WO2019168558A1 - Constructions liposomales d'acides nucléiques sphériques (sna) pour des inhibiteurs de survie de neurones moteurs (smn) - Google Patents

Constructions liposomales d'acides nucléiques sphériques (sna) pour des inhibiteurs de survie de neurones moteurs (smn) Download PDF

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WO2019168558A1
WO2019168558A1 PCT/US2018/045685 US2018045685W WO2019168558A1 WO 2019168558 A1 WO2019168558 A1 WO 2019168558A1 US 2018045685 W US2018045685 W US 2018045685W WO 2019168558 A1 WO2019168558 A1 WO 2019168558A1
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sna
oligonucleotide
core
smn2
mrna
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Richard Kang
Subbarao NALLAGATLA
Bart ANDERSON
Ekambar Kandimalla
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Exicure, Inc.
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Definitions

  • Provisional Application Serial No. 62/636,764 entitled“LIPOSOMAL SPHERICAL NUCLEIC ACID (SNA) CONSTRUCTS LOR SURVIVAL OL MOTOR NEURON (SMA) INHIBITORS” filed Lebruary 28, 2018, to United States Provisional Application Serial No. 62/664,055, entitled“LIPOSOMAL SPHERICAL NUCLEIC ACID (SNA) CONSTRUCTS LOR SURVIVAL OL MOTOR NEURON (SMA) INHIBITORS” filed April 27, 2018, to United States Provisional Application Serial No.
  • SMA Spinal muscular atrophy
  • SMA is an autosomal recessive neurodegenerative disorder characterized by progressive muscle wasting and loss of muscle function due to severe motor neuron dysfunction. SMA is the leading genetic cause of infant mortality. SMA is caused by low levels of Survival of Motor Neuron (SMN) due to deletion or loss of function of SMN1 gene.
  • SSN Motor Neuron
  • spherical nucleic acids are contemplated.
  • a SNA comprises a core and an antisense oligonucleotide comprised of 8 to 50 linked nucleosides in length targeted to ISS-N1 site of Survival of Motor Neuron 2 (SMN2) pre-mRNA, and wherein the antisense oligonucleotide is attached to the core and forms an oligonucleotide shell.
  • SNA2 Motor Neuron 2
  • the core is a lipid bilayer containing core and the
  • oligonucleotide is attached to the lipid bilayer core.
  • the antisense oligonucleotide is 18 nucleotides in length.
  • the ISS-N1 site of the SMN2 pre-mRNA comprises a nucleic acid sequence of SEQ ID NO: 15.
  • the internucleoside linkages are phosphodiester. In some embodiments, the antisense oligonucleotide has phosphorothioate internucleoside linkages. In some embodiments, less than all of the intemucleoside linkages are
  • the antisense oligonucleotide has 2 ⁇ (2-methoxyethyl) modifications. In some embodiments, less than all of the nucleotides include a 2 ⁇ (2- methoxyethyl) modification. In some embodiments, the antisense oligonucleotide has LNA modifications. In some embodiments, less than all of the nucleotides include a LNA modification. In some embodiments, the antisense oligonucleotide has morpholino
  • nucleotides include a morpholino modification.
  • the antisense oligonucleotide has 2 ⁇ methyl modifications. In some embodiments, less than all of the nucleotides include a 2 ⁇ methyl modification.
  • the antisense oligonucleotide has 2 ⁇ (2-methoxyethyl) modifications. In some embodiments, less than all of the nucleotides include a 2 ⁇ (2- methoxyethyl) modification.
  • the antisense oligonucleotide is comprised of 18 to 21 linked nucleosides. In other embodiments the antisense oligonucleotide is comprised of 1 to 10, 8- 20, 8-30, 10-20, 10-30, 10-40, 15-20, 15-30, 15-40, 18-20, 18-25, 18-30, 18-35, 18-40, 18-45 or 18-50 linked nucleosides.
  • the antisense oligonucleotides of the oligonucleotide shell are directly attached to the lipid containing core. In some embodiments, the antisense
  • oligonucleotides of the oligonucleotide shell are indirectly attached to the lipid containing core through a linker.
  • the linker comprises a molecular species at the 3’ or 5’ termin of the oligonucleotide, wherein the molecular species is positioned in a core and the oligonucleotide extends radially from the core.
  • the molecular species is linked to the oligonucleotide at the 5’ end of the oligonucleotide.
  • the molecular species is a hydrophobic group.
  • the hydrophobic group is selected from the group consisting of cholesterol, a cholesteryl or modified cholesteryl residue, tocopherol, adamantine, dihydrotesterone, long chain alkyl, long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic, oleoyl-cholenic, decane, dodecane, docosahexaenoyl, palmityl, C6-palmityl, heptadecyl, myrisityl, arachidyl, stearyl, behenyl, linoleyl, bile acids, cholic acid or taurocholic acid, deoxycholate, oleyl litocholic acid,
  • the linker moiety comprises a non-nucleotidic linker moiety linked to the molecular species.
  • the non-nucleotidic linker moiety is selected from the group consisting of an abasic residue (dSpacer), oligoethyleneglycol, triethyleneglycol, hexaethyleneglycol, polyethylene glycol, alkane-diol, or butanediol.
  • the non-nucleotidic linker moiety is a double linker.
  • the double linker is two oligoethyleneglycols. In some embodiments, the two oligoethyleneglycols are triethyleneglycol. In some embodiments, the two oligoethyleneglycols are triethyleneglycol. In some embodiments, the two oligoethyleneglycols are triethyleneglycol. In some embodiments, the two oligoethyleneglycols are triethyleneglycol. In some embodiments, the two oligoethyleneglycols.
  • oligoethyleneglycols are hexaethyleneglycol.
  • the double linker is two alkane-diols. In some embodiments, the two alkane-diols are butanediol. In some
  • the double linker is linked in the center by a phosphodiester, phosphorothioate, methylphosphonate, or amide linkage.
  • the non-nucleotidic linker moiety is a triple linker.
  • the triple linker is three oligoethyleneglycols.
  • the three oligoethyleneglycols are triethyleneglycol.
  • the three oligoethyleneglycols are triethyleneglycol.
  • oligoethyleneglycols are hexaethyleneglycol.
  • the triple linker is three alkane-diols. In some embodiments, the three alkane-diols are butanediol. In some embodiments, the triple linker is linked in between each single linker by a phosphodiester, phosphorothioate, methylphosphonate, or amide linkage.
  • the antisense oligonucleotides comprise the entire SNA such that no other structural components are part of the nanostructure and wherein the
  • oligonucleotide includes a molecular species and non-nucleotidic linker moiety that form the core, with the oligonucleotides extending radially from the core.
  • the SNA is free of lipids, cell penetrating peptides, cationic peptides, polymers or solid cores.
  • oligonucleotide shell has a density of 5-1,000 oligonucleotides per SNA. In some embodiments, the oligonucleotide shell has a density of 100-1,000 oligonucleotides per SNA. In some embodiments, the oligonucleotide shell has a density of 500-1,000 oligonucleotides per SNA.
  • the lipid bilayer containing core is comprised of one or more lipids selected from: sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, lysophosphatid
  • sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, l4-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, l4-demethyl-l4-dehydrlanosterol, sitostanol, campesterol, ether anionic lipids, ether cationic lipids, lanthanide chelating lipids, A-ring substituted oxysterols, B-ring substituted oxysterols, D-ring substituted oxysterols, side-chain substituted oxysterols, double substituted oxy
  • the lipid bilayer-containing core is comprised of DOPC.
  • the ratio of number of oligonucleotide molecules to nm DOPC is 30:20.
  • a SNA decribed herein comprises an oligonucleotide that comprises or consists of
  • the method comprises administering to a subject having SMA a spherical nucleic acid (SNA) of any one of claims 1-42, in an effective amount to increase expression levels of SMN2 protein over a baseline level in the subject in order to treat the disorder.
  • SNA spherical nucleic acid
  • the baseline level is the level of SMN2 protein in the subject prior to treatment with the SNA. In some embodiments, the baseline level is the level of SMN2 protein in a subject having SMA and treated with a linear antisense oligonucleotide targeted to ISS-N1 site of SMN2 pre-mRNA.
  • the SNA is delivered by a route selected from the group consisting of intrathecal, oral, nasal, sublingual, intravenous, subcutaneous, mucosal, respiratory, direct injection, and dermally.
  • SMA spinal muscular atrophy
  • the method comprises administering to a subject having SMA at least two doses of a spherical nucleic acid (SNA), in an effective amount to increase expression levels of Survival of Motor Neuron 2 (SMN2) protein over a baseline level in the subject in order to treat the disorder, wherein the second dose is administered about 3 months to 2 years after the first dose, and wherein the SNA comprises a core and an antisense oligonucleotide comprised of 10 to 40 linked nucleosides in length targeted to a region of SMN2 pre-mRNA, such that a level of exon 7-containing SMN2 mRNA relative to exon 7- deleted SMN2 mRNA in the subject is enhanced, and wherein the oligonucleotides are attached to the core and thus form an oligonucleotide shell.
  • SNA spherical nucleic acid
  • SMN2 Motor Neuron 2
  • the method comprises contacting the cell with a spherical nucleic acid (SNA) comprising a core and an antisense oligonucleotide comprised of 10 to 40 linked nucleosides in length targeted to a region of SMN2 pre-mRNA, such that the level of exon 7-containing SMN2 mRNA relative to exon 7-deleted SMN2 mRNA in the cell is enhanced.
  • SNA spherical nucleic acid
  • the antisense oligonucleotide comprises a sequence which is complementary to a portion of intron 7 of the SMN2 gene or the SMN2 pre-mRNA.
  • a spherical nucleic acid (SNA) comprising a core and an antisense oligonucleotide comprised of 8 to 50 linked nucleosides in length targeted to a regulator of splicing of Survival of Motor Neuron 2 (SMN2) pre-mRNA, and wherein the antisense oligonucleotide is attached to the core and forms an oligonucleotide shell is contemplated herein.
  • SNA spherical nucleic acid
  • the regulator of splicing of SMN2 pre-mRNA regulates inclusion of exon 7 in the SMN2 mRNA.
  • the regulator of splicing of SMN2 pre-mRNA is an RNA binding protein.
  • the RNA binding protein is RBM10.
  • the regulator of splicing of SMN2 pre-mRNA is a
  • SR serine/arginine
  • hnRNP heterogeneous ribonucleoprotein
  • the SR splicing factor is SRSF1, SRSF2, SRSF3, SRSF4, SRSF5,
  • the hnRNP protein is hnRNPAl, hnRNP A2B1, hnRNP C or hnRNP U.
