US20140315795A1 - Compositions and Methods for Selective Delivery of Oligonucleotide Molecules to Cell Types - Google Patents

Compositions and Methods for Selective Delivery of Oligonucleotide Molecules to Cell Types Download PDF

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US20140315795A1
US20140315795A1 US14/064,047 US201314064047A US2014315795A1 US 20140315795 A1 US20140315795 A1 US 20140315795A1 US 201314064047 A US201314064047 A US 201314064047A US 2014315795 A1 US2014315795 A1 US 2014315795A1
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receptor
group
neuropeptide
nucleic acid
conjugate according
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Maria del Carmen CARMONA OROZCO
Andrés Montefeltro
Gabriel Alvarado Urbina
Analía Bortolozzi Biassoni
Raquel Revilla-Sanchez
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Nlife Therapeutics SL
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Nlife Therapeutics SL
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Assigned to NLIFE THERAPEUTICS, S.L. reassignment NLIFE THERAPEUTICS, S.L. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: BIASSONI, ANALIA BORTOLOZZI, URBINA, GABRIEL ALVARADO, MONTEFELTRO, ANDRES, OROZCO, MARIA DEL CARMEN CARMONA, REVILLA-SANCHEZ, Raquel
Publication of US20140315795A1 publication Critical patent/US20140315795A1/en
Assigned to NLIFE THERAPEUTICS, S.L. reassignment NLIFE THERAPEUTICS, S.L. CORRECTIVE ASSIGNMENT TO CORRECT THE APPLICATION # 14064067 PREVIOUSLY RECORDED ON REEL 032924 FRAME 0229. ASSIGNOR(S) HEREBY CONFIRMS THE APPLICATION # 14064047. Assignors: BIASSONI, ANALIA BORTOLOZZI, URBINA, GABRIEL ALVARADO, MONTEFELTRO, ANDRES, OROZCO, MARIA DEL CARMEN CARMONA, REVILLA-SANCHEZ, Raquel
Priority to US15/360,195 priority patent/US20170073679A1/en
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    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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Definitions

  • the present invention relates to conjugates comprising a nucleic acid specific for a target of interest and a group which allows the delivery of the nucleic acid to specific cells by means of their affinity towards G-protein coupled receptors on the surface of said cells.
  • nucleic acids has proved effective for altering the state of a cell.
  • RNA interference is the general term given for regulating the expression of genes at the post-transcriptional level in diversified organisms.
  • RNAi gene silencing can be accomplished using homologous short (21-23 bp) dsRNA fragments known as short interfering or “siRNA.”
  • siRNA short interfering RNA
  • Dicer the cellular enzyme Dicer will cleave it into short interfering RNA (siRNA) molecules.
  • This short interfering RNA molecule is now called the guided RNA.
  • the guided RNA will guide the RNA-Induced-Silencing-Complex (RISC) to the homologous target mRNA. Once it forms a hybrid structure to the homologous mRNA sequence, the RISC will cleave the mRNA.
  • RISC RNA-Induced-Silencing-Complex
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • nucleic acid constructs which contain a nucleic acid specific for a given target molecule and a selective ligand of a receptor which can be endocytosed in response to the binding of the selective ligand. These constructs are shown to be particularly useful for the delivery of the nucleic acid of interest to the interior of a cell expressing the receptor. Without wishing to be bound by any theory, it is believed that the ligand will bind to the corresponding receptor in the surface of the cell wherein the receptor is expressed. This will in turn result in the translocation of the complex nucleic acid-inhibitor to the interior of the cell by means of receptor-mediated endocytosis.
  • the invention is not limited to conjugate for delivery to cells expressing growth hormone secretagogue receptor (GHS-R).
  • GLS-R growth hormone secretagogue receptor
  • the results provided in the present invention illustrate that the mechanism used by the cells to signal via surface receptor are adequate means for promoting delivery to cells of small molecules attached to molecules showing affinity for said receptors.
  • GHS-R1a Potent, orally active ghrelin receptor
  • FIG. 2 Specific targeting of Tabimorelin conjugated siRNA. Selective targeting of hypothalamic neurons with TAB-NS-Cy3 or NS-Cy3. Mice were icy infused with 30 ⁇ g of TAB-NS-Cy3 or NS-Cy3 and 1 hour later were sacrificed and brains processed for fluorescent microscopy. Red labeling (Cy3) can be only detected in the hypothalamus of TAB-NS-Cy3 treated mice.
  • FIG. 3 Cumulative body weight (BW) gain of obese animals after 12 days of treatment with different molecules. Cumulative body weight gain (g) of animals treated with vehicle (VH), TAB-NS-ASO4 (NS), TAB-SOCS3-ASO4 (SOCS3) or TAB-PTP1B-ASO4 (PTP1B) for 12 days. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 vs. VH. + p ⁇ 0.05; ++ p ⁇ 0.01; +++ p ⁇ 0.001; ++++ p ⁇ 0.0001 vs. NS. Data represent mean+ESD
  • FIG. 4 Cumulative food intake (FI) of obese animals after 12 days of treatment with different molecules. Cumulative FI gain (g) of animals treated with vehicle (VH), TAB-NS-ASO4 (NS), TAB-SOCS3-ASO4 (SOCS3) or TAB-PTP1B-ASO4 (PTP1B) for 12 days. *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001 vs. VH. + p ⁇ 0.05; ++ p ⁇ 0.01; +++ p ⁇ 0.001; ++++ p ⁇ 0.0001 vs. NS. Data represent mean+ESD
  • FIG. 5 Daily body weight (BW) gain and food intake (FI) of obese animals.
  • BW body weight
  • FI food intake
  • One-way ANOVA denoted significant differences between treatments (p ⁇ 0.0001). **p ⁇ 0.01; ***p ⁇ 0.001 vs. VH. ++ p ⁇ 0.01; +++ p ⁇ 0.001 vs. NS. Data represent mean+ESD
  • FIG. 6A-6R Ligand/Receptor tables organized by Area. Ligand/receptor pair is selected for delivery of the conjugated nucleotide to the interest area. The sequence of the oligonucleotide will modulate the expression of target gene of the disease table.
  • the present inventors have observed that it is possible to specifically target a nucleic acid to a cell of interest which expresses a receptor by covalently coupling said nucleic acid to a molecule which is capable of specifically binding to said receptor. Moreover, the present inventors have also observed that the nucleic acid can be internalized by the cells, thereby exerting its effects. Without wishing to be bound by any theory, it is believed that the conjugates are internalized by the receptor as the receptor is internalized in response to the binding of the selectivity agent.
  • animals treated with a conjugate containing a ligand for the growth hormone secretagogue receptor (tabimorelin) and a nucleic acid capable of specifically silencing either SOCS4 or PTP1B gain significantly less weight and show reduced food intake than control animals (VH and NS), thereby showing that the nucleic acids were effective in reaching the targeted hypothalamic areas.
  • a conjugate containing a ligand for the growth hormone secretagogue receptor (tabimorelin) and a nucleic acid capable of specifically silencing either SOCS4 or PTP1B gain significantly less weight and show reduced food intake than control animals (VH and NS), thereby showing that the nucleic acids were effective in reaching the targeted hypothalamic areas.
  • the invention relates to a conjugate comprising:
  • conjugate refers to any composition resulting from the covalent attachment of two or more individual compounds.
  • conjugate refers to a molecule comprising a nucleic acid and a selectivity agent which are covalently coupled, being said coupling direct or via a linking compound.
  • covalent coupling or “covalent attachment” mean that the nucleic acid and the selectivity agent are either directly covalently joined to one another, or else are indirectly covalently joined to one another through an intervening moiety or moieties, such as a linker, or a bridge, or a spacer, moiety or moieties.
  • selectivity agent which binds specifically to one or more of a receptor refers to any moiety which binds to a receptor, wherein the receptor undergoes endocytosis in response to the binding of said selectivity agent.
  • This binding specificity allows the delivery of a molecule which is attached to said selectivity agent to the cell, tissue or organ which expresses said receptor.
  • a conjugate carrying said selectivity agent will be directed specifically to said cells when administered to an animal or contacted in vitro with a population of cells of different types.
  • receptor denotes a cell-associated protein that binds to a bioactive molecule termed a “ligand.”
  • a selectivity agent according to the present invention may show a Kd for the target (the receptor) of at least about 10 ⁇ 4 M, alternatively at least about 10 ⁇ 5 M, alternatively at least about 10 ⁇ 6 M, alternatively at least about 10 ⁇ 7 M, alternatively at least about 10 ⁇ 8 M, alternatively at least about 10 ⁇ 9 M, alternatively at least about 10 ⁇ 10 M, alternatively at least about 10 ⁇ 11 M, alternatively at least about 10 ⁇ 12 M or greater.
  • Receptors which may be targeted by the selectivity agents of the invention also include, without limitation, a 5-hydroxytryptamine receptor, an adenosine receptor, an adrenoceptor receptor, an angiotensin receptors, a bombesin receptors, a bradykinin receptors, a calcitonin receptor, a chemokine receptor, a cholecystokinin receptor, a corticotropin-releasing factor receptor, a dopamine receptor, an endothelin receptor, en ephrin receptor, a formylpeptide receptor, a Frizzled receptor, a galanin receptor, a the growth hormone secretagogue receptor (Ghrelin) receptor, a Kisspeptin receptor, a melanocortin receptor, a melatonin receptors, Neuropeptide FF/neuropeptide AF receptor, a neuropeptide S receptor, a neuropeptide W/neuropeptide B receptor, a
  • the receptors that can be targeted by the selectivity agent of the conjugates according to the invention are as defined in Table 1.
  • the receptor is a G-protein coupled receptor.
  • G-protein coupled receptor refers to a target receptor that, when expressed by a cell, associates with a G-protein (e.g., a protein which hydrolyzes GTP).
  • GPCR G-protein coupled receptor
  • the GPCR is a “seven transmembrane segment receptor” (or “7 TMS receptor”), which refers to a protein that structurally comprises seven hydrophobic transmembrane spanning regions.
  • the receptor is expressed at one or more locations of the central nervous system.
  • said location of the central nervous system is selected from the group consisting of the hypothalamus, the brainstem, the cortex, the cerebellum, the striatum, the mesencephalon, the hippocampus, the glia and the spinal cord.
  • the selectivity agent within the conjugate is a ligand that binds to a receptor which is expressed in the hypothalamus.
  • the receptor which is expressed in the hypothalamus is selected from the group consisting of the growth hormone secretagogue receptor, the galanin GAL1 receptor, the calcitonin receptor-like, the neuropeptide FF/B NPBW2 receptor, the neuropeptide FF/B NPFF2 receptor, the neuropeptide Y Y2 receptor, the bombesin BB2 receptor, the bombesin BB3 receptor, the calcitonin AM1 receptor, the calcitonin AMY1 receptor, the calcitonin CGRP receptor the calcitonin receptor, the frizzled FZD2 receptor, the frizzled FZD5 receptor, the melanocortin MC2 receptor, the melanocortin MC3 receptor, the melanocortin MC4 receptor, the neuropeptide S receptor, the
  • the ligand which binds to the receptor is selected from the group of the ligands shown in FIG. 6 .
  • the selectivity agent is a GHS-R agonist.
  • the GHS-R agonist is tabimorelin or a structural analog thereof having the structure (I):
  • R 1 and R 2 independently of each other are hydrogen or C1-C6 alkyl or R1 and R 2 taken together form a C2-C5 alkylene group; J is a group
  • R 4 and R 5 independently of each other are hydrogen or C 1 -C 6 alkyl and R 6 is hydrogen or C 1 -C 6 alkyl, preferably hydrogen.
