: serotonin Reuptake Inhibitors (SRIs), selective Serotonin Reuptake Inhibitors (SSRIs), serotonin-adrenoceptor reuptake inhibitors (SNRIs), noradrenergic and specific serotonergic antidepressants (NASSA), noradrenergic Reuptake Inhibitors (NRIs), dopamine Reuptake Inhibitors (DRI), endogenous cannabinoid reuptake inhibitors (ecbris), adenosine reuptake inhibitors (AdoRI), excitatory Amino Acid Reuptake Inhibitors (EAARI), glutamate reuptake inhibitors (GluRI), GABA Reuptake Inhibitors (GRI), glycine reuptake inhibitors (GlyRI) and noradrenergic-dopamine reuptake inhibitors (NDRI).

37. The conjugate of claim 36, wherein the Selective Serotonin Reuptake Inhibitor (SSRI) is selected from the group consisting of: sertraline, sertraline structural analogues, fluoxetine, fluvoxamine, paroxetine, indacene, zimeldine, citalopram, dapoxetine, escitalopram and mixtures thereof.

38. The conjugate of claim 37, wherein when the cell targeting moiety is henhouse Qu Linshi, the conjugate has the structure:

39. the conjugate of claim 37, wherein when the cell targeting moiety is henhouse Qu Linshi, the conjugate has the structure:

40. the conjugate of claim 37, wherein when the cell targeting moiety is henhouse Qu Linshi, the conjugate has the structure:

41. the conjugate of claim 37, wherein when the cell targeting moiety is henhouse Qu Linshi, the conjugate has the structure:

42. the conjugate of any one of claims 30 to 33, wherein the cell targeting moiety specifically binds to a tumor-associated antigen, or to a molecule expressed or presented on a bone cell, muscle cell or gastrointestinal cell.

43. The composition of matter of any one of claims 1 to 29, or the conjugate of any one of claims 30 to 42, further comprising at least one of a cell penetrating moiety or a moiety for transport across the Blood Brain Barrier (BBB).

44. The composition of matter or conjugate of claim 43, further comprising a linker between the cell penetrating moiety or the moiety for transport across the BBB and the synthetic miR-135 molecule.

45. A pharmaceutical composition comprising the composition of matter of any one of claims 1 to 29 or 43 to 44, or the conjugate of any one of claims 30 to 44, and a pharmaceutically acceptable carrier.

46. A method of treating a Central Nervous System (CNS) -related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, the conjugate of any one of claims 30 to 41 or 43 to 44, or the pharmaceutical composition of claim 45, thereby treating the CNS-related disorder.

47. A method of treating a depression-related disorder in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, the conjugate of any one of claims 30 to 41 or 43 to 44, or the pharmaceutical composition of claim 45, thereby treating the depression-related disorder.

48. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, the conjugate of any one of claims 30 to 34 or 42 to 44, or the pharmaceutical composition of claim 45, thereby treating the cancer.

49. A method of promoting bone regeneration or muscle cell differentiation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, the conjugate of any one of claims 30 to 33 or 42 to 44, or the pharmaceutical composition of claim 45, thereby promoting bone regeneration or muscle cell differentiation.

50. A therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, or the conjugate of any one of claims 30 to 41 or 43 to 44, for use in treating a CNS-related disorder in a subject in need thereof.

51. A therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, or the conjugate of any one of claims 30 to 41 or 43 to 44, for use in treating a depression-related patient in a subject in need thereof.

52. A therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, or the conjugate of any one of claims 30 to 34 or 42 to 44, for use in treating cancer in a subject in need thereof.

53. A therapeutically effective amount of the composition of matter of any one of claims 1 to 29 or 43 to 44, or the conjugate of any one of claims 30 to 33 or 42 to 44, for use in treating a bone-related disease or disorder or a muscle-related disease or disorder in a subject in need thereof.