  • the regulator of splicing of SMN2 pre-mRNA is
  • HuR/ELAVLl HuR/ELAVLl, Puf60, Sam68, SF1, SON, U2AF35 or ZIS2/ZNF265.
  • the SNA has an average or number mean diameter (average or number mean diameter are used interchangeably herein) on the order of 10-100 nanometers.
  • the number mean diameter of the nanoparticle is from about 15 nm to about 100 nm, about 20 nm to about 100 nm, about 25 nm to about 100 nm, about 150 nm to about 50 nm, about 15 nm to about 50 nm, about 20 nm to about 50 nm, about 10 nm to about 70 nm, about 15 nm to about 70 nm about 20 nm to about 70 nm, about 10 nm to about 30 nm, about 15 nm to about 30 nm, about 20 nm to about 30 nm, about 10 nm to about 40 nm, about 15 nm to about 40 nm, about 20 nm to about 40 nm, about 10 nm to about 80 nm, about 15 nm to about 80 nm, or about 20 nm to about 80 nm in number mean diameter.
  • the invention is a spherical nucleic acid (SNA), comprising a core and a first antisense oligonucleotide comprised of 8 to 50 linked nucleosides in length targeted to a regulatory site of Survival of Motor Neuron 2 (SMN2) pre-mRNA, and a second antisense oligonucleotide comprised of 8 to 50 linked nucleosides in length targeted to a region of a lncRNA, and wherein the antisense oligonucleotides are attached to the core and form an oligonucleotide shell.
  • SNA spherical nucleic acid
  • the core in some embodiments has a minimal number mean diameter of about 8 nm, about 10 nm, or about 15 nm, or has a number mean diameter of about 10 nm to about 50 nm, about 20 nm to about 25 nm, or about 20 nm.
  • the core is a lipid bilayer containing core and the oligonucleotide is attached to the lipid bilayer core.
  • the lncRNA is SMN-AS1, GenBank accession # BC045789.1.
  • the second antisense oligonucleotide is selected from SEQ ID NO: 81 to SEQ ID NO: 160.
  • the second antisense oligonucleotide is selected from oligonucleotides having 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with oligonucleotides of SEQ ID NO: 81 to SEQ ID NO: 160.
  • the second antisense oligonucleotide has a 5-10-5 MOE gapmer design, wherein the central gap segment comprises of ten 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each.
  • Each nucleoside in the 5' wing segment and/or each nucleoside in the 3' wing segment may in some embodiments have a 2'-MOE modification.
  • the gapmers have mixed backbone, including phosphorothioate and phosphodiester internucleotide linkages.
  • one or more or all cytosine residues throughout each gapmer are 5- methylcytosines.
  • the invention is a method of increasing expression of full length SMN2 mRNA in a cell comprising contacting the cell with an SNA disclosed herein.
  • a method of increasing expression of full length SMN2 mRNA in a cell involves contacting the cell with a spherical nucleic acid (SNA) comprising a core and an antisense oligonucleotide comprised of 10 to 40 linked nucleosides in length targeted to a region of SMN2 pre-mRNA and another SNA comprising a core and an antisense oligonucleotide comprised of 10 to 40 linked nucleosides in length targeted to a region of SMN-AS1.
  • SNA spherical nucleic acid
  • FIGs. 1A-1B show the fold increase of SMN2 mRNA over SMN D7 mRNA following 48 hour treatment of SMA patient-derived fibroblasts with the compounds.
  • FIG. 1A Full-length SMN mRNA
  • FIG. 1B D7 SMN mRNA
  • FIGs. 2A-2B show SMN2 protein, mRNA detection and quantification (72 hours).
  • FIG. 2A Western blot showing total SMN protein and loading control GRP94.
  • FIG. 2B is a densitometric quantification of SMN western blot (solid bars) and qRT-PCR of full-length SMN mRNA (hashed bars) from identically treated wells.
  • FIGs. 3A-3B show Kaplan-Meier survival plots of SMA mice treated with a single intracerebro-ventricular (ICV) injection of SNA-ASO or linear ASO at 10, 20 or 30 pg doses at age P0 (post natal day 0). Finear represents linear ASO and SNA represents SNA-ASO.
  • FIG. 3A shows A7SMA mice treated with the 30pg dose Nusinersen-SNA had increased survival to a maximum of 82 days while scramble SNA has no effect on survival.
  • FIG. 3B shows that linear Nusinersen improved survival of D7 SMA mice to a maximum of 28 days.
  • FIGs. 4A-4B show increase in body weight of SMA mice treated with a single ICV injection of SNA-ASO at 10, 20 or 30 pg or linear ASO at 10 or 20 pg doses. Mice in 30 pg SNA-ASO group have not reached end point. Finear represents linear ASO and SNA represents SNA-ASO.
  • FIG. 4A shows that weights are similar in A7SMA mice treated with linear or Nusinersen-SNA treated mice.
  • FIG. 4B shows that weights are similar in A7SMA mice treated with morpholino to ISS-N1 or Nusinersen-SNA.
  • SNA ISS-N1 represents SNA-ASO.
  • SMA Spinal Muscular Atrophy
  • SMA is an autosomal recessive neurodegenerative disorder characterized by progressive muscle wasting and loss of muscle function due to severe motor neuron dysfunction. SMA is caused by low levels of Survival of Motor Neuron (SMN) due to deletion or loss of function of SMN 1 gene. Humans carry a second copy of SMN gene, SMN2. However, due to a mutation in exon 7, SMN2 exon 7 is inefficiently spliced producing a truncated protein SMNA7, which is unstable and only partially functional. While several additional splice isoforms are generated by alternative splicing of both SMN1 and SMN2, SMNA7 transcript appears to be the major isoform produced by SMN2.
  • SMN2 Due to the potential for SMN2 to produce full-length SMN protein, it has been the principal target for therapies designed to increase the production of functional SMN protein in SMA.
  • FDA Food and Drug Administration
  • splice modulating antisense oligonucleotides described herein are more potent when arranged in a SNA format. It was discovered that these splice modulating antisense oligonucleotides are more active in a SNA format relative to same linear splice modulating antisense oligonucleotides.
  • splice modulating antisense oligonucleotides comprised of a variety of lipid containing or other cores, oligonucleotide sequences, oligonucleotide lengths, and oligonucleotide densities are capable of enhancing the expression of a protein, whose low levels are associated with SMA.
  • the data presented herein show that having the splice modulating antisense oligonucleotide in a SNA structure enhanced the inclusion of an exon normally excluded from the SMN2 gene in SMA.
  • nusinersen is clinically administered to the CSF, the constructs were delivered to the CSF via intracerebral ventricular (ICV) injection in post-natal day 0 (P0) mice.
  • ICV intracerebral ventricular
  • Mice treated with 20pg of nusinersen had a median survival of 17 days, compared to 14 days in untreated mice.
  • lOpg of nusinersen-SNA increased median survival to 26 days whereas 20pg increased survival to 69 days.
  • Increasing the nusinersen dose to 30 pg resulted in toxicity and a median survival of 2 days.
  • nusinersen-SNA treatment resulted in substantially increased median survival over nusinersen at the same dose.
  • administration of nusinersen-SNA by ICV injection to the CSF at 30 pg dose did not lead to acute toxicity.
  • nusinersen-SNA treatment elicited more full length SMN mRNA compared to nusinersen.
  • the SNAs of the invention may improve the therapeutic window of existing splice modulating oligonucleotides and thus, may be used as novel therapies for CNS disorders.
  • the data show that the SNA of the invention demonstrated increased survival and decreased toxicity in a translationally-relevant SNA mouse model.
  • the data demonstrated prolonged survival by four-fold (maximal survival of 115 days compared to 28 days for nusinersen-treated mice), doubled the levels of healthy full-length SMN2 mRNA and protein in SMA patient fibroblasts when compared to nusinersen, doubled the quantity of healthy full-length SMN mRNA levels in spinal cord tissue compared to untreated mice and mitigated toxicity of nusinersen at the highest dose tested in mice.
  • Spherical nucleic acids are three-dimensional arrangements of nucleic acids, with densely packed and radially arranged oligonucleotides on a central nanoparticle core.
  • the SNA is composed of oligonucleotides and a core.
  • the core may be a hollow core which is produced by a 3-dminensional arrangement of molecules which form the outer boundary of the core.
  • the molecules may be in the form of a lipid layer or bilayer which has a hollow center.
  • the molecules may be in the form of lipids, such as amphipathic lipids, i.e., sterols which are linked to an end the oligonucleotide.
  • Sterols such as cholesterol linked to an end of an oligonucleotide may associate with one another and form the outer edge of a hollow core with the oligonucleotides radiating outward from the core.
  • the core may also be a solid or semi-solid core.
  • the oligonucleotides are associated with the core.
  • An oligonucleotide that is associated with the core may be covalently linked to the core or non-covalently linked to the core, i.e., potentially through hydrophobic interactions. For instance, when a sterol forms the outer edge of the core an oligonucleotide may be covalently linked to the sterol directly or indirectly.
  • the oligonucleotide may be covalently linked to the lipid or may be non-covalently linked to the lipids e.g., by interactions with the oligonucleotide or a molecule such as a cholesterol attached to the oligonucleotide directly or indirectly through a linker.
  • SNAs are taken up by cells to a greater extent than the same oligonucleotides that are not in the SNA format.
  • Nontoxic, biocompatible, and biodegradable lipid-containing SNAs that are useful for treating neurodegenerative diseases and disorders, such as spinal muscular atrophy (SMA) are disclosed herein.
  • Antisense technology is an effective means for modulating the expression of one or more specific gene products, including alternative splice products, and is uniquely useful in a number of therapeutic, diagnostic, and research applications.
  • the principle behind antisense technology is that an antisense compound, which hybridizes to a target nucleic acid, modulates gene expression activities such as transcription, splicing or translation through one of a number of antisense mechanisms.
  • the sequence specificity of antisense compounds makes them extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in disease.
  • antisense activity refers to any detectable and/or measurable change attributable to the hybridization of an antisense compound to its target nucleic acid related to splice modulating.
  • antisense activity is an increase in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid compared to target nucleic acid levels or target protein levels in the absence of the antisense compound.
  • antisense compound refers to a compound comprising a splice modulating antisense oligonucleotide in a spherical nucleic acid (SNA).
  • SNA spherical nucleic acid
  • antisense compound or“oligonucleotide” and“splice modulating compound” or “oligonucleotide” are used interchangeably to refer to a splice modulating oligonucleotide.
  • antisense oligonucleotide refers to an oligonucleotide having a nucleobase sequence that is at least partially complementary to a target nucleic acid. In some embodiments, the antisense oligonucleotide contains one or more additional features, or one or more additional modifications.