  • Compounds of Formula I can have one or more asymmetric centres, and any and all optical isomers in the form of separated, pure or partially purified optical isomers or racemic mixtures thereof are included within the scope of Formula I. Both E and Z geometric isomers (with respect to the olefinic double bond to the left in the structure of Formula I as depicted above) are likewise included within the scope of Formula I.
  • the invention provides conjugates comprising compound according to Formula I wherein R 1 and R 2 are both alkyl, preferably methyl. In one aspect, the invention provides conjugates comprising a compound according to Formula I wherein J is also or alternatively 2-naphthyl. In one aspect, m also or alternatively is one. In one aspect, R 3 is methyl. In another aspect, p is one. In another aspect, G is phenyl.
  • R 4 is methyl.
  • R 5 is hydrogen or methyl.
  • R 6 is hydrogen or methyl.
  • C1-6 alkyl is intended to include straight-chain (linear), branched and cyclic alkyl groups of from 1 to 6 carbon atoms.
  • Relevant linear C1-6 alkyl groups are methyl, ethyl, propyl, butyl, pentyl and hexyl.
  • Examples of branched C1-6alkyl groups are isopropyl, sec-butyl, tert-butyl, isopentyl and isohexyl.
  • Examples of cyclic groups (C3-6cycloalkyl groups) are cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
  • C1-6 alkyl in the present context likewise includes, for example, cycloalkyl-substituted alkyl groups having from 1 to 6 carbon atoms, examples of which include groups such as (cyclopropyl)methyl, (cyclopropyl)ethyl, (cyclopropyl)propyl, (cyclobutyl)methyl, (cyclobutyl)ethyl and (cyclopentyl) methyl.
  • Particularly suitable C1-6alkyl groups are often chosen among C1-3alkyl groups, i.e., methyl, ethyl, propyl, isopropyl and cyclopropyl.
  • C 2-5 alkylene group i.e. C 2-5 alkandiyl group
  • C 2-5 alkandiyl group is intended to include both straight-chain (linear) and branched alkandiyl groups of from 2 to 5 carbon atoms.
  • Relevant linear groups are: —CH 2 —CH 2 —; —CH 2 —CH 2 —CH 2 —; —CH 2 —(CH 2 ) 2 —CH 2 —; and —CH 2 —(CH 2 ) 3 —CH 2 —.
  • Suitable branched groups include: —CH 2 —CH(CH 3 )—; —CH 2 —CH(CH 3 )—CH 2 —; —CH 2 — CH 2 —CH(CH 3 )—; —CH 2 —(CH 2 ) 2 —CH(CH 3 )—; and —CH 2 —CH 2 —CH(CH 3 )—CH 2 —.
  • halogen includes Cl, F, Br and I. Particularly suitable halogens in the context of Formula I are Cl and F.
  • the GSH-R agonist is N-[(2E)-5-amino-5-methylhex-enoyl]-N-methyl-3-(2-naphthyl)alanyl-N,N ⁇ -dimethyl-D-phenylalaninamide (tabimorelin)
  • the selectivity agent specifically binds to a receptor which is expressed in the mesencephalon.
  • the receptor which is expressed in the mesencephalon is selected from the group consisting of the growth hormone secretagogue receptor, the bombesin BB1 receptor, the bradykinin B2 receptor, the galanin GAL2 receptor, neuropeptide FF/B NPBW2 receptor, the neuropeptide FF/B NPFF2 receptor, the neuropeptide Y Y1 receptor, the neurotensin NTSR1 receptor, the neurotensin NTSR2 receptor, the neuropeptide S receptor, the orexin OX2 receptor, the 5-HT1D receptor, the angiotensin AT2a receptor, the angiotensin AT2b receptor, the calcitonin AM2 receptor, the calcitonin AMY3 receptor, the frizzled FZD6 receptor, the kisspeptin receptor, the melatonin MT1 receptor, the neuropeptide FF/B NPB
  • the selectivity agent specifically binds to a receptor which is expressed in the brainstem.
  • the receptor which is expressed in the brainstem is selected from the group consisting of the 5-HT 3 receptor, the galanin receptor 1, the melanocortin MC1 receptor, the melanocortin MC2 receptor, the melanocortin MC3 receptor, the melanocortin MC4 receptor, the calcitonin receptor-like receptor, the CRF2 receptor, the neuropeptide FF/B NPBW2 receptor, the 5-HT1 A receptor, the neuropeptide Y Y2 receptor, the neurotensin NTSR1 receptor, the opioid mu receptor, the orexin OX1 receptor, the orexin OX2 receptor and the dopamine D2 receptor.
  • the selectivity agent specifically binds to a receptor which is expressed in the cortex.
  • the receptor which is expressed in the cortex is selected from the group consisting of the 5-HT 3 receptor, the melanocortin MC1 receptor, the CRF1 receptor, the 5-HT2 A receptor, the alpha1 adrenergic receptor, the bombesin BB1 receptor, the frizzled FZD3 receptor, the bombesin BB3 receptor, the bradykinin B2 receptor, the calcitonin receptor-like receptor, the cholecystokinin CCK2 receptor, the CRF1 receptor, the CRF2 receptor, the galanin GAL2 receptor, the galanin GAL3 receptor, the neuropeptide FF/B NPBW2 receptor, the neuropeptide FF/B NPFF2 receptor, the neuropeptide Y Y1 receptor, the neuropeptide Y Y5 receptor, the neuropeptide Y Y2 receptor, the neurotensin NTSR2 receptor
  • the selectivity agent specifically binds to a receptor which is expressed in the cerebellum.
  • the receptor which is expressed in the cerebellum is selected from the group consisting of the CRF1 receptor, the 5-HT 1B receptor, the frizzled FZD4 receptor, the frizzled FZD10 receptor, the frizzled FZD7 receptor, the bradikinin B2 receptor, the galanin GAL3 receptor, the neurotensin NTSR2 receptor, the endothelin ETb receptor, the formylpeptide FPR1 receptor, the formylpeptide FPR2 receptor, the melatonin MT2 receptor, the vasopressin V1A receptor, the angiotensin AT2a receptor, the angiotensin AT2b receptor, the kisspeptin receptor and the melatonin MT1 receptor.
  • the selectivity agent specifically binds to a receptor which is expressed in the striatum.
  • the receptor which is expressed in the striatum is selected from the group consisting of the 5-HT2A receptor, the cholecystokinin CCK2 receptor, the CRF1 receptor, the neuropeptide FF/B NPBW2 receptor, the neuropeptide FF/B NPFF2 receptor, the somatostatin sst5 receptor, the vasopressin V1B receptor, the 5-HT6 receptor, the adenosine A2 receptor, the adenosine A2A receptor, the dopamine D1 receptor, the dopamine D2 receptor, the peptide P518 receptor, the tachykinin NK1 receptor, the tachykinin NK2 receptor and the tachykinin NK3 receptor.
  • the selectivity agent specifically binds to a receptor which is expressed in the hippocampus.
  • the receptor which is expressed in the hippocampus is selected from the group consisting of the 5-HT 3 receptor, the bradykinin B2 receptor, the CRF2 receptor, the frizzled FZD3 receptor, the galanin GAL3 receptor, the neuropeptide FF/B NPBW2 receptor, the neuropeptide Y Y1 receptor, the neuropeptide Y Y5 receptor, the neurotensin NTSR2 receptor, the opioid delta receptor, the somatostatin sst3 receptor, the somatostatin sst5 receptor, the 5-HT1A receptor, the adenosine A1 receptor, the endothelin ETa receptor, the endothelin ETb receptor, the formylpeptide FPR1 receptor, the formylpeptide FPR3 receptor, the frizzled FZD8 receptor, the frizzled FZD9 receptor, the melaton
  • the selectivity agent specifically binds to a receptor which is expressed in the medulla.
  • the receptor which is expressed in the medulla is selected from the group consisting of the EphA1 receptor, the EphA2 receptor, the EphA3 receptor, the EphA4 receptor, the EphAB1 receptorm, the EphAB2 receptor, the EphAB3 receptor, the opiuoid mu receptor, the GlyT1 transporer, the DP1 prostanoid receptor, the tachykinin NK1, NK2 or NK3 receptors, the CXCR4 chemokine receptor and the VEGFR1, VEGFR2 or VEGFR3 receptor.
  • the selectivity agent specifically binds to a receptor which is expressed in the glia.
  • the receptor which is expressed in the glia is selected from the group consisting of the formylpeptide FPR1 receptor, the formylpeptide FPR2 receptor, the formylpeptide FPR3 receptor and TLR7.
  • the selectivity agent which binds to the receptor is as shown in Table 2.
  • 5-HT3 1-Phenylbiguanide hydrochloride, B-HT 920, m-Chlorophenylbiguanide, SR 57227 hydrochloride, 2- Methyl-5-Hydroxytryptamine, Quipazine, RS-56812 5-HT4 5-Methoxytryptamine, BIMU-8, Cinitapride, Cisapride, Dazopride, Metoclopramide, Mosapride, Prucalopride, RS-67333, Renzapride, Tegaserod and Zacopride 5-HT5a 5-carboxamidotryptamine maleate, ergotamine 5-HT6 Ro 60-0175 fumarate, EMD-386,088 5-HT7 5-carboxamidotryptamine maleate, AS-19, E-55888 and RA-7 GAL1 galanin, galanin-like peptide and M617 GAL2 AR-M 1896, galanin, galanin-like peptide, M11
  • a further component of the conjugates according to the present invention is a nucleic acid which is capable of specifically binding to a target molecule which is expressed in the same cell as the receptor.
  • the nucleic acid of the invention is capable of inhibiting the function of the target molecule.
  • the nucleic acid typically a siRNA, a shRNA or an antisense nucleic acid
  • the target nucleic acid is protein
  • the nucleic acid typically an aptamer acts by inhibiting the activity of the protein.
  • nucleic acid refers to a polymer having two or more deoxyribonucleotide, ribonucleotide and/or nucleotide analog molecules as well as molecules that are structurally similar to a native nucleic acid, but differ from the native nucleic acid (e.g., through chemical modification) at one or more of the nucleic acid backbone (e.g., phosphate in native nucleic acids), nucleic acid sugar (e.g., deoxyribose for native DNA and ribose in native RNA), and nucleic acid base (e.g., adenosine, cytosine, guanine, thymidine, or purine in native nucleic acids).
  • nucleic acid backbone e.g., phosphate in native nucleic acids
  • nucleic acid sugar e.g., deoxyribose for native DNA and ribose in native RNA
  • nucleic acid base e.g., a
  • the oligonucleotide can be a double stranded or single stranded oligonucleotide including, without limitation, small interfering RNAs (siRNA), small hairpin RNAs (shRNA), microRNAs (miRNA), antisense oligonucleotides or ribozymes. If double stranded nucleic acids are used, these comprise a first sense strand which is complementary to the target nucleic acid and a second antisense strand which is complementary to the sense, which allows the formation of the double stranded DNA by base pairing between the first and second strand.
  • siRNA small interfering RNAs
  • shRNA small hairpin RNAs
  • miRNA microRNAs
  • antisense strand refers to the strand of a double stranded nucleic acid which includes a region that is substantially complementary to a target sequence Where the region of complementarity is not fully complementary to the target sequence, the mismatches are most tolerated outside nucleotides 2-7 of the 5′ terminus of the antisense strand.
  • sense strand refers to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand.
  • siRNA small interfering RNA
  • siRNA molecules are not limited to RNA molecules but further encompass nucleic acids with one or more chemically modified nucleotides, such as morpholinos.
  • RNA or “short hairpin RNA” as used herein refers to a dsRNA where the two strands are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′ end of the respective other strand to form a duplex structure.
  • miRNA refers to short single-stranded RNA molecules, typically of about 21-23 nucleotides in length capable of regulating gene expression. miRNAs may be synthetic (i.e., recombinant) or natural. Natural miRNAs are encoded by genes that are transcribed from DNA and processed from primary transcripts (“pri-miRNA”) to short stem-loop structures (“pre-miRNA”), and finally to mature miRNA. Mature miRNA molecules are partially complementary to one or more mRNA molecules, and downregulate gene expression via a process similar to RNA interference, or by inhibiting translation of mRNA.
  • pri-miRNA primary transcripts
  • pre-miRNA short stem-loop structures
  • an “antisense sequence,” as used herein includes antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences.
  • RNA or DNA single-stranded nucleic acid sequence
  • the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen, Cancer Res. 48:2659, (1988) and van der Krol et al., BioTechniques 6:958, (1988).
  • ribozyme or “RNA enzyme” or “catalytic RNA” refers to an RNA molecule that catalyzes a chemical reaction. Many natural ribozymes catalyze either the hydrolysis of one of their own phosphodiester bonds, or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome, the ligase activity of a DNA ligase, and a number of other chemical reactions performed by conventional protein enzymes.
  • an “aptamer” as used herein refers to a nucleic acid ligand that binds to more than one site on a target molecule where binding is not “complementary,” i.e., is not due to base-pair formation between a nucleic acid ligand and a target nucleic acid sequence.
  • An aptamer can be designed which binds to any envisionable target, including polypeptides. Aptamers offer the utility for biotechnological and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies.
  • aptamers offer advantages over antibodies as they can be engineered completely in a test tube, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • Aptamers can be synthesized through repeated rounds of in vitro partition, selection and amplification, a methodology known in the state of the art as “SELEX”, (Systematic Evolution of Ligands by Exponential Enrichment) (Shamah et al, Acc. Chem. Res. 2008, 41 pp. 130-8). Alternatively, they can be synthesized, for example, by step-wise solid phase.
  • the nucleic acid of the invention may contain one or more modifications in the nucleobases, in the sugars and/or in the internucleotide linkages.
  • Modifications to one or more backbone residues of the nucleic acids may comprise one or more of the following: 2′ sugar modifications such as 2′-O-methyl (2′-OMe), 2′-O-methoxyethyl (2′-MOE), 2′-O-methoxyethoxy, 2′-Fluoro (2′-F), 2′-Allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 2′-O-(N-methylcarbamate); 4′ sugar modifications including 4′-thio, 4′-CH 2 —O-2′-bridge, 4-(CH 2 ) 2 -O-2′-bridge; Locked Nucleic Acid (LNA); Peptide Nucleic Acid (PNA); Intercalating nucleic acid (INA); Twisted intercalating nucleic acid (TINA); Hexitol nucleic acids (HNA); arabinonucleic acid (ANA); cyclohexane nucleic acids (CNA); cycl
  • RNA Aptamers regulated with antidotes on the subject of the specific RNA aptamer (ref. Oney S, Oligonucleotides. 2007 Fall; 17(3):265-74.) or any combinations thereof.
  • Modifications to one or more internucleoside linkages of the nucleic acids may comprise one or more of the following: Phosphorothioate, phosphoramidate, phosphorodiamidate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate and phosphoranilidate, or any combinations thereof.
  • a Locked Nucleic Acid is a modified RNA nucleotide.
  • the ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2′ and 4′ carbons (02′,C4′-methylene bridge).
  • the bridge “locks” the ribose in the 3′-endo structural conformation, which is often found in the A-form of DNA or RNA.
  • LNA nucleotides can be mixed with DNA or RNA bases in the nucleic acid whenever desired. Such oligomers are commercially available.
  • the locked ribose conformation enhances base stacking and backbone pre-organization.
  • PNA Peptide Nucleic Acid
  • DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • the various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds.
  • PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
  • Intercalating nucleic acid is a modified nucleic acid analogue comprised of normal deoxyribonucleotides covalently linked to hydrophobic insertions.
  • INA has high affinity for complementary DNA with stabilization of up to 11 degrees for each modification.
  • INA has a higher specificity for a fully matched target over mismatched targets than normal DNA. Utilizing that INAs have higher affinity for DNA makes it possible to use shorter probes and thereby enhance specificity even further.
  • INA is a DNA selective oligonucleotide analogue, with a unique ability to discriminate between DNA and RNA. Even though INAs have high affinities for complementary DNA, it has a lower affinity for a complementary sequence of complementary INAs. Twisted intercalating nucleic acids are denoted TINA.
  • Hexitol nucleic acids are oligonucleotides built up from natural nucleobases and a phosphorylated 1,5-anhydrohexitol backbone. Molecular associations between HNA and RNA are more stable than between HNA and DNA and between natural nucleic acids (dsDNA, dsRNA, DNA/RNA).
  • Other synthetically modified oligonucleotides comprise ANA (arabinonucleic acid), CNA (cyclohexane nucleic acids), CeNA (cyclohexenylnucleic acid) and TNA (threosyl nucleic acid).
  • Morpholinos are synthetic molecules which are the product of a redesign of the natural nucleic acid structure. Structurally, the difference between morpholinos and DNA or RNA is that while Morpholinos have standard nucleobases, those bases are bound to 6-membered morpholine rings instead of deoxyribose/ribose rings and non-ionic phosphorodiamidate intersubunit linkages replace anionic phosphodiester linkages. Morpholinos are sometimes referred to as PMO (phosphorodiamidate morpholino oligonucleotide). The 6-membered morpholine ring has the chemical formula O—(CH 2 —CH 2 ) 2 -NH.
  • Gapmers are RNA-DNA-RNA chimeric oligonucleotide probes, where windows or ‘gaps’ of DNA are inserted into an otherwise normal or modified RNA oligonucleotide. This modification increases oligonucleotide stability in vivo and the avidity of the interaction of the probe with the target, so that shorter probes can be used effectively.
  • the nucleic acid of the conjugates of the invention are capable of specifically binding to a target molecule which is expressed in the same cell as the neurotransmitter transporter.
  • the binding of the nucleic acid to the target molecule can occur via Watson-Crick interactions wherein the target molecule is a nucleic acid which contains a sequence which is complementary to the sequence of the nucleic acid.
  • the target molecule is a polypeptide
  • the nucleic acid of the conjugates of the invention can also interact with said molecule, in which case the nucleic acid is acting as an aptamer.
  • nucleic acid which forms part of the conjugates of the invention is complementary to the nucleic acid sequence of the target mRNA
  • different criteria are available to the skilled person for selecting the most adequate nucleic acid.
  • the nucleic acid forming part of the conjugate is a siRNA
  • this can be selected by scanning the mRNA sequence of the target for AA dinucleotides and recording the 19 nucleotides immediately downstream of the AA.
  • Other methods can also been used to select the nucleic acid targets.
  • the selection of the siRNA target sequence is purely empirically determined (see, e.g., Sui G et al., Proc. Natl. Acad. Sci.
  • the hairpin siRNA expression cassette is constructed to contain the sense strand of the target, followed by a short spacer, the antisense strand of the target, and 5-6 Ts as transcription terminator.
  • the order of the sense and antisense strands within the siRNA expression constructs can be altered without affecting the gene silencing activities of the hairpin siRNA. In certain instances, the reversal of the order may cause partial reduction in gene silencing activities.
  • the length of nucleotide sequence being used as the stem of siRNA expression cassette can range, for instance, from 19 to 29.
  • the loop size can range from 3 to 23 nucleotides. Other lengths and/or loop sizes can also be used.
  • a 5′ overhang in the hairpin siRNA construct can be used, provided that the hairpin siRNA is functional in gene silencing.
  • the 5′ overhang includes about 6 nucleotide residues.
  • the target sequence for RNAi is a 21-mer sequence fragment.
  • the 5 end of the target sequence has dinucleotide “NA”, where “N” can be any base and “A” represents adenine.
  • the remaining 19-mer sequence has a GC content of between 35% and 55%.
  • the remaining 19-mer sequence does not include any four consecutive A or T (i.e., AAAA or TTT), three consecutive G or C (i.e., GGG or CCC), or seven “GC” in a row.
  • RNAi target sequences can be selected to have low sequence homology to other genes.
  • potential target sequences are searched by BLASTN against NCBI's human UniGene cluster sequence database.
  • the human UniGene database contains non-redundant sets of gene-oriented clusters. Each UniGene cluster includes sequences that represent a unique gene. 19-mer sequences producing no hit to other human genes under the BLASTN search can be selected. During the search, the e-value may be set at a stringent value (such as “1”).
  • siRNA sequences as well as any other RNAi sequence derived according to the present invention in silencing expression of the target gene, can be evaluated using various methods known in the art.
  • the degree of inhibition is usually expressed in terms of:
  • the degree of inhibition may be given in terms of a reduction of a parameter that is functionally linked to target gene expression, e.g., the amount of protein encoded by a target gene or the number of cells displaying a certain phenotype.
  • target genome silencing may be determined in any cell expressing the target, either constitutively or by genomic engineering, and by any appropriate assay.
  • the assay provided in the Examples below and those known in the art shall serve as such reference.
  • expression of a target gene is suppressed by at least about 5 percent, 10 percent, 15 percent, 20 percent, 25 percent, 30 percent, 35 percent, 40 percent, 45 percent, or 50 percent by administration of the double-stranded oligonucleotide.
  • a target gene is suppressed by at least about 60 percent, 70 percent, or 80 percent by administration of the double-stranded oligonucleotide.
  • the target gene is suppressed by at least about 85 percent, 90 percent, or 95 percent by administration of the double-stranded oligonucleotide.
  • the nucleic acid sequence according to the present invention can be introduced into a cell that expresses the target gene.
  • the mRNA level of the target gene in the cell can be detected by using RT-PCR, Northern blot or any other standard methods).
  • the level of the polypeptide encoded by the target mRNA can be measured using Western blot, ELISA or any other immunological or non-immunlogical method.
  • a substantial change in the expression level of mRNA or of the protein encoded by the target gene after the introduction of the siRNA sequence is indicative of the effectiveness of the siRNA sequence in suppressing the expression of the target gene.
  • the expression levels of other genes are also monitored before and after the introduction of the siRNA sequence.
  • siRNA sequence which has inhibitory effect on target gene expression but does not significantly affect the expression of other genes can be selected.
  • multiple siRNA or other RNAi sequences can be introduced into the same target cell. These siRNA or RNAi sequences specifically inhibit target gene expression but not the expression of other genes.
  • siRNA or other RNAi sequences that inhibit the expression of the target gene and other gene or genes can be used.
  • nucleic acid molecule which is incorporated into the conjugates of the invention will depend on the type of selectivity agent present in the conjugate.
  • the nucleic acid will be specific for a target molecule which is expressed in the cells which express the neurotransmitter transporter which is specifically bound by the selectivity agent.
  • the nucleic acid acts by base-pairing with the target molecule, in which case the target molecule is an mRNA. If the nucleic acid is an aptamer, the target molecule is the polypeptide encoded by said mRNA.