  • Splice-switching or splice modulating oligonucleotides direct pre-mRNA splicing by binding sequence elements and blocking access to the transcript by the spliceosome and other splicing factors. They can be applied to (1) restore correct splicing of an aberrantly spliced transcript, (2) produce a novel splice variant that is not normally expressed, or (3) manipulate alternative splicing from one splice variant to another. Through the latter mechanism, splice switching oligonucleotides may therefore downregulate a deleterious transcript while simultaneously upregulating expression of a preferred transcript. Notably, their activity is enhanced with increased target gene expression because this enables increased production of the preferred splice variant. This is in contrast to traditional anti-sense approaches and small- interfering RNA, which exhibit decreased potency with increased target gene expression.
  • an antisense oligonucleotide refers to an antisense
  • oligonucleotide that comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 below.
  • the antisense oligonucleotide refers to the nucleic acid sequence of ISIS 396443.
  • ISIS 396443 refers to an oligonucleotide having the following structure:
  • m C indicates 5-methyl cytosine;“e” indicates a 2'-MOE modification;“C” indicates cytidine,“T” indicates thymidine,“A” indicates adenosine,“G” indicates guanosine, and“s” indicates phosphorothioate linkage.
  • Isis 396443 is also referred to in the art as Nusinersen, which is the International Nonproprietary Name (INN), and as Ionis- SMNRx.
  • “MOE” refers to methoxyethyl.“2'-MOE” means a - OCH 2 CH 2 OCH 3 group at the 2’ position of a furanosyl ring.
  • the antisense oligonucleotide refers to an antisense
  • oligonucleotide that comprises or consists of the nucleic acid sequence of SEQ ID NO: 17 below.
  • each base of the antisense oligonucleotide of SEQ ID NO: 17 is modified with morpholino chemistry groups.
  • A“morpholino oligomer” or“PMO” refers to an oligonucleotide having a backbone which supports a nucleobase capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose Sugar backbone moiety, but instead contains a morpholino ring.
  • An exemplary“morpholino oligomer comprises morpholino subunit structures linked together by phosphoramidate or
  • each subunit comprising a purine or pyrimidine nucleobase effective to bind, by base-specific hydrogen bonding, to a base in a
  • Morpholino oligomers are detailed, for example, in U.S. Pat. Nos. 5,698,685: 5,217, 866; 5,142,047; 5,034,506; 5,166,315;
  • each base of the antisense oligonucleotide of SEQ ID NO: 17 is modified with locked nucleic acid (LNA), in which the 2'-hydroxyl group of the RNA is linked to the 4' carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.
  • LNA locked nucleic acid
  • the linkage is in certain aspects is a methylene (— CH2— )n group bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1, 2 or 3.
  • LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.
  • each base of the antisense oligonucleotide of SEQ ID NO: 17 is a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone. See, for example US Patent Nos. 5,539,082; 5,714,331; and 5,719,262, and Nielsen et ah, Science, 1991, 254, 1497-1500, the disclosures of which are herein incorporated by reference.
  • the present invention provides antisense compounds, which comprise or consist of an oligomeric compound comprising an antisense oligonucleotide, having a nucleobase sequences complementary to that of a target nucleic acid.
  • antisense compounds are single- stranded.
  • Such single- stranded antisense compounds typically comprise or consist of an oligomeric compound that comprises or consists of a modified oligonucleotide and optionally a conjugate group.
  • antisense compounds are double- stranded.
  • Such double-stranded antisense compounds comprise a first oligomeric compound having a region complementary to a target nucleic acid and a second oligomeric compound having a region complementary to the first oligomeric compound.
  • the first oligomeric compound of such double stranded antisense compounds typically comprises or consists of a modified oligonucleotide and optionally a conjugate group.
  • the oligonucleotide of the second oligomeric compound of such double - stranded antisense compound may be modified or unmodified. Either or both oligomeric compounds of a double- stranded antisense compound may comprise a conjugate group.
  • the oligomeric compounds of double-stranded antisense compounds may include non
  • oligomeric compounds of antisense compounds are capable of hybridizing to a target nucleic acid, resulting in at least one antisense activity.
  • antisense compounds selectively affect one or more target nucleic acid.
  • Such selective antisense compounds comprises a nucleobase sequence that hybridizes to one or more target nucleic acid, resulting in one or more desired antisense activity and does not hybridize to one or more non-target nucleic acid or does not hybridize to one or more non target nucleic acid in such a way that results in significant undesired antisense activity.
  • hybridization of an antisense compound to a target nucleic acid results in alteration of processing, e.g., splicing, of the target precursor transcript. In some embodiments, hybridization of an antisense compound to a target precursor transcript results in inhibition of a binding interaction between the target nucleic acid and a protein or other nucleic acid. In certain such embodiments, hybridization of an antisense compound to a target precursor transcript results in alteration of translation of the target nucleic acid.
  • Antisense activities may be observed directly or indirectly.
  • observation or detection of an antisense activity involves observation or detection of a change in an amount of a target nucleic acid or protein encoded by such target nucleic acid, a change in the ratio of splice variants of a nucleic acid or protein, and/or a phenotypic change in a cell or animal.
  • antisense compounds and/or oligomeric compounds comprise antisense oligonucleotides that are complementary to the target nucleic acid over the entire length of the oligonucleotide. In some embodiments, such oligonucleotides are 99% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 95% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 90% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 85% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 80% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 99% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 99% complementary to the target nucleic acid. In some embodiments, such oligonucleotides are 95% complementary to the target nucleic
  • antisense oligonucleotides are at least 80% complementary to the target nucleic acid over the entire length of the oligonucleotide and comprise a region that is 100% or fully complementary to a target nucleic acid.
  • the region of full complementarity is from 6 to 20 nucleobases in length. In certain such embodiments, the region of full complementarity is from 10 to 18 nucleobases in length. In certain such embodiments, the region of full complementarity is from 18 to 20 nucleobases in length.
  • oligomeric compounds and/or antisense compounds comprise one or more mismatched nucleobases relative to the target nucleic acid.
  • antisense activity against the target is reduced by such mismatch, but activity against a non-target is reduced by a greater amount.
  • selectivity of the antisense compound is improved.
  • the mismatch is specifically positioned within an oligonucleotide having a gapmer motif.
  • the mismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5'-end of the gap region.
  • the mismatch is at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3 '-end of the gap region.
  • the mismatch is at position 1, 2, 3, or 4 from the 5'-end of the wing region.
  • the mismatch is at position 4, 3, 2, or 1 from the 3'-end of the wing region.
  • oligomeric compounds comprise or consist of a modified oligonucleotide that is complementary to a target precursor transcript.
  • the target precursor transcript is a target pre-mRNA.
  • contacting a cell with a compound complementary to a target precursor transcript modulates processing of the target precursor transcript.
  • the resulting target processed transcript has a different nucleobase sequence than the target processed transcript that is produced in the absence of the compound.
  • the target precursor transcript is a target pre-mRNA and contacting a cell with a compound complementary to the target pre-mRNA modulates splicing of the target pre-mRNA.
  • the resulting target mRNA has a different nucleobase sequence than the target mRNA that is produced in the absence of the compound.
  • an exon is excluded from the target mRNA.
  • an exon is included in the target mRNA.
  • the exclusion or inclusion of an exon induces or prevents nonsense mediated decay of the target mRNA, removes or adds a premature termination codon from the target mRNA, and/or changes the reading frame of the target mRNA.
  • double- stranded antisense compound refers to an antisense compound comprising two oligomeric compounds that are complementary to each other and form a duplex, and wherein one of the two said oligomeric compounds comprises an antisense oligonucleotide.
  • hybridization refers to the pairing or annealing of complementary oligonucleotides and/or nucleic acids. While not limited to a particular mechanism, the most common mechanism of hybridization involves hydrogen bonding, which may be Watson- Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases.
  • inhibiting the expression or activity refers to a reduction or blockade of the expression or activity relative to the expression of activity in an untreated or control sample and does not necessarily indicate a total elimination of expression or activity.
  • “lower”,“reduced”,“reduction” or“decrease” or“inhibit” means a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (i.e. absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.
  • decrease or “inhibition” is used in the context of the level of expression or activity of a gene or a protein, it refers to a reduction in protein or nucleic acid level or activity in a cell, a cell extract, or a cell supernatant. For example, such a decrease may be due to reduced RNA stability,
  • “up-regulate”,“increase” or“higher” means an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or a 100% increase or more, or any increase between 10-100% as compared to a reference level, or an increase greater than 100%, for example, an increase at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and lO-fold or greater as compared to a reference level.
  • “increase” refers to a positive change in protein or nucleic acid level or activity in a cell, a cell extract, or a cell supernatant.
  • such an increase may be due to increased RNA stability, transcription, or translation, or decreased protein degradation.
  • this increase is at least 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 80%, at least about 100%, at least about 200%, or even about 500% or more over the level of expression or activity under control conditions.
  • oligonucleotide refers to a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified.
  • the length of an oligonucleotide described herein, such as an antisense oligonucleotide is of 2-500 linked nucleosides.
  • the length of an oligonucleotide described herein is of 2-200, 2-195, 2-190, 2- 185, 2-180, 2-175, 2-170, 2-165, 2-160, 2-155, 2-150, 2-145, 2-140, 2-135, 2-130, 2-125, 2- 120, 2-115, 2-110, 2-105, 2-100, 2-95, 2-90, 2-85, 2-80, 2-75, 2-70, 2-65, 2-60, 2-55, 2-50, 2- 45, 2-40, 2-39, 2-38, 2-37, 2-36, 2-35, 2-34, 2-33, 2-32, 2-31, 2-30, 2-29, 2-28, 2-27, 2-26, 2-
  • modified oligonucleotide means an oligonucleotide, wherein at least one nucleoside or intemucleoside linkage is modified.
  • unmodified oligonucleotide means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.
  • modified oligonucleotides having one or more modified sugar moieties at the 2’ position have enhanced pharmacologic activity for modulation of SMN2 pre-mRNA, including increasing the percentage of SMN2 transcripts containing exon 7.
  • mismatch or“non-complementary” means a nucleobase of a first oligonucleotide that is not complementary with the corresponding nucleobase of a second oligonucleotide or target nucleic acid when the first and second oligomeric compound are aligned.
  • naturally occurring means found in nature.
  • ameliorate in reference to a treatment improvement in at least one symptom relative to the same symptom in the absence of the treatment.
  • the treatment is of a neurodegenerative disorder described herein, such as treatment of spinal muscular atrophy (SMA).
  • SMA spinal muscular atrophy
  • amelioration is the reduction in the severity or frequency of a symptom or the delayed onset or slowing of progression in the severity or frequency of a symptom associated with a neurodegenerative disorder, such as SMA.