  • nucleic acid of the invention specific towards a target mRNA can be selected using any of the methods mentioned above and tested for its ability to induce a substantial decrease in the levels of the corresponding mRNA.
  • regions correspond to regions which are highly conserved among different species or regions corresponding to non-coding regions of the primary transcript in order to avoid potential interference with translation complexes inside the coding region.
  • Methods for pairwise alignment of two given nucleic acid sequences are widely known to the skilled person and can be carried out by standard algorithms of the type BLASTN [BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990)] using the default parameters.
  • Methods for the alignment of multiple nucleic acid sequences can be carried out using standard algorithms of the type CLUSTALW (Thompson J D et al, Nucleic Acids Res, 1994, 22:4673-4680) using the default parameters.
  • the nucleic acid is specific for the mRNA of the suppressor of cytokine signaling 3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B), CB1, interleukin beta 1 (IL1B), neuropeptide Y (NPY), neuropeptide Y1, neuropeptide Y5 and the ghrelin receptor.
  • SOCS3 cytokine signaling 3
  • PTP1B protein-tyrosine phosphatase 1B
  • IL1B interleukin beta 1
  • NPY neuropeptide Y
  • neuropeptide Y1 neuropeptide Y1
  • neuropeptide Y5 neuropeptide Y5 and the ghrelin receptor
  • SOCS3 refers to naturally occurring or recombinant forms of the polypeptide “Suppressor of cytokine signaling-3”, which is involved in transducing the signaling by leptin by means of its binding to the phosphorylated leptin receptor through its SH2 domain and inhibiting Jak tyrosine kinase activity through its N-terminal kinase inhibitory region, which functions as pseudosubstrate.
  • Different orthologs of the SOSC3 polypeptide are shown in the NCBI database under accession numbers NP — 003946, NP — 031733, and NP — 446017 for human, mouse and rat protein sequences, respectively.
  • Suitable SOCS3-specific siRNA that can be used in the present invention are, e.g. those shown in WO2012082765.
  • Suitable SOCS3-specific antisense nucleic acids that can be used in the present invention are, e.g. those shown in US2010135952 (incorporated by reference), US2004087530 (incorporated by reference), in Raghavendra Rao et al., (J Neurochem., 2002, 83, 1072-1086), Fox et al., (J. Immunol., 2003, 170, 3679-3687).
  • the nucleic acid specific for SOCS3 is a gapmer having the sequence GUGGCG CTGGTCCG AGCTGT , wherein the underlined blocks correspond to 2′-O-methyl modified nucleotides.
  • the nucleic acid specific for SOCS3 is a siRNA having the a sense strand with the sequence cuuuucgcugcagagugacTT and an antisense strand having the sequence gucacucugcagcgaaaagTT.
  • PTP1B refers to protein tyrosine phosphatase IB which has been identified as a negative regulator of the insulin response. Isolated PTP-1B dephosphorylates the insulin receptor in vitro (Tonks, N. K., 1988, J. Biol. Chem., 263: 6731-6737). PTP-1B dephosphorylation of multiple phosphotyrosine residues of the insulin receptor proceeds sequentially and with specificity for the three tyrosine residues that are critical for receptor autoactivation (Ramachandran, C. 1992, Biochemistry, 31: 4232-4238).
  • PTP-1B In addition to insulin receptor dephosphorylation, PTP-1B also dephosphorylates the insulin related substrate 1 (IRS-1), a principal substrate of the insulin receptor (Lammers, R., 1993, J. Biol. Chem. 268: 22456-22462).
  • IMS-1 insulin related substrate 1
  • the human ortholog of the PTP-1B polypeptide is shown in the NCBI database under Genbank Accession number NM — 002827.
  • Suitable PTP-1B sequences that can be targeted by the nucleic acids according to the invention are shown in Table 2 of WO200307088 (incorporated by reference).
  • Suitable PTP 1B-specific siRNAs are shown in Table 2 of WO200307088 (incorporated by reference).
  • the nucleic acid specific for PTP-1B is a gapmer having the sequence GCUCC TTCCACTGAT CCUGC , wherein the underlined blocks correspond to 2′-O-methyl modified nucleotides and the bold block corresponds to a nucleotides connected by phosphorothioate.
  • the nucleic acid specific for PTP-1B is a siRNA having the a sense strand with the sequence ccgcaucauggagaaaggcTT and an antisense strand having the sequence gccuuucuccaugaugcggTT, wherein the capitalized sequences correspond to the overhangs in the duplex siRNA.
  • the nucleic acid is specific for dopamine D1, D2 or D3 receptor, for the dopamine transporter or for alpha-synuclein.
  • the nucleic acid is specific for the mRNA selected from the group consisting of the serotonin 5-HT1A receptor, the serotoine 5-HT2C receptor, the SERT (serotonin transporter), the alpha1A-adrenoceptor, the angiotensin converting enzyme, G protein ⁇ 3, the 5-HT2C receptor, interleukin 10, monoamine oxidase A, the cannabinoid CB1 receptor and ⁇ -synuclein.
  • the nucleic acid is specific for a mRNA selected from the group consisting of a gene product encoded in chromosome 21, for amyloid precursor protein, presenilin 1, presenilin 2, Apolipoprotein E, cyclin-dependent kinase 5, glycogen synthase kinase 3, the microtubule affinity-regulating kinase A2, dopamine D4 receptor, acetylcholinesterase, adenosine A2 receptor, CB1, catechol-O-methyl transferase, histamine N-methyltransferase, H3, 5-HT6, phosphodiesterase 10A, phosphodiesterase 1B, phosphodiesterase 1C, phosphodiesterase 2A, phosphodiesterase 4A, phosphodiesterase 4B, phosphodiesterase 4D, phosphodiesterase 7A, phosphodiesterase 7B, phosphodiesterase
  • the nucleic acid is specific for a mRNA selected from the group consisting of atrophin-1, Fragile X Mental retardation 1, G-protein coupled receptor 55, 1p36, ataxin 1, ataxin 10, tubulin kinase 2, PPP2R2B, Kv3.3 channel, Protein kinase C gamma, inositol receptor, Ataxin 17, interferon-related developmental regulator gene 1, Ataxin 19, Ataxin 2, Ataxin 20, Ataxin 21, Ataxin 22, dynorphin, Ataxin 25, Ataxin 26, Fibroblast growth factor14, mitochondrial metalloprotease complex, ataxin 29, ataxin 3, ataxin 30, Thymidine kinase 2 & Brain expressed associated with NEDD4, ataxin 32, ataxin 33, gene product encoded by 16p12.3-q16.2, transglutaminase, Nuclear Protein 56, A
  • the nucleic acid is specific for a mRNA selected from the group consisting of the dopamine D1, D2 or D3 receptors, the serotonin 5-HT2C receptor, the adenosine A2A receptor, Huntingtin, and the dopamine transporter.
  • the nucleic acid is specific for a mRNA selected from the group consisting of a gene product encoded in chromosome 21, the amyloid precursor protein, presenilin 1, presenilin 2, Apolipoprotein E, cyclin-dependent kinase 5, glycogen synthase kinase 3, the microtubule affinity-regulating kinase, the serotonin 5-HT1A receptor, the adenosine A1 receptor, acetylcholinesterase, cannabinoid CB1 receptor, catechol-O-methyl transferase, histamine N-methyltransferase, H3, 5-HT6, nitric oxide synthase, phosphodiesterase 10A, phosphodiesterase 1B, phosphodiesterase 1C, phosphodiesterase 2A, phosphodiesterase 4A, phosphodiesterase 4B, phosphodiesterase
  • the nucleic acid is specific for a mRNA selected from the group consisting of superoxide dismutase 1, Alsin, Probable helicase senataxin, RNA-binding protein FUS, cesicle-associated membrane protein-associated protein B/C, Angiogenin, TAR DNA-binding protein 43, Polyphosphoinositide phosphatase, Optineurin, Ataxin-2, valosin-containing protein, reticulon 4, Nav1.7, Nav1.8, Cav2.2, COX-2, kappa and Survival motor neuron protein.
  • a mRNA selected from the group consisting of superoxide dismutase 1, Alsin, Probable helicase senataxin, RNA-binding protein FUS, cesicle-associated membrane protein-associated protein B/C, Angiogenin, TAR DNA-binding protein 43, Polyphosphoinositide phosphatase, Optineurin, Ataxin-2, valosin-containing protein, reticulon 4,
  • the nucleic acid and the selectivity agent may be directly coupled. However, it is preferred that both moieties are linked by a connecting group.
  • connecting group refers to an organic moiety that connects two parts of a compound.
  • the selectivity agent can be attached to any sense or antisense nucleotide within the nucleic acid, but it can be preferably coupled through the 3′ terminal nucleotide and/or 5′ terminal nucleotide.
  • An internal conjugate may be attached directly or indirectly through a linker to a nucleotide at a 2′ position of the ribose group, or to another suitable position.
  • the conjugate can be attached to the sense 3′ terminal nucleotide, the sense 5′ terminal nucleotide, the antisense 3′ terminal nucleotide, and/or the antisense 5′ terminal nucleotide.
  • the length of the linker is described by counting the number of atoms that represent the shortest distance between the atom that joins the selectivity agent to the linker and the oxygen atom of the terminal phosphate moiety associated with the oligonucleotide through which the linker is attached to the oligonucleotide.
  • counting the atoms around the ring that represent the shortest path is preferred.
  • Suitable linker groups for use in the present invention include, without limitation, modified or unmodified nucleotides, nucleosides, polymers, sugars, carbohydrates, polyalkylenes such as polyethylene glycols and polypropylene glycols, polyalcohols, polypropylenes, mixtures of ethylene and propylene glycols, polyalkylamines, polyamines such as polylysin and spermidine, polyesters such as poly(ethyl acrylate), polyphosphodiesters, aliphatics, and alkylenes.
  • polyalkylenes such as polyethylene glycols and polypropylene glycols, polyalcohols, polypropylenes, mixtures of ethylene and propylene glycols, polyalkylamines, polyamines such as polylysin and spermidine, polyesters such as poly(ethyl acrylate), polyphosphodiesters, aliphatics, and alkylenes.
  • linkers/linker chemistries that are based on omega-amino-1,3-diols, omega-amino-1,2-diols, hydroxyprolinols, omega-amino-alkanols, diethanolamines, omega-hydroxy-1,3-diols, omega-hydroxy-1,2-diols, omega-thio-1,3-diols, omega-thio-1,2-diols, omega-carboxy-1,3-diols, omega-carboxy-1,2-diols, co-hydroxy-alkanols, omega-thio-alkanols, omega-carboxy-alkanols, functionalized oligoethylene glycols, allyl amine, acrylic acid, allyl alcohol, propargyl amine, propargyl alcohol, and more, can be applied in this context to generate linkers of the appropriate length.
  • the linker may also confer other desirable properties on the oligonucleotide conjugate improved aqueous solubility, optimal distance of separation between the conjugate moiety and the oligonucleotide, flexibility (or lack thereof), specific orientation, branching, and others.
  • said connecting group has the following structure
  • 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 of m+n+p is an integer number selected from 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 and wherein k is 0 or 1.
  • p is 5, n is 2, k is 1 and m is 6 giving a linker having the structure:
  • p is 5
  • n and k are 0 and m is 6 giving a linker having the structure:
  • the linker comprises more than one coupling for the selectivity agent.
  • the linker is a bivalent or trivalent linker, i.e. 2 or 3 molecules of selectivity agent can be coupled, respectively.
  • said molecules can represent the same or different selectivity agents.
  • the bivalent or trivalent linker has the following formula:
  • m, m′, m′′, n, n′, n′′, p, p′, p′′, r, r′, r′′, s, 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 are independently selected from CH 2 , O, S, NH, CO, C(O)O and C(O)NH.
  • branched linkers can be symmetrical or asymmetrical.
  • the linker is a bivalent linker as shown above wherein p and p′ are 5, n and n′ are 2, k and k′ are l and m and m′ are 6. In a particular embodiment, the linker is a bivalent linker wherein p and p′ are 5, n, n′, k and k′ are 0 and m and m′ are 6.
  • the linker is a bivalent linker as shown above wherein r and r′ are 4, s and s′ are 1, t and v are 0 and X 1 and X 2 represent C(O)NH.
  • the linker is a bivalent linker wherein r is 2, r′ is 0, s is 1, s′ is 0, t and v are 0 and X 1 and X 2 represent CH 2 .
  • the linker is a bivalent linker wherein p and p′ are 5, n and n′ are 2, k and k′ are 1, m and m′ are 6, r and r′ are 4, s and s′ are 1, t and v are 0 and X 1 and X 2 represent C(O)NH.
  • the linker is a bivalent linker wherein p and p′ are 5, n and n′ are 2, k and k′ are 1, m and m′ are 6, r is 2, r′ is 0, s is 1, s′ is 0, t and v are 0 and X 1 and X 2 represent CH 2 .
  • the linker is a bivalent linker wherein p and p′ are 5, n, n′, k and k′ are 0 and m and m′ are 6, r and r′ are 4, s and s′ are 1, t and v are 0 and X 1 and X 2 represent C(O)NH.
  • the linker is a bivalent linker wherein p and p′ are 5, n, n′. k and k′ are 0 and m and m′ are 6, r is 2, r′ is 0, s is 1, s′ is 0, t and v are 0 and X 1 and X 2 represent CH 2 .
  • the linker is a trivalent linker as shown above wherein p, p′ and p′′ are 5, n, n′ and n′′ are 2, k, k′ and k′′ are l and m, m′ and m′′ are 6.
  • the linker is a trivalent linker wherein p, p′ and p′′ are 5, n, n′, n′′, k, k′ and k′′ are 0 and m, m′ and m′′ are 6.
  • the linker is a trivalent linker as shown above wherein r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, v is 0 and X 1 , X 2 and X 3 represent O.
  • the linker is a trivalent linker wherein r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, u is 3, v is 1 and X 1 , X 2 and X 3 represent O.
  • the linker is a trivalent linker wherein p, p′ and p′′ are 5, n, n′ and n′′ are 2, k, k′ and k′′ are 1, m, m′ and m′′ are 6, r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, v is 0 and X 1 , X 2 and X 3 represent O.
  • the linker is a trivalent linker wherein p, p′ and p′′ are 5, n, n′ and n′′ are 2, k, k′ and k′′ are 1, m, m′ and m′′ are 6, r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, u is 3, v is 1 and X 1 , X 2 and X 3 represent O.
  • the linker is a trivalent linker wherein p, p′ and p′′ are 5, n, n′, n′′, k, k′ and k′′ are 0, m, m′ and m′′ are 6, r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, v is 0 and X 1 , X 2 and X 3 represent O.
  • the linker is a trivalent linker wherein p, p′ and p′′ are 5, n, n′, n′′, k, k′ and k′′ are 0, m, m′ and m′′ are 6, r, r′ and r′′ are 3, s, s′ and s′′ are 1, t is 1, u is 3, v is 1 and X 1 , X 2 and X 3 represent 0.
  • a particular preferred linking group according to the present invention has the following structure:
  • a and B represent monomer units independently selected from the group consisting of a monosaccharide, a (C 1 -C 50 )alkyl and a (C 2 -C 20 ) alkylene glycol; a and b are integers ranging from 0 to 50; c is an integer ranging from 0 and 30; L1, L2 and L3 are linking compounds independently selected from the group consisting of phosphodiester, phosphorothioate, carbamate, carbonyl (C ⁇ O), methylphosphonate, guanidinium, sulfamate, sulfamide, formacetal, thioformacetal, sulfone, amide and mixtures thereof; and d is 0 or 1.
  • a and B independently represent a (C 1 -C 20 )alkyl.
  • (C 1 -C 50 )alkyl refers to a straight chain or branched alkyl group having between 1 and 50 carbon atoms.
  • (C 1 -C 20 )alkyl refers to a straight chain or branched alkyl group having between 1 and 20 carbon atoms. Examples of alkyl groups include but are not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, and hexyl.
  • the linking group has the structure:
  • the linking group has the structure:
  • A is (C 1 -C 50 )alkyl
  • L1 is a carbonyl
  • L2 is a phosphodiester
  • conjugates of the invention involve chemically linking to the nucleic acid or to the protecting group one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the nucleic acid.
  • moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al, Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid (Manoharan et al, Biorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al, Ann. N.Y. Acad.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides and Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et ai, Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
  • the moiety capable of enhancing cellular distribution may be a a low molecular weight compound or polypeptide which is capable of being specifically translocated across biological barriers by the use of receptor-mediated endocytosis using specific transporters present in said biological barriers.
  • a wide array of uptake receptors and carriers, with a even wider number of receptor-specific ligands, are known in the art.
  • Preferred ligands for receptors that mediates endocytosis and/or transcytosis for use in accordance with present invention include e.g.
  • ligands for, or that specifically bind to the thiamine transporter, folate receptor, vitamin B 12 receptors, a sialoglycoprotein receptors, alpha(2,3)-sialoglycoprotein receptor (with e.g., the FC5 and FC44 nanobodies consisting of llama single-domain antibodies (sdAbs) as receptor-specific ligands), transferrin-1 and -2 receptors, scavenger receptors (class A or B, types I, II or III, or CD36 or CD163), low-density lipoprotein (LDL) receptor, LDL-related protein 1 receptor (LRP1, type B), the LRP2 receptor (also known as megalin or glycoprotein 330), diphtheria toxin receptor (DTR, which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor (HB-EGF)), insulin receptor, insulin-like growth factors (IGF) receptors, leptin receptors, substance P receptor, glutathione
  • Preferred ligands that bind to these receptors include e.g. ligands selected from the group consisting of: lipoprotein lipase (LPL), alpha2-macroglobulin (alpha2M), receptor associated protein (RAP), lactoferrin, desmoteplase, tissue- and urokinase-type plasminogen activator (tPA/uPA), plasminogen activator inhibitor (PAI-I), tPA/uPA:PAT-1 complexes, melanotransferrin (or P97), thrombospondin 1 and 2, hepatic lipase, factor VIIa/tissue-factor pathway inhibitor (TFPI), factor Villa, factor IXa, Abeta1-40, amyloid-beta precursor protein (APP), C1 inhibitor, complement C3, apolipoprotein E (apoE), pseudomonas exotoxin A, CRM66, HIV-I Tat protein, rhinovirus, matrix metallo
  • LPL lipoprotein lipa
  • the conjugate of the invention further comprises a group that facilitates the transport across biological membranes of the conjugate.
  • the group is amphipathic.
  • An exemplary agents include, without limitation, penetratin, the fragment of the Tat protein comprising amino acids 48-60, the signal sequence based peptide, PVEC, transportan, amphiphilic model peptide, Arg9, bacterial cell wall permeating peptide, LL-37, cecropin P1, ⁇ -defensin, ⁇ -defensin, bactenectin, PR-39 and indolicidin.
  • the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids.
  • the helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.
  • the ligand can be a peptide or peptidomimetic.
  • a peptidomimetic also referred to herein as an oligopeptidomimetic is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide.
  • the peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.
  • the conjugate of the invention further comprises an endosomolytic ligand.
  • Endosomolytic ligands promote the lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell.
  • the endosomolytic ligand may be a polyanionic peptide or peptidomimetic which shows pH-dependent membrane activity and fusogenicity. In certain embodiments, the endosomolytic ligand assumes its active conformation at endosomal pH.
  • the “active” conformation is that conformation in which the endosomolytic ligand promotes lysis of the endosome and/or transport of the composition of the invention, or its components, from the endosome to the cytoplasm of the cell.
  • exemplary endosomolytic ligands include the GAL4 peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk et al., Biochem. Biophys.
  • the endosomolytic component may contain a chemical group (e.g., an amino acid) which will undergo a change in charge or protonation in response to a change in pH.
  • the endosomolytic component may be linear or branched.
  • the nucleic acids forming part of the conjugates of the invention have to be preserved from degrading factors, such as nucleases (endo/exonucleases), during their transport through the different fluids and compartments of the organism.
  • degrading factors such as nucleases (endo/exonucleases)
  • the oligonucleotides are designed to resist the enzymatic digestion, and to improve the in vivo stability and bioavailability of the oligonucleotide.
  • the nucleic acids are chemically modified by the presence of a group which prevents nuclease-mediated degradation.
  • cap structure or “protecting group” shall be understood to mean chemical modifications, which have been incorporated at either terminus of the oligonucleotide.
  • Non-limiting examples of the 5′-cap include inverted abasic residue (moiety), 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclic nucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide, 3′-3
  • the 3′-cap includes, for example, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide: 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide; phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-di
  • the cap structure which is attached to the nucleic acid sequence of the conjugates of the invention has the following general structure:
  • M is H, a lipid moiety or a targeting group as defined above;
  • a and B represent monomer units independently selected from the group consisting of a monosaccharide and a (C 2 -C 20 ) alkylene glycol;
  • L1, L2 and L3 are linking compounds independently selected from the group consisting of phosphodiester, phosphorothioate, carbamate, methylphosphonate, guanidinium, sulfamate, sulfamide, formacetal, thioformacetal, sulfone, amide and mixtures thereof;
  • a and b are integers ranging from 0 to 50;
  • c is an integer ranging from 0 and 30;
  • d is an integer which is at least 1.
  • a lipid moiety refers to a group of organic compounds that has lipophilic or amphipathic properties, including, but not limited to, fats, fatty oils, essential oils, waxes, steroids, sterols, phospholipids, glycolipids, sulpholipids, aminolipids, chromolipids (lipochromes), and fatty acids,
  • lipid encompasses both naturally occurring and synthetically produced lipids. Lipid moieties usually increase lipophilic properties of the oligonucleotide and facilitate the intracellular uptake in vivo of the oligonucleotide construction.
  • Suitable lipids that can be used include fatty acids; fats; oils; waxes; cholesterol; sterols; fat-soluble vitamins, such as vitamins A, D, E and K; monoglycerides; diglycerides, and phospholipids.
  • Preferred fatty acids are those selected from the group consisting of lauroic acid (C12), myristic acid (C14), palmitic acid (C16), stearic acid (C18), docosanoic acid (C22), and hybrid of lithocholic acid and oleylamine (lithocholic-oleyamine, C43).
  • the lipid may be selected by the skilled person according to the circumstances by taking into consideration the target tissue, the target cell, the administration route, the pathway that the oligonucleotide is expected to follow, etc.
  • Preferred sugar moieties for this conjugation group are selected from the group consisting of of furanose, fructose, glucose, galactose, mannose, a modified monosaccharide, sialic acid and eritrose and mixtures thereof.
  • the monosaccharides may be in its lineal or cyclic forms (hemiacetalic cyclic isomers).
  • the furanose is any simple sugar containing a five-membered furan-based ring, such as a D-ribose or a fructose residue (D-( ⁇ )-fructofuranose). With the combination of the monosaccharides, multiple sugar structures can be attained.