  • a“cell-targeting moiety” refers to a conjugate group or portion of a conjugate group that results in improved uptake to a particular cell type and/or distribution to a particular tissue relative to an oligomeric compound lacking the cell-targeting moiety.
  • “complementary” to an oligonucleotide described means that at least 70% of the nucleobases of such oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions.
  • Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another.
  • Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5 -methyl cytosine ( m C) and guanine (G).
  • oligonucleotides and/or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.
  • “fully complementary” or “100% complementary” in reference to an oligonucleotide described herein means that such oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.
  • internucleoside linkage refers to a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide.
  • a“modified intemucleoside linkage” refers to any intemucleoside linkage other than a naturally occurring, phosphate intemucleoside linkage or phosphodiester linkage. Non phosphate linkages are referred to herein as modified intemucleoside linkages.
  • the intemucleoside linkage is a phosphorothioate linkage.
  • “phosphorothioate linkage” refers to a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom.
  • a phosphorothioate intemucleoside linkage is a modified intemucleoside linkage.
  • all or 100% of the intemucleoside linkages of an antisense oligonucleotide described herein are phosphodiesters.
  • less than all or less than 100% of the intemucleoside linkages of an antisense oligonucleotide described herein are phosphodiester linkages.
  • 5-20%, 5-50%, 5-75%, 5-100%, 10-20%, 10-50%, 10-75% or 10-100% of the intemucleoside linkages are phosphodiester linkages. In some embodiments, 5-20%, 5-50%, 5-75%, 5-100%, 10-20%, 10-50%, 10-75% or 10-100% of the intemucleoside linkages of an antisense oligonucleotide described herein are phosphorothioate linkages.
  • phosphodiester intemucleoside linkage means a phosphate group that is covalently bonded to two adjacent nucleosides of a modified oligonucleotide.
  • an antisense oligonucleotide described herein is attached or inserted in to the surface of the lipid-containing core through conjugation to one or more linkers.
  • linkers contemplated herein include: tocopherols, sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2- hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as phosphatidylcholines, lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA,
  • a spherical nucleic acid (SNA) can be functionalized in order to attach a
  • polynucleotide Alternatively or additionally, the polynucleotide can be functionalized.
  • One mechanism for functionalization is the alkanethiol method, whereby oligonucleotides are functionalized with alkanethiols at their 3’ or 5’ termini prior to attachment to gold nanoparticles or nanoparticles comprising other metals, semiconductors or magnetic materials.
  • alkanethiol method whereby oligonucleotides are functionalized with alkanethiols at their 3’ or 5’ termini prior to attachment to gold nanoparticles or nanoparticles comprising other metals, semiconductors or magnetic materials.
  • Oligonucleotides can also be attached to nanoparticles using other functional groups such as phosophorothioate groups, as described in and incorporated by reference from US Patent No. 5,472,881, or substituted alkylsiloxanes, as described in and incorporated by reference from Burwell, Chemical Technology, 4, 370-377 (1974) and Matteucci and Caruthers, J. Am. Chem. Soc., 103, 3185-3191 (1981).
  • polynucleotides are attached to nanoparticles by terminating the polynucleotide with a 5’ or 3’ thionucleoside.
  • an aging process is used to attach polynucleotides to nanoparticles as described in and incorporated by reference from US Patent Nos. 6,361,944, 6,506, 569, 6,767,702 and 6,750,016 and PCT Publication Nos. WO 1998/004740, WO 2001/000876, WO 2001/051665 and WO 2001/073123.
  • the oligonucleotide is attached or inserted in the SNA.
  • a spacer sequence can be included between the attachment site and the oligonucleotide.
  • a spacer sequence comprises or consists of an oligonucleotide, a peptide, a polymer or an oligoethylene glycol.
  • precursor transcript means a coding or non-coding RNA that undergoes processing to form a processed or mature form of the transcript.
  • Precursor transcripts include but are not limited to pre-mRNAs, long non-coding RNAs, pri-miRNAs, and intronic RNAs.
  • processing in reference to a precursor transcript means the conversion of a precursor transcript to form the corresponding processed transcript.
  • Processing of a precursor transcript includes but is not limited to nuclease cleavage events at processing sites of the precursor transcript.
  • oligonucleotide and“nucleic acid” are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)).
  • a substituted pyrimidine e.g., cytosine (C), thymidine (T) or uracil (U)
  • a substituted purine e.g., adenine (A) or guanine (G)
  • oligonucleotides are also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base containing polymer.
  • Oligonucleotides can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).
  • a polynucleotide of the nanoscale construct and optionally attached to a nanoparticle core can be single stranded or double stranded.
  • a double stranded polynucleotide is also referred to herein as a duplex.
  • Double- stranded oligonucleotides of the invention can comprise two separate complementary nucleic acid strands.
  • duplex includes a double- stranded nucleic acid molecule(s) in which complementary sequences are hydrogen bonded to each other.
  • the complementary sequences can include a sense strand and an antisense strand.
  • the antisense nucleotide sequence can be identical or sufficiently identical to the target gene to mediate effective target gene inhibition (e.g., at least about 98% identical, 96% identical, 94%, 90% identical, 85% identical, or 80% identical) to the target gene sequence.
  • a double- stranded polynucleotide can be double-stranded over its entire length, meaning it has no overhanging single-stranded sequences and is thus blunt-ended.
  • the two strands of the double-stranded polynucleotide can have different lengths producing one or more single- stranded overhangs.
  • a double- stranded polynucleotide of the invention can contain mismatches and/or loops or bulges. In some embodiments, it is double-stranded over at least about 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the length of the oligonucleotide.
  • the double- stranded polynucleotide of the invention contains at least or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mismatches.
  • Polynucleotides associated with the invention can be modified such as at the sugar moiety, the phosphodiester linkage, and/or the base.
  • “sugar moieties” includes natural, unmodified sugars, including pentose, hexose, conformationally flexible sugars, conformationally locked sugars, arabinose, ribose and deoxyribose, modified sugars and sugar analogs. Modifications of sugar moieties can include replacement of a hydroxyl group with a halogen, a heteroatom, or an aliphatic group, and can include functionalization of the hydroxyl group as, for example, an ether, amine or thiol.
  • Modification of sugar moieties can include 2’-0-methyl nucleotides, which are referred to as“methylated.”
  • polynucleotides associated with the invention may only contain modified or unmodified sugar moieties, while in other instances, polynucleotides contain some sugar moieties that are modified and some that are not.
  • modified nucleomonomers include sugar- or backbone-modified ribonucleotides.
  • Modified ribonucleotides can contain a non-naturally occurring base such as uridines or cytidines modified at the 5’-position, e.g., 5’ -(2-amino )propyl uridine and 5’- bromo uridine; adenosines and guanosines modified at the 8-position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; and N-alkylated nucleotides, e.g., N6- methyl adenosine.
  • sugar- modified ribonucleotides can have the 2’ -OH group replaced by an H, alkoxy (or OR), R or alkyl, halogen, SH, SR, amino (such as NH2, NHR, NR2,), or CN group, wherein R is lower alkyl, alkenyl, or alkynyl.
  • modified ribonucleotides can have the phosphodiester group connecting to adjacent ribonucleotides replaced by a modified group, such as a phosphorothioate group.
  • 2'-0-methyl modifications can be beneficial for reducing undesirable cellular stress responses, such as the interferon response to double-stranded nucleic acids.
  • the sugar moiety can also be a hexose or arabinose.
  • alkyl includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups.
  • straight-chain alkyl groups e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl,
  • a straight chain or branched chain alkyl has 6 or fewer carbon atoms in its backbone (e.g., C1-C6 for straight chain, C3-C6 for branched chain), and more preferably 4 or fewer.
  • preferred cycloalkyls have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C1-C6 includes alkyl groups containing 1 to 6 carbon atoms.
  • alkyl includes both“unsubstituted alkyls” and “substituted alkyls,” the latter of which refers to alkyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and
  • alkylarylamino examples include alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
  • Cycloalkyls can be further substituted, e.g., with the substituents described above.
  • An“alkylaryl” or an“arylalkyl” moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)).
  • the term“alkyl” also includes the side chains of natural and unnatural amino acids.
  • the term“n-alkyl” means a straight chain (i.e., unbranched) unsubstituted alkyl group.
  • alkenyl includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond.
  • the term“alkenyl” includes straight-chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched- chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups.
  • a straight chain or branched chain alkenyl group has 6 or fewer carbon atoms in its backbone (e.g., C2- C6 for straight chain, C3-C6 for branched chain).
  • cycloalkenyl groups may have from 3-8 carbon atoms in their ring structure, and more preferably have 5 or 6 carbons in the ring structure.
  • C2-C6 includes alkenyl groups containing 2 to 6 carbon atoms.
  • alkenyl includes both“unsubstituted alkenyls” and“substituted alkenyls,” the latter of which refers to alkenyl moieties having independently selected substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • substituents can include, for example, alkyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,
  • aryloxycarbonyloxy carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl,
  • aminocarbonyl alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.
  • hydrophobic modifications refers to modification of bases such that overall hydrophobicity is increased and the base is still capable of forming close to regular Watson -Crick interactions.
  • base modifications include 5-position uridine and cytidine modifications like phenyl, 4-pyridyl, 2-pyridyl, indolyl, and isobutyl, phenyl (C6H50H); tryptophanyl (C8H6N)CH2CH(NH2)CO), Isobutyl, butyl, aminobenzyl; phenyl; naphthyl,
  • heteroatom includes atoms of any element other than carbon or hydrogen. In some embodiments, preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorus.
  • hydroxy or“hydroxyl” includes groups with an -OH or -O- (with an appropriate counterion).
  • halogen includes fluorine, bromine, chlorine, iodine, etc.
  • perhalogenated generally refers to a moiety wherein all hydrogens are replaced by halogen atoms.
  • substituted includes independently selected substituents which can be placed on the moiety and which allow the molecule to perform its intended function.
  • substituents include alkyl, alkenyl, alkynyl, aryl, (CR'R")0-3NR'R", (CR'R")0- 3CN, N02, halogen, (CR'R")0-3C(halogen)3, (CR'R")0-3CH(halogen)2, (CR'R")0- 3CH2(halogen), (CR'R")0-3CONR'R", (CR'R")0-3S(O)l-2NR'R", (CR'R")0-3CHO, (CR'R")0-30(CR'R")0-3H, (CR'R")0-3S(0)0-2R', (CR'R")0-30(CR'R")0-3H, (CR'R")0- 3COR', (CR'R")0-3CO2R', or (CR'R")0-3OR' groups; wherein each R' and R" are each independently hydrogen, a C1-C5 alkyl, C2-C5 alkenyl, C2-C5 alkynyl, or (CR
  • amine or“amino” includes compounds or moieties in which a nitrogen atom is covalently bonded to at least one carbon or heteroatom.