  • the fructooligosaccharides (FOS) and the galactooligosaccharides (GOS) are combinations of special interest, as well as the disaccharides sacarose or lactose; or the polysaccharides inulin, dextrin, starch or glycogen.
  • alkylene glycol encompasses a family of polyether polymers which share the general formula
  • n represents the number of repeating units, and therefore represents the size or length of the polymer.
  • the term includes. without limitation, ethylene glycol, propylene glycol, dialkylene glycol (for example, diethylene glycol), trialkylene glycol (for example, triethylene glycol), and glycols such as corresponding mono- and di-alkyl ethers of the aforementioned glycols, wherein the alkyl ethers are lower alkyl ethers having 1 to 6 carbon atoms (for example, methyl, ethyl, propyl ether and the like)
  • the group of formula (I) has a (C 2 -C 20 )alkylene glycol monomer unit, which may be any linear or branched molecules from 2 to 20 carbon atoms, or, depending on the values of a and b, a polyalkylene glycol polymer with several (C 2 -C 20 ) alkylene glycol monomer units.
  • the alkylene glycol group is selected from C 16 -C 20 alkylene glycol. Still more preferably, the alkylene glycol group is a C 18 alkylene glycol.
  • PEG and“sugar” are used essentially as described above and include furanose as sugar and a PEG selected from the group of C3, C9 and C18 spacers.
  • the conjugate further comprises a protecting group attached to one end or to both ends of the nucleic acid which is not attached to the selectivity agent.
  • the selectivity agent may be coupled to the 5′ end and/or to the 3′ end of the nucleic acid.
  • the selectivity agent is coupled to the 5′ end of the nucleic acid.
  • the nucleic acid and the selectivity agent may be directly linked or may be connected by a linker.
  • the linker may be coupled to the 5′ end and/or to the 3′ end of the nucleic acid.
  • the linker is coupled to the 5′ end of the nucleid acid.
  • the conjugate of the invention may contain more than one nucleic acid chain that modulates the expression of the target molecule.
  • a construction of this invention can contain up to five different nucleic acids joined in tandem through phosphodiester bonds targeted at different regions of a given target molecule.
  • the selectivity agent may be coupled to the sense and/or to the antisense strand and may be directly coupled or connected by a linker group.
  • the nucleic acid of the invention contains a single nucleic acid chain, the possible arrangements are:
  • the different selectivity agents may be the same or different. Wherein the selectivity agents are different, they may bind the same or different receptors.
  • the receptors may be expressed either at the same cell or in different cell types. Wherein the receptors are expressed in different cell types, the receptors may be located at the same or at different locations of the central nervous system. In a preferred embodiment, the selectivity agents found within a single conjugate can bind to the following locations:
  • the selectivity agent of the conjugate according to the invention is a ligand of a growth hormone secretagogue receptor and the nucleic acid is specific for the SOCS3 or PTP 1B mRNAs or for the corresponding polypeptides.
  • the selectivity agent is tabimorelin.
  • the nucleic acid is a single stranded nucleic and the conjugate contains two tabimorelin groups attached to the 5′ end of the nucleic acid by the use of a bifunctional linker.
  • the nucleic acid is a single stranded nucleic acid and the conjugate contains two tabimorelin groups attached to the 3′ end of the nucleic acid by the use of a bifunctional linker attached to the 3′ end of the nucleic acid.
  • the nucleic acid is a single stranded nucleic acid and the conjugate contains two tabimorelin groups attached, respectively, to the 5′ and 3′ ends of the nucleic acid.
  • the nucleic acid is a double stranded nucleic acid and the conjugate contains two tabimorelin groups attached to the 5′ end of the sense strand or to the 5′ end of the antisense strand by the use of a bifunctional linker.
  • the nucleic acid is a double stranded nucleic acid and the conjugate contains two tabimorelin groups, the first one attached to the 5′ end of the sense strand and the second one attached to the 5′ end of the antisense strand.
  • the conjugates according to the invention contain different selectivity agents which are targeted to different receptors expressed in the same area of the central nervous system.
  • the selectivity ligands may be directed to different receptors expressed in the hypothalamus.
  • the first and second selectivity agent are directed to any pairwise combination of receptors expressed in the hypothalamus selected from the group consisting of growth hormone secretagogue receptor, the galanin GAL1 receptor, the calcitonin receptor-like, the neuropeptide FF/B NPBW2 receptor, the neuropeptide FF/B NPFF2 receptor, the neuropeptide Y Y2 receptor, the bombesin BB2 receptor, the bombesin BB3 receptor, the calcitonin AM1 receptor, the calcitonin AMY1 receptor, the calcitonin CGRP receptor the calcitonin receptor, the frizzled FZD2 receptor, the frizzled
  • the nucleic acids forming part of the conjugates of the invention have to be preserved from degrading factors, such as nucleases (endo/exonucleases), during their transport through the different fluids and compartments of the organism.
  • degrading factors such as nucleases (endo/exonucleases)
  • the oligonucleotides are designed to resist the enzymatic digestion, and to improve the in vivo stability and bioavailability of the oligonucleotide.
  • Cellular exonucleases use free 5′ ends as targets.
  • the selectivity agent may act as a stabilizing moiety when coupled to the 5′ end of the nucleic acid.
  • the conjugate may further comprise an stabilising moiety or cap structure which is usually a group which prevents degradation of the nucleic acid by the activity of exonucleases.
  • an stabilising moiety or cap structure which is usually a group which prevents degradation of the nucleic acid by the activity of exonucleases.
  • the nucleic acid is a double stranded RNA wherein the selectivity agent is linked to the 5′ end of the antisense strand and the protecting group is linked to the 5′ end of the sense strand.
  • the protecting group has the structure
  • M is H, d is 0, A is a C18 spacer of polyethylene glycol, B is a furanose, a is 2, b and c are 1 and L2 and L3 are phosphodiester bonds
  • the nucleic acids forming part of the conjugates of the invention have to be protected from degrading factors, such as nucleases (endo/exonucleases), during their transport through the different fluids and compartments of the organism.
  • degrading factors such as nucleases (endo/exonucleases)
  • the oligonucleotides are designed to resist the enzymatic digestion, and to improve the in vivo stability and bioavailability of the oligonucleotide.
  • Cellular exonucleases use free 5′ ends as targets.
  • the selectivity agent may act as a stabilizing moiety when coupled to the 5′ end of the nucleic acid.
  • the conjugate may further comprise an stabilising moiety or cap structure which is usually a group which prevents degradation of the nucleic acid by the activity of exonucleases.
  • an stabilising moiety or cap structure which is usually a group which prevents degradation of the nucleic acid by the activity of exonucleases.
  • the conjugate of the invention has the structure (I)
  • R 1 and R 2 independently of each other are hydrogen or C1-C6 alkyl or R1 and R 2 taken together form a C2-C5 alkylene group; J is a group
  • R 4 and R 5 independently of each other are hydrogen or C1-C6 alkyl and R 6 is hydrogen or C1-C6 alkyl, preferably hydrogen, and wherein the oligonucleotide comprises a sequence specific for the SOCS3 mRNA or for the PTP-1B mRNA.
  • the conjugate has the structure:
  • the conjugates of the invention have the ability of modulating the expression of the nucleic acid which is targeted by the nucleic acid sequences of the conjugates. For instance, in the case of conjugates comprising a nucleic acid specific for the SOCS3 or PTP1B and a ligand for the growth hormone secretagogue receptor, when the construction is administered to a subject, it can effectively induce a specific knock-down of SOCS3 and PTP1B in the subjects midbrain raphe nuclei.
  • the conjugates of the invention are adequate for the treatment of diseases which may benefit from the reduction in the expression levels of the genes which are targeted by the nucleic acids present in the conjugates of the invention.
  • the invention relates to a conjugate according to the invention for use in medicine.
  • the invention also relates to a pharmaceutical composition comprising a conjugate according to the invention and a pharmaceutically-acceptable excipient.
  • Appropriate amounts of oligonucleotide constructions of the invention can be formulated with pharmaceutically acceptable excipients and/or carriers to obtain a pharmaceutical composition.
  • a composition that includes a conjugate according to the invention can be delivered to a subject by a variety of routes. Exemplary routes include intrastriatal, intracerebroventricular, intrathecal, intraparenchymal (e.g., in the striatum), intranasal, and ocular delivery.
  • the composition can also be delivered systemically, e.g., by intravenous, subcutaneous or intramuscular injection, which is particularly useful for delivery of the conjugates to peripheral neurons.
  • conjugates of the invention intranasally which allows systemic administration by a non-aggressive mode of administration.
  • intraventricular administration may also be adequate.
  • a preferred route of delivery is directly to the brain, e.g., into the ventricles or the hypothalamus of the brain, or into the lateral or dorsal areas of the brain.
  • the conjugates are formulated in accordance with standard procedure as a pharmaceutical composition adapted for delivered administration to human beings and other mammals.
  • compositions for intravenous or intraventricular administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic to ameliorate any pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • the composition can contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the composition can be a liquid solution, suspension, emulsion, gel, polymer, or sustained release formulation.
  • the composition can be formulated with traditional binders and carriers, as would be known in the art.
  • Formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharide, cellulose, magnesium carbonate, etc., and inert carriers having well established functionality in the manufacture of pharmaceuticals.
  • Various delivery systems are known and can be used to administer a therapeutic of the present invention including encapsulation in liposomes, microparticles, microcapsules and the like.
  • therapeutics containing the conjugates of the invention can be formulated as neutral or salt forms.
  • Pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids and the like, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine or similar.
  • the clinical condition that can be treated with the conjugates of the invention will depend on the specificity of the nucleic acid which forms part of the conjugates.
  • the conjugates of the invention can be used for the treatment of any disease which can be improved by knocking down a gene of interest in a cell that expresses a neurotransmitter transporter.
  • the skilled person will understand that the conjugates are useful for the treatment of diseases characterized by abnormal expression of a protein in a cell or for diseases wherein the target protein is expressed at normal levels but which can be improved by decreasing the expression of said target protein.
  • the invention relates to a method for the treatment of a diseases in a subject in need thereof comprising administering to said subject a conjugate according to the invention wherein the conjugate contains a nucleic acid which is capable of silencing the expression of a target gene involved in said disease and a ligand which is specific for a receptor which is expressed in the cells wherein said target gene is expressed.
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 3 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 3 (left hand-column).
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 4 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 4 (left hand-column).
  • Target polypeptides expressed in the mesencephalon and diseases which can be treated by silencing said polypeptides
  • Target polypeptide Disease D1, D2, D3, DAT Addiction alpha-synuclein Synucleinopathies
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 5 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 5 (left hand-column).
  • Target polypeptides expressed in the brainstem and diseases which can be treated by silencing said polypeptides Target polypeptide Disease 5-HT1A, 5-HT2C, SERT Anxiety alpha1A-adrenoceptor, angiotensin Depression converting enzyme, G protein ⁇ 3, 5- HT2C, interleukin 1 ⁇ , monoamine oxidase A, SERT CB1 Eating disorders.
  • 5-HT1A Migraine alpha-synuclein Synucleinopathies SERT Phobias, post-traumatic stress disorder.
  • 5-HT1A, 5-HT1A2C Psychotic disorders
  • 5-HT1A Sleep disorders 5-HT1A Sleep disorders
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 6 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 6 (left hand-column).