  • alkyl amino includes groups and compounds wherein the nitrogen is bound to at least one additional alkyl group.
  • dialkyl amino includes groups wherein the nitrogen atom is bound to at least two additional alkyl groups.
  • ether includes compounds or moieties which contain an oxygen bonded to two different carbon atoms or heteroatoms.
  • alkoxyalkyl refers to an alkyl, alkenyl, or alkynyl group covalently bonded to an oxygen atom which is covalently bonded to another alkyl group.
  • base includes the known purine and pyrimidine heterocyclic bases, deazapurines, and analogs (including heterocyclic substituted analogs, e.g., aminoethyoxy phenoxazine), derivatives (e.g., l-alkyl-, l-alkenyl-, heteroaromatic- and l-alkynyl derivatives) and tautomers thereof.
  • purines include adenine, guanine, inosine, diaminopurine, and xanthine and analogs (e.g., 8-oxo-N6-methyladenine or 7-diazaxanthine) and derivatives thereof.
  • Pyrimidines include, for example, thymine, uracil, and cytosine, and their analogs (e.g., 5-methylcytosine, 5-methyluracil, 5-(l-propynyl)uracil, 5-(l- propynyl)cytosine and 4,4-ethanocytosine).
  • suitable bases include non- purinyl and non-pyrimidinyl bases such as 2-aminopyridine and triazines.
  • the nucleomonomers of a polynucleotide of the invention are RNA nucleotides, including modified RNA nucleotides.
  • nucleoside includes bases which are covalently attached to a sugar moiety, preferably ribose or deoxyribose.
  • examples of preferred nucleosides include ribonucleosides and deoxyribonucleosides.
  • Nucleosides also include bases linked to amino acids or amino acid analogs which may comprise free carboxyl groups, free amino groups, or protecting groups. Suitable protecting groups are well known in the art (see P. G. M. Wuts and T. W. Greene,“Protective Groups in Organic Synthesis”, 2nd Ed., Wiley-Interscience, New York, 1999).
  • nucleotide includes nucleosides which further comprise a phosphate group or a phosphate analog.
  • linkage includes a naturally occurring, unmodified phosphodiester moiety (-0-(P02-)-0-) that covalently couples adjacent nucleoside monomers.
  • substitute linkage includes any analog or derivative of the native phosphodiester group that covalently couples adjacent nucleomonomers.
  • Substitute linkages include phosphodiester analogs, e.g., phosphorothioate,
  • phosphorodithioate and P-ethyoxyphosphodiester, P-ethoxyphosphodiester, P- alkyloxyphosphotriester, methylphosphonate, and nonphosphorus containing linkages, e.g., acetals and amides.
  • Such substitute linkages are known in the art (e.g., Bjergarde et al. 1991. Nucleic Acids Res. 19:5843; Caruthers et al. 1991. Nucleosides Nucleotides. 10:47).
  • non-hydrolysable linkages are preferred, such as phosphorothioate linkages.
  • polynucleotides of the invention comprise 3' and 5' termini (except for circular oligonucleotides).
  • the 3' and 5' termini of a polynucleotide can be substantially protected from nucleases, for example, by modifying the 3' or 5' linkages (e.g., U.S. Pat. No. 5,849,902 and WO 98/13526).
  • Oligonucleotides can be made resistant by the inclusion of a “blocking group.”
  • blocking group refers to substituents (e.g., other than OH groups) that can be attached to oligonucleotides or nucleomonomers, either as protecting groups or coupling groups for synthesis (e.g., FITC, propyl (CH2-CH2-CH3), glycol (-0-CH2-CH2-0-) phosphate (P032-), hydrogen phosphonate, or phosphoramidite).
  • Blocking groups also include“end blocking groups” or“exonuclease blocking groups” which protect the 5' and 3' termini of the oligonucleotide, including modified nucleotides and non-nucleotide exonuclease resistant structures.
  • Exemplary end-blocking groups include cap structures (e.g., a 7-methylguanosine cap), inverted nucleomonomers, e.g., with 3 '-3' or 5 '-5' end inversions (see, e.g., Ortiagao et al. 1992. Antisense Res. Dev. 2:129), methylphosphonate, phosphoramidite, non-nucleotide groups (e.g., non-nucleotide linkers, amino linkers, conjugates) and the like.
  • the 3' terminal nucleomonomer can comprise a modified sugar moiety.
  • the 3' terminal nucleomonomer comprises a 3'-0 that can optionally be substituted by a blocking group that prevents 3'- exonuclease degradation of the oligonucleotide.
  • the 3 '-hydroxyl can be esterified to a nucleotide through a 3' 3' internucleotide linkage.
  • the alkyloxy radical can be methoxy, ethoxy, or isopropoxy, and preferably, ethoxy.
  • the 3 ' 3 'linked nucleotide at the 3' terminus can be linked by a substitute linkage.
  • the 5' most 3' 5' linkage can be a modified linkage, e.g., a
  • the two 5' most 3' 5' linkages are modified linkages.
  • the 5' terminal hydroxy moiety can be esterified with a phosphorus containing moiety, e.g., phosphate, phosphorothioate, or P- ethoxypho sphate .
  • modified oligonucleotides comprise one or more nucleoside comprising an unmodified nucleobase. In some embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase.
  • modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines.
  • modified nucleobases are selected from: 2-aminopropyladenine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine, 2-prop yladenine , 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl (-CoC-CH3) uracil, 5- propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7- methylguanine, 7-
  • nucleobases include tricyclic pyrimidines, such as l,3-diazaphenoxazine-2-one, l,3-diazaphenothiazine-2-one and 9-(2-aminoethoxy)-l,3- diazaphenoxazine-2-one (G-clamp).
  • Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza- adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
  • Further nucleobases include those disclosed in Merigan et ah, U.S. 3,687,808, those disclosed in The Concise
  • oligonucleotides comprise modified and/or unmodified nucleobases arranged along the oligonucleotide or region thereof in a defined pattern or motif.
  • each nucleobase is modified. In some embodiments, none of the nucleobases are modified.
  • each purine or each pyrimidine is modified.
  • each adenine is modified.
  • each guanine is modified.
  • each thymine is modified.
  • each uracil is modified.
  • each cytosine is modified. In some embodiments, some or all of the cytosine nucleobases in a modified oligonucleotide are 5- methylcytosines.
  • modified oligonucleotides comprise a block of modified nucleobases.
  • the block is at the 3 '-end of the oligonucleotide.
  • the block is within 3 nucleosides of the 3 '-end of the oligonucleotide. In some embodiments, the block is at the 5'-end of the oligonucleotide. In certain embodiments,
  • the block is within 3 nucleosides of the 5 '-end of the oligonucleotide.
  • oligonucleotides having a gapmer motif comprise a nucleoside comprising a modified nucleobase.
  • one nucleoside comprising a modified nucleobase is in the central gap of an oligonucleotide having a gapmer motif.
  • the sugar moiety of said nucleoside is a 2'-deoxyribosyl moiety.
  • the modified nucleobase is selected from: a 2-thiopyrimidine and a 5- propynepyrimidine.
  • polynucleotides can comprise both DNA and RNA.
  • At least a portion of the contiguous polynucleotides are linked by a substitute linkage, e.g., a phosphorothioate linkage.
  • a substitute linkage e.g., a phosphorothioate linkage.
  • nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage.
  • the two main classes of intemucleoside linking groups are defined by the presence or absence of a phosphorus atom.
  • Modified intemucleoside linkages compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide.
  • intemucleoside linkages having a chiral atom can be prepared as a racemic mixture, or as separate enantiomers.
  • Representative chiral intemucleoside linkages include but are not limited to alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous -containing and non-phosphorous -containing intemucleoside linkages are well known to those skilled in the art.
  • Further neutral intemucleoside linkages include nonionic linkages comprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S.
  • Further neutral intemucleoside linkages include nonionic linkages comprising mixed N, O, S and CH2 component parts.
  • unmodified sugar moiety means a 2'-OH(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2'-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”).
  • Unmodified sugar moieties have one hydrogen at each of the , 3', and 4' positions, an oxygen at the 3' position, and two hydrogens at the 5' position.
  • modified sugar moiety or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate.
  • modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety.
  • a modified furanosyl sugar moiety is a 2'- substituted sugar moiety.
  • Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.
  • sugar surrogate means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an intemucleoside linkage, conjugate group, or terminal group in an oligonucleotide.
  • Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to
  • modified oligonucleotides comprise one or more modified nucleoside comprising a modified sugar. In some embodiments, modified oligonucleotides comprise one or more modified nucleosides comprising a modified nucleobase. In some embodiments, modified oligonucleotides comprise one or more modified intemucleoside linkage. In such embodiments, the modified, unmodified, and differently modified sugar moieties, nucleobases, and/or intemucleoside linkages of a modified oligonucleotide define a pattern or motif. In some embodiments, the patterns of sugar moieties, nucleobases, and intemucleoside linkages are each independent of one another. Thus, a modified
  • oligonucleotide may be described by its sugar motif, nucleobase motif and/or intemucleoside linkage motif (as used herein, nucleobase motif describes the modifications to the
  • nucleobases independent of the sequence of nucleobases.
  • oligonucleotides comprise one or more type of modified sugar and/or unmodified sugar moiety arranged along the oligonucleotide or region thereof in a defined pattern or sugar motif.
  • sugar motifs include but are not limited to any of the sugar modifications discussed herein.
  • modified oligonucleotides comprise or consist of a region having a gapmer motif, which comprises two external regions or "wings" and a central or internal region or "gap."
  • the three regions of a gapmer motif (the 5 '-wing, the gap, and the 3 '-wing) form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap.
  • the sugar moieties of the nucleosides of each wing that are closest to the gap are modified sugar moieties and differ from the sugar moieties of the neighboring gap nucleosides, which are unmodified sugar moieties, thus defining the boundary between the wings and the gap (i.e., the wing/gap junction).
  • the sugar moieties within the gap are the same as one another.
  • the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap.
  • the sugar motifs of the two wings are the same as one another (symmetric gapmer).
  • the sugar motif of the 5 '-wing differs from the sugar motif of the 3 '-wing (asymmetric gapmer).
  • the wings of a gapmer comprise 1-5 nucleosides. In some embodiments, the wings of a gapmer comprise 2-5 nucleosides. In some embodiments, the wings of a gapmer comprise 3-5 nucleosides. In some embodiments, the nucleosides of a gapmer are all modified nucleosides.