  • Target polypeptides expressed in the cortex and diseases which can be treated by silencing said polypeptides Target polypeptide Disease gene product encoded in chromosome 21 Alzheimer's disease associated with Down syndrome amyloid precursor protein, presenilin 1, Early onset Alzheimer's presenilin 2 disease Apolipoprotein E, cyclin-dependent Late onset, early onset or kinase 5, glycogen synthase kinase 3, the sporadic Alzheimer's disease. microtubule affinity-regulating kinase A2, D4 Attention deficit hyperactivity disorder. acetylcholinesterase, A2, CB1, catechol- Cognitive impairment.
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 7 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 7 (left hand-column).
  • Target polypeptides expressed in the cerebellum and diseases which can be treated by silencing said polypeptides
  • Target polypeptide Disease Atrophin-1 Dentatorubral Atrophy
  • Fragile X Mental retardation
  • Fragile X-associated Tremor/Ataxia Syndrome FXTAS
  • G-protein coupled receptor 55 Movement disorders FXTAS
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 8 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 8 (left hand-column).
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 9 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 9 (left hand-column).
  • Target polypeptides expressed in the hippocampus and diseases which can be treated by silencing said polypeptides Target polypeptide Disease gene product encoded in Alzheimer's disease associated with chromosome 21 Down syndrome amyloid precursor protein, Early onset Alzheimer's disease presenilin 1, presenilin 2 Apolipoprotein E, cyclin- Late onset, early onset or sporadic dependent kinase 5, glycogen Alzheimer's disease synthase kinase 3, the microtubule affinity-regulating kinase 5-HT1A Anxiety A1 Attention Deficit Hyperactivity Disorder (ADHD) 5-HT1A Bipolar disorder acetylcholinesterase, A1, CB1, Cognitive impairment catechol-O-methyl transferase, histamine N-methyltransferase, H3, 5-HT6, nitric oxide synthase, phosphodiesterase 10A, phosphodiesterase 1B, phosphodiesterase 1C, phosphodiesterase 2A, phosphodiesterase
  • the invention relates to the use of a conjugate for the treatment of the diseases shown in Table 10 (right-hand column) wherein the nucleic acid is specific for the target polypeptide shown in Table 10 (left hand-column).
  • Target polypeptides expressed in the spinal cord and diseases which can be treated by silencing said polypeptides
  • Target polypeptide Disease Superoxide dismutase 1, Alsin, Probable Amyotrophic lateral sclerosis helicase senataxin, RNA-binding protein (ALS)/Lou Gehrig's disease FUS, Vesicle-associated membrane protein-associated protein B/C, Angiogenin, TAR DNA-binding protein 43, Polyphosphoinositide phosphatase, Optineurin, Ataxin-2, valosin-containing protein reticulon 4 Ataxia, Brown-Séquard Sindrome and Neuropathy, Pain, Paralysis, Sipanl Cord injury Nav1.7, Nav1.8, Cav2.2, COX-2, kappa Neuropathy, Pain Survival motor neuron protein Spinal cord injury, spinal cord atrophy (SMA)
  • SMA spinal cord atrophy
  • the amount of the conjugate of the present invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques, well established in the administration of therapeutics.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and the patient's needs.
  • Suitable dose ranges for intracranial administration are generally about 10 3 to 10 15 infectious units of viral vector per microliter delivered in 1 to 3000 microliters of single injection volume.
  • Addition amounts of infections units of vector per micro liter would generally contain about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , or 10 14 infectious units of viral vector delivered in about 10, 50, 100, 200, 500, 1000, or 2000 microliters. Effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.
  • multiple catheters having access ports can be implanted in a given patient for a complete therapy.
  • the patient's neurologist can perform a course of therapy consisting of repeated bolus injections of the conjugates over a period of weeks to months, along with monitoring for therapeutic effect over time.
  • the devices can remain implanted for several months or years for a full course of therapy.
  • the access ports might optionally be explanted, and the catheters can be sealed and abandoned, or explanted as well.
  • the device material should not interfere with magnetic resonance imaging, and, of course, the small interfering RNA preparations must be compatible with the access port and catheter materials and any surface coatings.
  • the conjugates of the invention are typically synthesized using standard procedures in organic synthesis. The skilled person will appreciate that the exact steps of the synthesis will depend on the exact structure of the conjugate which has to be synthesized. For instance, if the conjugate comprises a single nucleic acid strand conjugated to the selectivity agent through its 5′ end, then the synthesis is usually carried out as explained below by contacting an activated oligonucleotide and a reactive activated selectivity reagent.
  • the conjugate comprises a double stranded nucleic acid
  • the sense and antisense strands are synthesized separately and annealed in vitro using standard molecular biology procedures.
  • the first nucleic acid strand carries the selectivity agent and the second nucleic acid strand carries a protecting group.
  • the selectivity agent 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 the attachment of the selectivity agent or of the protecting group can also be carried out at the 3′ ends of the nucleic acid strands.
  • the conjugates of the invention can be prepared using techniques known by those skilled in the art.
  • the synthesis of conjugates may involve the selective protection and deprotection of functional groups.
  • Suitable protecting groups are well known to the skilled person in the art.
  • a general review of protecting groups in organic chemistry is provided by Wuts, P.G.M. and Greene T.W. in Protecting Groups in Organic Synthesis (4 th Ed. Wiley-Interscience), and by Kocienski P. J. in Protecting Groups (3 rd Ed. Georg Thieme Verlag).
  • C 1 -C 6 alkyl relates to a linear or branched hydrocarbon radical consisting of carbon and hydrogen atoms, which does not contain unsaturation, having one to six, preferably one to three (C 1 -C 3 alkyl), carbon atoms and which is joined to the rest of the molecule by a single bond.
  • alkyl groups include but are not limited to alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl and hexyl.
  • alkyl refers to methyl.
  • halogen refers to bromo, chloro, iodo or fluoro.
  • haloalkyl refers to an alkyl group as defined above wherein at least one hydrogen atom has been replaced by halogen.
  • haloalkyl groups include but are not limited to CF 3 , CCl 3 , CHF 2 , CF 2 CF 3 .
  • haloalkyl refers to CF 3 .
  • C 6 -C 10 aryl refers to an aromatic group having between 6 and 10 carbon atoms, comprising 1 or 2 aromatic nuclei, bound by means of a carbon-carbon bond or fused, including for example phenyl, naphthyl and diphenyl.
  • aryl refers to phenyl.
  • the compounds of the present invention represented by the above described formula (I) may include stereoisomers depending on the presence of chiral centres.
  • the single isomers, enantiomers or diastereoisomers and mixtures thereof fall within the scope of the present invention.
  • the compounds used in the invention are intended to include compounds that only differ in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the substitution of a hydrogen with deuterium or tritium, or the substitution of a carbon with a 13 C- or 14 C-enriched carbon or a 15 N-enriched nitrogen are within the scope of this invention.
  • the conjugate of the invention is obtained by the conjugation of an amino-derivatized selectivity agent and a carboxyl-derivatized oligonucleotide.
  • the conjugate of the invention has the structure (I):
  • R 1 and R 2 independently of each other are hydrogen or C1-C6 alkyl or R1 and R 2 taken together form a C2-C5 alkylene group; J is a group
  • R 4 and R 5 independently of each other are hydrogen or C1-C6 alkyl and R 6 is hydrogen or C1-C6 alkyl, preferably hydrogen and wherein the oligonucleotide is a nucleic acid which is capable of specifically binding to a target molecule wherein said target molecule is the mRNA of a polypeptide as defined in Table 3 (left-hand column).
  • the process of synthesis of a conjugate having the structure of (III) comprises reacting a compound having the structure of (V):
  • the invention also relates to a compound having the structure (VI) wherein the oligonucleotide is a nucleic acid which is capable of specifically binding to a target molecule wherein said target molecule is the mRNA of a polypeptide as defined in Table 3 (left-hand column).
  • the oligonucleotide in the compound having the structure (VI) is an antisense gapmer.
  • said gapmer comprises a central block of 10 deoxynucleotides flanked by blocks of 5 2′-O-methyl modified ribonucleotides.
  • the nucleic acid is the sense or antisense strand of a siRNA.
  • the carboxymodified oligonucleotide should be deprotected for further conjugation with the selectivity agent.
  • all the remaining protecting groups in the oligonucleotide are removed as follows. 500 ⁇ l of a mixture containing 20% v/v of methylamine (aqueous solution 40% w/v) and 80% v/v of a saturated ammonia solution, (containing 30-32% w/v of NH 3 ) were added to an Eppendorf tube with the oligonucleotide (200 nmole scale). The tube was hermetically closed and heated for 45 minutes to a temperature of 65° C.
  • the oligonucleotide comprised by the conjugate synthesized by the method of the invention is an antisense gapmer.
  • the gapmer comprises a central block of 10 deoxynucleotides flanked by blocks of 5 2′-O-methyl modified ribonucleotides.
  • the carboxyl-activaded oligonucleotide is then reacted with the activated derivative of a selectivity agent of formula (V) as defined above.
  • a compound is obtained having the general formula (I) as shown above.
  • this compound (I) comprises an oligonucleotide which is capable of specifically binding to a target molecule wherein said target molecule is a polypeptide as defined in Table 3 or the mRNA encoding said polypeptide.
  • the oligonucleotide in the compound having the structure (III) is an antisense gapmer.
  • said gapmer comprises a central block of 10 deoxynucleotides flanked by blocks of 5 2′-O-methyl modified ribonucleotides.
  • the nucleic acid is the sense or antisense strand of a siRNA.
  • the invention provides a conjugate comprising a
  • selectivity agent and “receptor” have been described in detail above and can be understood equally for the diagnostic conjugates of the invention. Any of the selectivity agents mentioned above can be used in the conjugates for imaging according to the invention.
  • imaging agent and “contrast agent”, are used herein interchangeably and refer to a biocompatible compound, the use of which facilitates the differentiation of different parts of the image, by increasing the “contrast” between those different regions of the image.
  • contrast agents thus encompasses agents that are used to enhance the quality of an image that may nonetheless be generated in the absence of such an agent (as is the case, for instance, in MRI), as well as agents that are prerequisites for the generation of an image (as is the case, for instance, in nuclear imaging).
  • Suitable contrast agent include, without limitation, contrast agents for Radionuclide imaging, for computerized tomography, for Raman spectroscopy, for Magnetic resonance imaging (MRI) and for optical imaging.
  • Contrast agents for radionuclide imaging include radiopharmaceuticals and are commonly labeled with positron-emitters such as 11 C, 13 N, 15 O, 18 F, 82 Rb, 62 Cu and 68 Ga.
  • SPECT radiopharmaceuticals are commonly labeled with positron emitters such as 94 mTc, 201 Tl and 67 Ga.
  • Radionuclide imaging modalities positron emission tomography (PET); single photon emission computed tomography (SPECT)) are diagnostic cross-sectional imaging techniques that map the location and concentration of radionuclide-labeled radiotracers. PET and SPECT can be used to localize and characterize a radionuclide by measuring metabolic activity.
  • PET and SPECT provide information pertaining to information at the cellular level, such as cellular viability.
  • a patient ingests or is injected with a slightly radioactive substance that emits positrons, which can be monitored as the substance moves through the body.
  • patients are given glucose with positron emitters attached, and their brains are monitored as they perform various tasks. Since the brain uses glucose as it works, a PET image shows where brain activity is high.
  • a cell is labeled ex vivo for PET or SPECT imaging in vivo. Closely related to PET is single-photon emission computed tomography, or SPECT. The major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits low-energy photons.