  • the gap of a gapmer comprises 7-12 nucleosides. In some embodiments, the gap of a gapmer comprises 7-10 nucleosides. In some embodiments, the gap of a gapmer comprises 8-10 nucleosides. In some embodiments, the gap of a gapmer comprises 10 nucleosides. In certain embodiment, each nucleoside of the gap of a gapmer is an unmodified 2'-deoxy nucleoside.
  • the gapmer is a deoxy gapmer.
  • the nucleosides on the gap side of each wing/gap junction are unmodified 2'-deoxy nucleosides and the nucleosides on the wing sides of each wing/gap junction are modified nucleosides.
  • each nucleoside of the gap is an unmodified 2 '-deoxy nucleoside.
  • each nucleoside of each wing is a modified nucleoside.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif.
  • each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety.
  • each nucleoside in the entire modified oligonucleotide comprises a modified sugar moiety.
  • modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif.
  • a fully modified oligonucleotide is a uniformly modified oligonucleotide.
  • each nucleoside of a uniformly modified oligonucleotide comprises the same 2 '-modification. In some embodiments, each nucleoside of a uniformly modified oligonucleotide comprises a 2'-0-(N-alkyl acetamide) group. In some embodiments, each nucleoside of a uniformly modified oligonucleotide comprises a 2'-0-(N-methyl acetamide) group.
  • the invention provides oligomeric compounds, which consist of an oligonucleotide (modified or unmodified) and optionally one or more conjugate groups and/or terminal groups.
  • Conjugate groups consist of one or more conjugate moiety and a conjugate linker which links the conjugate moiety to the oligonucleotide. Conjugate groups may be attached to either or both ends of an oligonucleotide and/or at any internal position. In some embodiments, conjugate groups are attached to the 2'-position of a nucleoside of a modified oligonucleotide. In some embodiments, conjugate groups that are attached to either or both ends of an oligonucleotide are terminal groups.
  • conjugate groups or terminal groups are attached at the 3' and/or 5 '-end of oligonucleotides. In certain such embodiments, conjugate groups (or terminal groups) are attached at the 3'-end of oligonucleotides. In some embodiments, conjugate groups are attached near the 3 '-end of oligonucleotides. In some embodiments, conjugate groups (or terminal groups) are attached at the 5 '-end of oligonucleotides. In some embodiments, conjugate groups are attached near the 5 '-end of oligonucleotides.
  • terminal groups include but are not limited to conjugate groups, capping groups, phosphate moieties, protecting groups, abasic nucleosides, modified or unmodified nucleosides, and two or more nucleosides that are independently modified or unmodified.
  • oligonucleotides are covalently attached to one or more conjugate groups.
  • conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, tissue distribution, cellular distribution, cellular uptake, charge and clearance.
  • conjugate groups impart a new property on the attached oligonucleotide, e.g. , fluorophores or reporter groups that enable detection of the oligonucleotide.
  • Certain conjugate groups and conjugate moieties have been described previously, for example: cholesterol moiety (Letsinger et ah, Proc. Natl. Acad. Sci.
  • a tocopherol group (Nishina et ah, Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et ah, Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g., WO2014/ 179620).
  • conjugate groups may be selected from any of a C22 alkyl, C20 alkyl, C16 alkyl, CIO alkyl, C21 alkyl, C19 alkyl, Cl 8 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, Cl 1 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, C5 alkyl, C22 alkenyl, C20 alkenyl, C16 alkenyl, CIO alkenyl, C21 alkenyl, C19 alkenyl, C18 alkenyl, C15 alkenyl, C14 alkenyl, C13 alkenyl, C12 alkenyl, Cl 1 alkenyl, C9 alkenyl, C8 alkenyl, C7 alkenyl, C6 alkenyl, or C5 alkenyl.
  • conjugate groups may be selected from any of C22 alkyl, C20 alkyl, C16 alkyl, CIO alkyl, C21 alkyl, C19 alkyl, Cl 8 alkyl, C15 alkyl, C14 alkyl, C13 alkyl, C12 alkyl, Cl 1 alkyl, C9 alkyl, C8 alkyl, C7 alkyl, C6 alkyl, and C5 alkyl, where the alkyl chain has one or more unsaturated bonds.
  • conjugate moieties include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine,
  • intercalators include, without limitation, intercalators, reporter molecules, polyamines, polyamides, peptides, carbohydrates (e.g., GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, lipophilic groups, phospholipids, biotin, phenazine,
  • phenanthridine anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores, and dyes.
  • a conjugate moiety comprises an active drug substance, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, ( ⁇ S)- (+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • an active drug substance for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fen-bufen, ketoprofen, ( ⁇ S)- (+)-pranoprofen, car
  • Antisense oligonucleotide SNAs are nanoscale constructs composed of: (1) a lipid- containing core, which is formed by arranging non-toxic carrier lipids into a small hollow structure, (2) a shell of oligonucleotides, which is formed by arranging oligonucleotides such that they point radially outwards from the core, and (3) optionally a hydrophobic (e.g. lipid) anchor group attached to either the 5’- or 3’-end of the oligonucleotide, depending on whether the oligonucleotides are arranged with the 5’- or 3’-end facing outward from the core.
  • the anchor drives the insertion into the liposome and to anchor the oligonucleotides to the lipid-containing core.
  • a liposomal core as used herein refers to a centrally located core compartment formed by a component of the lipids or phospholipids that form a lipid bilayer.
  • “Liposomes” are artificial, self closed vesicular structure of various sizes and structures, where one or several membranes encapsulate an aqueous core. Most typically liposome membranes are formed from lipid bilayers membranes, where the hydrophilic head groups are oriented towards the aqueous environment and the lipid chains are embedded in the lipophilic core. Liposomes can be formed as well from other amphiphilic monomeric and polymeric molecules, such as polymers, like block copolymers, or polypeptides.
  • Unilamellar vesicles are liposomes defined by a single membrane enclosing an aqueous space.
  • oligo- or multilamellar vesicles are built up of several membranes.
  • the membranes are roughly 4 nm thick and are composed of amphiphilic lipids, such as phospholipids, of natural or synthetic origin.
  • the membrane properties can be modified by the incorporation of other lipids such as sterols or cholic acid derivatives.
  • the lipid bilayer is composed of two layers of lipid molecules. Each lipid molecule in a layer is oriented substantially parallel to adjacent lipid bilayers, and two layers that form a bilayer have the polar ends of their molecules exposed to the aqueous phase and the non polar ends adjacent to each other.
  • the central aqueous region of the liposomal core may be empty or filled fully or partially with water, an aqueous emulsion, oligonucleotides, or other therapeutic or diagnostic agent.
  • the lipid-containing core can be constructed from a wide variety of lipids known to those in the art including but not limited to: sphingolipids such as sphingosine, sphingosine phosphate, methylated sphingosines and sphinganines, ceramides, ceramide phosphates, 1-0 acyl ceramides, dihydroceramides, 2-hydroxy ceramides, sphingomyelin, glycosylated sphingolipids, sulfatides, gangliosides, phosphosphingolipids, and phytosphingosines of various lengths and saturation states and their derivatives, phospholipids such as
  • phosphatidylcholines lysophosphatidylcholines, phosphatidic acids, lysophosphatidic acids, cyclic LPA, phosphatidylethanolamines, lysophosphatidylethanolamines,
  • phosphatidylglycerols phosphatidylglycerols, lysophosphatidylglycerols, phosphatidylserines,
  • lysophosphatidylserines phosphatidylinositols, inositol phosphates, LPI, cardiolipins, lysocardiolipins, bis(monoacylglycero) phosphates, (diacylglycero) phosphates, ether lipids, diphytanyl ether lipids, and plasmalogens of various lengths, saturation states, and their derivatives, sterols such as cholesterol, desmosterol, stigmasterol, lanosterol, lathosterol, diosgenin, sitosterol, zymosterol, zymostenol, l4-demethyl-lanosterol, cholesterol sulfate, DHEA, DHEA sulfate, l4-demethyl-l4-dehydrlanosterol, sitostanol, campesterol, ether anionic lipids, ether cationic lipids, lanthanide chel
  • the oligonucleotides may be positioned on the exterior of the core, within the walls of the core and/or in the center of the core.
  • An oligonucleotide that is positioned on the core is typically referred to as coupled to the core. Coupled may be direct or indirect. In some embodiments at least 5, 10, 15, 25, 50, 75, 100, 200, 300, 400, 500, 600,
  • oligonucleotides or any range combination thereof are on the exterior of the core.
  • 1-1000, 10-500, 50-250, or 50-300 oligonucleotides are present on the surface.
  • the oligonucleotides of the oligonucleotide shell may be oriented in a variety of directions. In some embodiments the oligonucleotides are oriented radially outwards. The orientation of these oligonucleotides can be either 5’ distal/3’ terminal in relation to the core, or 3’ distal/5’terminal in relation to the core, or laterally oriented around the core. In one embodiment one or a multiplicity of different oligonucleotides are present on the same surface of a single SNA. In all cases, at least 1 oligonucleotide is present on the surface but up to 10,000 can be present.
  • the oligonucleotides may be linked to the core or to one another and/or to other molecules such an active agents either directly or indirectly through a linker.
  • oligonucleotides may be conjugated to a linker via the 5’ end or the 3’ end. Some or all of the oligonucleotides of the nanostructure may be linked to one another or the core either directly or indirectly through a covalent or non-covalent linkage. The linkage of one oligonucleotide to another oligonucleotide may be in addition to or alternatively to the linkage of that oligonucleotide to liposomal core.
  • the oligonucleotide shell may be anchored to the surface of the core through one or multiple of linker molecules, including but not limited to: any chemical structure containing one or multiple thiols, such as the various chain length alkane thiols, cyclic dithiol, lipoic acid, or other thiol linkers known to those skilled in the art.
  • linker molecules including but not limited to: any chemical structure containing one or multiple thiols, such as the various chain length alkane thiols, cyclic dithiol, lipoic acid, or other thiol linkers known to those skilled in the art.
  • the exterior of the lipid-containing core has an oligonucleotide shell.
  • oligonucleotide shell can be constructed from a wide variety of nucleic acids including, but not limited to: single-stranded deoxyribonucleotides, ribonucleotides, and other single- stranded oligonucleotides incorporating one or a multiplicity of modifications known to those in the art, double- stranded deoxyribonucleotides, ribonucleotides, and other double-stranded oligonucleotides incorporating one or a multiplicity of modifications known to those in the art, oligonucleotide triplexes incorporating deoxyribonucleotides, ribonucleotides, or oligonucleotides that incorporate one or a multiplicity of modifications known to those in the art.
  • the SNAs described herein are constructed from
  • the surface density of the oligonucleotides may depend on the size and type of the core and on the length, sequence and concentration of the oligonucleotides.
  • a surface density adequate to make the nanoparticles stable and the conditions necessary to obtain it for a desired combination of nanoparticles and oligonucleotides can be determined empirically.
  • a surface density of at least 100 oligonucleotides per particle will be adequate to provide stable core-oligonucleotide conjugates.
  • the surface density is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 100, 200, 300, 400,
  • the surface density is 1-10,000, 1-9,000, 1-8,000, 1-7,000, 1-6,000, 1- 5,000, 1-4,000, 1-3,000, 1-2,000, 1-1,000, 5-10,000, 5-9,000, 5-8,000, 5-7,000, 5-6,000, 5- 5,000, 5-4,000, 5-3,000, 5-2,000, 5-1,000, 100-10,000, 100-9,000, 100-8,000, 100-7,000, 100-6,000, 100-5,000, 100-4,000, 100-3,000, 100-2,000,100-1,000, 500-10,000, 500-9,000, 500-8,000, 500-7,000, 500-6,000, 500-5,000, 500-4,000, 500-3,000, 500-2,000, 500-1,000, 10-10,000, 10-500, 50-10,000, 50-300, or 50-250 oligonucleotides per SNA.
  • the surface density is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,200,
  • the surface density is 1-10,000, 1-9,000, 1-8,000, 1-7,000, 1-6,000, 1-5,000, 1- 4,000, 1-3,000, 1-2,000, 1-1,000, 5-10,000, 5-9,000, 5-8,000, 5-7,000, 5-6,000, 5-5,000, 5- 4,000, 5-3,000, 5-2,000, 5-1,000, 100-10,000, 100-9,000, 100-8,000, 100-7,000, 100-6,000, 100-5,000, 100-4,000, 100-3,000, 100-2,000,100-1,000, 500-10,000, 500-9,000, 500-8,000, 500-7,000, 500-6,000, 500-5,000, 500-4,000, 500-3,000, 500-2,000, 500-1,000, 10-10,000, 10-500, 50-10,000, 50-300, or 50-250 oligonucleotides per 20 nm liposome.
  • a SNA described herein has an average or number mean diameter on the order of nanometers (i.e., between about 1 nm and about 1 micrometer).
  • the number mean diameter of the nanoparticle is from about 1 nm to about 250 nm in number mean diameter, about 1 nm to about 240 nm in number mean diameter, about 1 nm to about 230 nm in number mean diameter, about 1 nm to about 220 nm in number mean diameter, about 1 nm to about 210 nm in number mean diameter, about 1 nm to about 200 nm in number mean diameter, about 1 nm to about 190 nm in number mean diameter, about 1 nm to about 180 nm in number mean diameter, about 1 nm to about 170 ran in number mean diameter, about 1 nm to about 160 nm in number mean diameter, about 1 nm to about 150 nm in number mean diameter, about 1 nm to about 140 nm in number
  • a SNA described herein has a ratio of number of
  • oligonucleotide molecules to nm of lipid of 30:20 In some embodiments, the ration of number of oligonucleotide molecule to nm of lipid is 30:5, 30:10, 30:15, 30:20, 30:25, 1:1, 30:35, 30:40, 30:45, 30:50, 30:55, 1:2, 30:65, 30:70, 30:75, 30:80, 30:85, 1:3, 30:95, 30:100, 1:5, 30:200, or 30:300.
  • a SNA containing a first oligonucleotide, such as a first antisense oligonucleotide, described herein is co-administered with one or more
  • the second oligonucleotide is designed to treat the same disease, disorder, or condition as the first oligonucleotide described herein.
  • the first oligonucleotide e.g., first antisense oligonucleotide
  • the second oligonucleotide e.g., second antisense
  • the oligonucleotide are in the same SNA.
  • the first oligonucleotide is more abundant in the SNA than the second oligonucleotide.
  • the second oligonucleotide is more abundant in the SNA than the first oligonucleotide.
  • the SNA contains about the same amounts of the first oligonucleotide and the second oligonucleotide.
  • the first oligonucleotide affects a first region of the SMN2 pre-mRNA and the second oligonucleotide affects a second region of the SMN2 pre-mRNA.
  • the first region of the SMN2 pre-mRNA is ISS-N1.
  • the second region of the SMN2 pre-mRNA comprises the genetic region upstream of SMN2 exon 7 called Element 1 (El).
  • El Element 1
  • the nucleotide sequence for El corresponds to the nucleic acid sequence of SEQ ID NO: 10:
  • the first region or second region of the SMN2 gene is a 3’ splice site of exon 8, also known as ex8 3’ss.
  • the first region or second region of the SMN2 gene is ISS+100. (See e.g., Pao et al., Molecular Therapy (2014) 22(4):855-6l).
  • the first oligonucleotide is in a first SNA and the second oligonucleotide is in a second SNA.
  • a plurality of different oligonucleotides are in one SNA. In some embodiments, a plurality of different
  • oligonucleotides are in more than one SNA.
  • a SNA containing a first oligonucleotide, such as a first antisense oligonucleotide, described herein is co-administered with one or more secondary agents, such as a drug or compound.
  • one or more of secondary oligonucleotides or agents are co administered with the first oligonucleotide to produce a combinational effect.
  • second oligonucleotides are co-administered with the first oligonucleotide to produce a synergistic effect.
  • the co-administration of the first and second oligonucleotides permits use of lower dosages than would be required to achieve a therapeutic or prophylactic effect if the oligonucleotides were administered as independent therapy.
  • inclusion of exon 7 in the SMN2 pre-mRNA is achieved through targeting a regulator of SMN2 pre-mRNA splicing.
  • an oligonucleotide targeting a regulator of mRNA splicing such as an oligonucleotide that regulates exon 7 inclusion, is in a SNA described herein.
  • the oligonucleotide improves exon 7 inclusion in the SMN2 pre-mRNA through downregulation of an RNA binding protein.
  • the RNA binding protein is RBM10.
  • RBM10 is downregulated using an siRNA of SEQ ID NO: 18, targeting exon 7 or SEQ ID NO: 19, targeting exon 23:
  • the regulator of mRNA splicing is a serine/arginine (SR) splicing factor or a heterogeneous ribonucleoprotein (hnRNP) protein.
  • SR serine/arginine
  • hnRNP heterogeneous ribonucleoprotein
  • an oligonucleotide in an SNA described herein improves exon 7 inclusion in the SMN2 pre-mRNA through downregulation of an SR splicing factor or a hnRNP protein.
  • the SR splicing factor is SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7 or SRSF11.
  • SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7 or SRSF11. See e.g., Cartegni et al. American journal of human genetics (2006) 78:63-77; Kashi m a et al. Nature genetics (2003) 34:460-3; Young et al. (2002) Hum Mol Genet 11: 577-87; and Cartegni et al. Nat Genet (2002) 30: 377-84).
  • the hnRNP protein is hnRNPAl, hnRNP A2B1, hnRNP C or hnRNP FT.
  • the regulator of mRNA splicing is HuR/ELAVLl, Puf60, Sam68, SF1, SON, U2AF35 or ZIS2/ZNF265. (See e.g., Wee et al., PLoS ONE (2014) 9(l2):el 15205).
  • an oligonucleotide in an SNA described herein improves exon 7 inclusion in the SMN2 pre-mRNA through downregulation of
  • HuR/ELAVLl HuR/ELAVLl, Puf60, Sam68, SF1, SON, U2AF35 or ZIS2/ZNF265.
  • the regulator of mRNA splicing is targeted with one or more oligonucleotides, such as one or more of the siRNAs disclosed in Table 1 below. (See e.g., See e.g., Wee et al., PLoS ONE (2014) 9(l2):el 15205).
  • the one or more oligonucleotides are in one or more SNAs described herein.
  • an oligonucleotide targeting a regulator of mRNA splicing such as an oligonucleotide that regulates exon 7 inclusion, is in a SNA described herein.
  • an oligonucleotide targeting a regulator of mRNA splicing and one or more oligonucleotides targeting a region of the SMN2 pre-mRNA are in different SNAs.
  • an oligonucleotide targeting a regulator of mRNA splicing and one or more oligonucleotides targeting a region of the SMN2 pre-mRNA are in the same SNA.
  • the second oligonucleotide targets a long non-coding RNA (lncRNA), which results in an increase in SMN expression in vitro and in vivo.
  • the second oligonucleotide is an antisense oligonucleotide (traditional antisense) that targets a lncRNA by binding to the lncRNA, forming a duplex that is susceptible to RNAse H cleavage or siRNA that leads to RISC-catalyzed mRNA degradation.
  • the second oligonucleotide is siRNA that targets a lncRNA.
  • the lncRNA is SMN-AS1, GenBank accession # BC045789.1 (d’Ydewalle et al., 2017, Neuron 93, 66-79).
  • the second oligonucleotide is chosen from
  • the second oligonucleotide has a 5-10-5 MOE gapmer design, wherein the central gap segment comprises of ten 2'- deoxynucleosides and is flanked by wing segments on the 5' direction and the 3' direction comprising five nucleosides each. Each nucleoside in the 5' wing segment and/or each nucleoside in the 3' wing segment may have a 2'-MOE modification.
  • the intemucleoside linkages throughout each gapmer are
  • the gapmers have mixed backbone, including phosphorothioate and phosphodiester intemucleotide linkages.
  • one or more or all cytosine residues throughout each gapmer are 5- methylcytosines.
  • the first oligonucleotide and the second oligonucleotides are in the same SNA. In some embodiments, the first oligonucleotide and the second
  • oligonucleotide are in separate SNAs, where such SNAs can be administered as a mixture, or one SNA after the other.
  • the SNA contains more than two distinct oligonucleotides.
  • the SNA contains oligonucleotides that target more than two distinct targets.
  • a modification to one or more of the nucleotides of an oligonucleotide or antisense oligonucleotide described herein decreases or prevents RNAse H-catalyzed mRNA degradation.
  • the modification is a 2’- methoxyethyl (2’-MOE) modification, such as the 2’-MOE modification used in Spinraza (nusinersen).
  • the modification is a 2’-0-methyl modification.
  • other modifications such as modifications known to one of ordinary skill in the art, decrease or prevent RNAse H catalyzed mRNA degradation.
  • oligonucleotides or antisense oligonucleotides such as the
  • oligonucleotides or antisense oligonucleotides described herein, that are less prone or completely protected from RNAse H-catalyzed mRNA degradation are useful in therapy that modifies mRNA splicing.
  • the modification is used in combination with traditional antisense/siRNA therapy.
  • traditional antisense/siRNA therapy relates to RNAse H dependent cleavage of mRNA; traditional antisense/siRNA therapy is the RISC-catalyzed mRNA degradation.
  • exon modulation or splice modulation the aim is not to degrade the target mRNA. In some embodiments, only the splicing patterns are altered.