  • Contrast agents for CT imaging include, for example, iodinated or brominated contrast media. Examples of these agents include iothalamate, iohexyl, diatrizoate, iopamidol, ethiodol and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent (see, e.g., Henson et al., 2004). For example, gadopentate agents has been used as a CT contrast agent (discussed in Strunk and Schild, 2004). Computerized tomography (CT) is contemplated as an imaging modality in the context of the present invention.
  • CT Computerized tomography
  • CT By taking a series of X-rays, sometimes more than a thousand, from various angles and then combining them with a computer, CT made it possible to build up a three-dimensional image of any part of the body.
  • a computer is programmed to display two-dimensional slices from any angle and at any depth.
  • intravenous injection of a radiopaque contrast agent such as those described herein can assist in the identification and delineation of soft tissue masses when initial CT scans are not diagnostic.
  • Contrast agents for optical imaging include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green, rhodamine green, a derivative of rhodamine green, an eosin, an erythrosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye and the various other fluorescent compounds disclosed herein.
  • the contrast agent is a compound that is able to be imaged by a magnetic resonance imaging apparatus.
  • Contrast agents which can be imaged by a magnetic resonance imaging apparatus differ from those used in other imaging techniques. Their purpose is to aid in distinguishing between tissue components with identical signal characteristics and to shorten the relaxation times (which will produce a stronger signal on T1-weighted spin-echo MR images and a less intense signal on T2-weighted images).
  • Examples of MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles.
  • the MRI contrast agent is 19 F. Both CT and MRI provide anatomical information that aid in distinguishing tissue boundaries.
  • CT Magnetic resonance imaging
  • MRI Magnetic resonance imaging
  • MRI contrast agents include complexes of metals selected from the group consisting of chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).
  • the compound that is able to be imaged by a magnetic resonance imaging apparatus is a gadolinium-based compound.
  • gadolinium-based compound shall mean, where used with respect to brain imaging, any gadolinium-containing substance administrable to a subject which results in an intravascular enhancement.
  • the gadolinium-containing contrast agent is selected from the group consisting of gadolinium, gadolinium pentate, and gadodiamide.
  • the amount of the gadolinium-containing contrast agent to be administered varies in an amount of about 10 mg per kg body weight.
  • the second magnetic resonance image is acquired about 45 minutes after administering the gadolinium-containing contrast agent.
  • This invention also provides the above-described method further comprising the step of intraperitoneally administering a saline solution (e.g. Ringer's solution) to the subject, which administering follows either step (c) or step (d).
  • a saline solution e.g. Ringer's solution
  • the invention also provides the use of a conjugate as defined above as diagnostic agent and methods for the detection of cells expressing the neurotransmitter transporter on their surface.
  • the invention also provides multimodal imaging methods. Certain embodiments of the present invention pertain to methods of imaging a subject, or a site within a subject using multiple imaging modalities that involve measuring multiple signals. In certain embodiments, the multiple signals result from a single label on, or in a cell. As set forth above, any imaging modality known to those of ordinary skill in the art can be applied in these embodiments of the present imaging methods.
  • the imaging modalities are performed at any time during or after administration of the labeled composition, e.g., labeled cell.
  • the imaging studies may be performed during administration of the labeled cell of the present invention, i.e., to aid in guiding the delivery to a specific location, or at any time thereafter.
  • Additional imaging modalities may be performed concurrently with the first imaging modality, or at any time following the first imaging modality. For example, additional imaging modalities may be performed about 1 sec, about 1 hour, about 1 day, or any longer period of time following completion of the first imaging modality, or at any time in between any of these stated times. In certain embodiments of the present invention, multiple imaging modalities are performed concurrently such that they begin at the same time following administration of the labeled cell or agent.
  • One of ordinary skill in the art would be familiar with performance of the various imaging modalities contemplated by the present invention.
  • the same imaging device is used to perform a first imaging modality and a second imaging modality.
  • different imaging devices are used to perform the different imaging modalities.
  • One of ordinary skill in the art would be familiar with the imaging devices that are available for performance of the imaging modalities described herein.
  • the instant invention provides methods for imaging cells using one or more imaging modalities.
  • the cells are labeled with multiple imaging agents, and in other aspects the cells are labeled with a single labeling agent.
  • the single labeling agent is a multimode-detectable agent.
  • the invention provides conjugates comprising a liposome and a selectivity agent which binds specifically to a receptor which can be internalized by the cell upon binding of said selectivity agent.
  • the invention provides conjugates comprising a dendrimer and a selectivity agent which binds specifically to a receptor which can be internalized by the cell upon binding of said selectivity agent.
  • the conjugates By encapsulating a therapeutical compound within the dendrimer or liposome, the conjugates allows the selective delivery of said compound to cells which express said neurotransmitter transporter.
  • the selectivity agent is specific for a receptor as defined in Table 1 (left-hand column). In another embodiment, the selectivity agent is specific for a receptor as defined in Table 1 (right-hand column). In another embodiment, the selectivity agent is any agent as defined in Table 2. In a more preferred embodiment, the selectivity agent is growth-hormone secretagogue receptor. In a still more preferred embodiment, the selectivity agent has the structure
  • Liposomes and nanoparticles are exemplary forms of nanocontainers that are commonly used for encapsulation of drugs.
  • the liposomes preferably have diameters of less than 200 nanometers. Liposomes having diameters of between 50 and 150 nanometers are preferred. Especially preferred are liposomes or other nanocontainers having external diameters of about 80 nanometers.
  • Suitable types of liposomes are made with neutral phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), diphosphatidyl phosphocholine, distearoylphosphatidylethanolamine (DSPE), or cholesterol, along with a small amount (1 percent) of cationic lipid, such as didodecyldimethylammonium bromide (DDAB) to stabilize the DNA within the liposome.
  • neutral phospholipids such as 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC), diphosphatidyl phosphocholine, distearoylphosphatidylethanolamine (DSPE), or cholesterol
  • POPC 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine
  • DSPE distearoylphosphatidylethanolamine
  • DDAB didodecyldimethyl
  • the liposome can be replaced with a nanoparticle or any other molecular nanocontainer with a diameter less than 200 nm that can encapsulate the DNA and protect the nucleic acid from nucleases while the formulation is still in the blood or in transit from the blood to the intracellular compartment of the target cell.
  • conjugation agents such as PEG strands
  • one or more other polymeric substances such as sphingomylein, can be attached to the surface of the liposome or nanocontainer and serve the dual purpose of providing a scaffold for conjugation of the “transportable peptide” and for delaying the removal of the formulation from blood and optimizing the plasma pharmacokinetics.
  • the present invention contemplates delivery of DNA to any group of cells or organs which have specific target receptors.
  • the liposomes may be used to deliver DNA to organs, such as liver, lung and spleen.
  • dendrimers refers to a macromolecule having a core and having multiple shells of branching structures emanating from the core.
  • the shape and size of a dendritic carrier can vary. In some instances, the dendritic carrier can be approximately spherical or globular in shape. Furthermore, the dendritic carrier can have a diameter in the range of about 15 angstroms (A) to about 250 A, with a corresponding range of molecular weights, e.g., from about 500 Daltons to about 2 million Daltons.
  • Dendrimers can be obtained commercially from various sources (e.g., Dendritech.
  • Dendritic molecules can roughly be divided into the low-molecular weight and the high-molecular weight species.
  • the first category includes dendrimers and dendrons whereas the second encompasses dendronized polymers, hyperbranched polymers, and brush-polymers (also called bottle-brushes).
  • Dendrimers and dendrons are repeatedly branched, monodisperse, and usually highly symmetric compounds. There is no apparent difference in defining dendrimer and dendron.
  • a dendron usually contains a single chemically addressable group that is called the focal point. Because of the lack of the molar mass distribution high-molar-mass dendrimers and dendrons are macromolecules but not polymers.
  • dendrimers are dominated by the functional groups on the molecular surface. Dendritic encapsulation of functional molecules allows for the isolation of the active site, a structure that mimics the structure of active sites in biomaterials because dendritic scaffolds separate internal and external functions.
  • a dendrimer can be water-soluble when its end-group is a hydrophilic group, like a carboxyl group.
  • Dendrimers may be generally characterised by the following features: (i) an initiator core (I) which may have one or more reactive sites and be point-like or of significant size so as to effect the final topology of the dendrimer; (ii) one or more layers of branched repeating units attached to the initiator core; (iii) functional terminal groups, such as anionic or cationic groups, attached, optionally through linking groups, to the surface of the dendrimer.
  • Dendrimers contemplated herein may comprise lysine, or lysine analogue building units.
  • lysine analogue refers to a molecule which has a single apex carboxyl group for attachment to the previous layer of building units, and two or three primary amine groups to which can be attached further building units, blocking groups, linkers or aryl acid groups. Examples of “lysine analogues” contemplated herein are described in PCT/AU2007/000352, for example glycyl-lys.
  • the dendrimer comprises only lysine or one type of lysine analogue as the building unit.
  • dendrimers contemplated herein include those comprising polyamidoamine (PAMAM), poly(etherhydroxylamine) (PEHAM) or polypropyleneimine building units.
  • PAMAM polyamidoamine
  • PEHAM poly(etherhydroxylamine)
  • PEHAM polypropyleneimine building units.
  • the dendrimer has only polyamidoamine (PAMAM), poly(etherhydroxylamine) (PEHAM) or polypropyleneimine as the building unit.
  • the core moiety may contain only 1 point of attachment for a building unit or may contain 2, 3 or more points, which may or may not be further utilized for the attachment of building units. Typically, the point of attachment is a free amino group.
  • Core moieties may consist of, comprise or be derived from a building unit or may be a molecule different to the building units. Exemplary core moieties are illustrated herein and described in PCT/AU2007/000352.
  • the liposomes and dendrimers may be combined with any suitable pharmaceutical carrier for intravenous administration.
  • Intravenous administration of the composition is the preferred route since it is the least invasive. Other routes of administration are possible, if desired.
  • Suitable pharmaceutically acceptable carriers include saline, Tris buffer, phosphate buffer, or any other aqueous solution. An appropriate dosage can be established by procedures well known to those of ordinary skill in the art.
  • the following molecules were tested for their ability to silence target genes in brain areas expressing the growth hormone secretagogue receptor (hypothalamus)
  • mice were treated with a single dose of 30 ⁇ g of a non-coding siRNA sequence conjugated to tabimorelin and labeled with Cy3 (long linker) (TAB-NS-Cy3). Unconjugated molecule was used as control (NS-Cy3). Molecules were administered at the lateral ventricle. 1 hour later animals were sacrificed, brains removed and processed for visualizing under fluorescent microscopy ( FIG. 2 ).
  • Cy3 labeling was detected in the specific brain regions, in the hypothalamic area, only in mice treated with the conjugated molecule (TAB-NS-Cy3) compared with the unconjugated control (NS-Cy3), demonstrating that tabimorelin can direct nucleic acid molecules to specific brain areas.
  • FIG. 3 shows cumulative body weight gain during treatment. As shown, animals treated with either TAB-SOCS3-ASO4 or TAB-PTP1B-ASO4 gained significantly less weight than control animals (VH and NS).
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US9084825B2 (en) 2012-10-26 2015-07-21 Nlife Therapeutics, S.L. Compositions and methods for the treatment of parkinson disease by the selective delivery of oligonucleotide molecules to specific neuron types
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US20150315575A1 (en) 2015-11-05
WO2014064258A1 (fr) 2014-05-01
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