  • the present disclosure provides administration of a first SNA into the cerebrospinal fluid (CSF), in combination with systemic delivery of a second SNA.
  • Systemic administration and CSF administration can occur simultaneously, separately or sequentially.
  • a subject receives a first dose of a SNA in the CSF and subsequently receives a second dose of a SNA through a different route of administration.
  • a subject receives a first dose of a SNA in the CSF and subsequently receives a second dose of an antisense compound systemically.
  • the SNA administered into the CSF comprises the oligonucleotide of SEQ ID NO:l or SEQ ID NO: 16.
  • a target precursor transcript is associated with a disease or condition.
  • an oligomeric compound comprising or consisting of a modified oligonucleotide that is complementary to the target precursor transcript is used to treat the disease or condition.
  • the compound modulates processing of the target precursor transcript to produce a beneficial target processed transcript.
  • the disease or condition is associated with aberrant processing of a precursor transcript.
  • the disease or condition is associated with aberrant splicing of a pre-mRNA.
  • a SNA described herein is used for the treatment of a disease or disorder associated with a decrease in survival motor neuron (SMN) protein or a disease or disorder associated with a deletion of the SMN 1 gene that results in reduced or eliminated SMN protein expression.
  • SMA spinal muscular atrophy
  • SMA is a genetic disorder characterized by degeneration of spinal motor neurons. SMA is caused by the loss of both functional copies of the survival motor neuron 1 (SMN1) gene, which may also be known as SMN Telomeric, a protein that is part of a multi-protein complex thought to be involved in snRNP biogenesis and recycling.
  • SMN2 survival motor neuron
  • SMN 1 and SMN2 have the potential to code for the same protein
  • SMN2 contains a translationally silent mutation at position +6 of exon 7, which results in inefficient inclusion of exon 7 in SMN2 transcripts.
  • SMNA7 truncated version, lacking exon 7
  • SMN2 gene results in approximately 10-20% of the SMN protein and 80- 90% of the unstable/non-functional SMNA7 protein.
  • SMN protein plays a well-established role in assembly of the spliceosome and may also mediate mRNA trafficking in the axon and nerve terminus of neurons.
  • therapeutic compounds capable of modulating SMN2 splicing such that the percentage of SMN2 transcripts containing exon 7 is increased would be useful for the treatment of SMA.
  • SMA is caused by a reduction of the SMN protein.
  • SMA is caused by a mutation in the SMN 1 gene.
  • the type of SMA can be SMA1, SMA2, SMA3, SMA4, SMARD, SBMA, or DSMA.
  • SMA1 also known as Werdnig -Hoffmann disease
  • SMA1 is believed to be the most common form. It causes severe muscle weakness, which can result in problems moving, eating, breathing and swallowing. These symptoms are usually apparent at birth or during the first few months of life.
  • the muscles of babies with SMA1 are thin and weak. They're usually unable to raise their head or sit without support. Breathing problems can be caused by weakness in the baby's chest muscles, and difficulty swallowing can be made worse by weakness of the muscles in the tongue and throat. Because of the high risk of serious respiratory problems, most children with SMA1 die in the first few years of life.
  • SMA2 Symptoms of SMA2 usually appear when an infant is 7-18 months old. The symptoms are less severe than SMA1, but become more noticeable in older children. Infants with SMA2 are usually able to sit, but cannot stand or walk unaided. They may also have the following symptoms: breathing problems, weakness in their arms and, particularly, their legs, swallowing or feeding problems, and/or a slight tremor (shaking) of their fingers. In some cases, deformities of the hands, feet, chest and joints develop as the muscles shrink. As they grow, many children with SMA2 develop scoliosis. This is an abnormal curvature of the spine caused by the muscles supporting the bones of the spine becoming weaker. A child with SMA2 has weak respiratory muscles, which can make it difficult for them to cough effectively. This can make them more vulnerable to respiratory infections. Although SMA2 may shorten life expectancy, improvements in care standards mean most people can live long, fulfilling and productive lives. The majority of children with SMA2 are now expected to survive into adulthood.
  • SMA3 (also known as Kugelberg-Welander disease) is the mildest form of childhood SMA. Symptoms of muscle weakness usually appear after 18 months of age, but this is very variable and sometimes the symptoms may not appear until late childhood or early adulthood. Most children with SMA3 are able to stand unaided and walk, although many find walking or getting up from a sitting position difficult. They may also have: balance problems, difficulty walking, difficulty running or climbing steps, and /or a slight tremor (shaking) of their fingers. Over time, the muscles of children with SMA3 become weaker, resulting in some children losing the ability to walk when they get older. Breathing and swallowing difficulties are very rare and the condition doesn't usually affect life expectancy.
  • SMA4 is a less common form that begins in adulthood. The symptoms are usually mild to moderate, and may include: muscle weakness in the hands and feet, difficulty walking, and/or muscle tremor (shaking) and twitching. SMA4 doesn't affect life expectancy.
  • SMARD Spinal muscular atrophy with respiratory distress
  • Kennedy's syndrome or spinobulbar muscular atrophy (SBMA)
  • SBMA spinobulbar muscular atrophy
  • the initial symptoms of Kennedy's syndrome may include tremor (shaking) of the hands, muscle cramps on exertion, and/or muscle twitches and weakness of the limb muscles. As the condition progresses, it may cause other symptoms, including: weakness of the facial and tongue muscles, which may cause difficulty swallowing (dysphagia) and slurred speech, and/or recurring pneumonia (infection of lung tissue).
  • Some people with Kennedy's syndrome also develop enlarged male breasts (gynaecomastia), diabetes, and a low sperm count or infertility. Kennedy's syndrome doesn't usually affect life expectancy.
  • DSMA Distal spinal muscular atrophy
  • a SNA described herein is used for the treatment of a genetic disorder.
  • Non-liminting examples include achondroplasia, alpha- 1 antitrypsin deficiency, antiphospholipid syndrome, autism, autosomal dominant polycystic kidney disease, breast cancer, charcot-marie-tooth, colon cancer, cri du chat, crohn's disease, cystic fibrosis, dercum disease, down syndrome, duane syndrome, duchenne muscular dystrophy, factor v leiden, thrombophilia, familial hypercholesterolemia, familial mediterranean fever, fragile x syndrome, gaucher disease, hemochromatosis, hemophilia, holoprosencephaly, huntington's disease, klinefelter syndrome, marfan syndrome, myotonic dystrophy, neurofibromatosis, noonan syndrome, osteogenesis imperfecta, parkinson's disease, phenylketonuria, poland anomaly, porphyria, prog
  • aspects of the invention relate to delivery of SNAs to a subject for therapeutic and/or diagnostic use.
  • the SNAs may be administered alone or in any appropriate pharmaceutical carrier, such as a liquid, for example saline, or a powder, for administration in vivo.
  • the SNAs can also be co-delivered with larger carrier particles or within administration devices.
  • the SNAs may be formulated.
  • the formulations of the invention can be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. It should be appreciated that any method of delivery of SNAs known in the art may be compatible with aspects of the invention.
  • a“patient,”“individual,”“subject” or“host” refers to either a human, a nonhuman animal or a mammal.
  • the mammal is a vertebrate animal including but not limited to a dog, cat, horse, cow, pig, sheep, goat, turkey, chicken, primate, e.g., monkey, and fish (aquaculture species), e.g. salmon.
  • the invention can also be used to treat diseases or disorders in non-human subjects.
  • a SNA described herein is administered in one dose to treat a subject with SMA in an effective amount to increase expression levels of SMN over a baseline level in the subject in order to treat the disorder.
  • a baseline level is the level of SMN in the subject prior to treatment with a SNA described herein.
  • a subject having SMA is administered at least two doses of a SNA, in an effective amount to increase expression levels of SMN over a baseline level in the subject in order to treat the disorder.
  • the second dose is administered about 3 months, 6 months, 9 months, one year, 15 months, 18 months, 21 months or two years after the first dose.
  • “pharmaceutically acceptable carrier or diluent” refers to any substance suitable for use in administering to an animal. Certain such carriers enable pharmaceutical compositions to be formulated as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspension and lozenges for the oral ingestion by a subject.
  • a pharmaceutically acceptable carrier or diluent is sterile water; sterile saline; or sterile buffer solution.
  • “pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of compounds, such as oligomeric compounds, i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
  • “pharmaceutical composition” means a mixture of substances suitable for administering to a subject.
  • a pharmaceutical composition may comprise an antisense compound and a sterile aqueous solution.
  • a pharmaceutical composition shows activity in free uptake assay in certain cell lines.
  • an effective amount of the SNAs can be administered to a subject by any mode that delivers the SNAs to the desired cell.
  • Administering pharmaceutical compositions may be accomplished by any means known to the skilled artisan. Routes of administration include but are not limited to oral, parenteral, intramuscular, intravenous, subcutaneous, mucosal, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, dermal, rectal, and by direct injection.
  • an effective amount is used interchangeably with the term "therapeutically effective amount” and refers to the amount of at least one SNA described herein, at dosages and for periods of time necessary to achieve the desired therapeutic result, for example, to reduce or stop at least one symptom of SMA, for example a symptom of decreased muscle mass, known as muscle wasting, in the subject.
  • an effective amount using the methods as disclosed herein would be considered as the amount sufficient to reduce a symptom of SMA by at least 10%.
  • An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease.
  • the term “effective amount” or“therapeutically effective amount” as used herein refers to the amount of a pharmaceutical composition described herein to alleviate at least one symptom of SMA.
  • “therapeutically effective amount” of an antisense oligonucleotide SNA as disclosed herein is the amount of SNA which exerts a beneficial effect on, for example, the symptoms of SMA.
  • the dosage administered, as single or multiple doses, to an individual will vary depending upon a variety of factors, including pharmacokinetic properties of the muscarinic acetylcholine receptor inhibitor, the route of administration, conditions and characteristics (sex, age, body weight, health, size) of subjects, extent of symptoms, concurrent treatments, frequency of treatment and the effect desired.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects.
  • the effective amount in each individual case can be determined empirically by a skilled artisan according to established methods in the art and without undue experimentation. In general, the phrases
  • terapéuticaally-effective and “effective for the treatment, prevention, or inhibition”, are intended to qualify the antisense oligonucleotide SNA as disclosed herein which will achieve the goal of reduction in the severity of at least one symptom of SMA.
  • parenteral administration and“administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracap sular, intraorbital, intracardiac, intradermal,
  • systemic administration means the administration of a pharmaceutical composition comprising at least an muscarinic acetylcholine receptor inhibitor as disclosed herein such that it enters the animal's system and, thus, is subject to metabolism and other like processes, for example,
  • a SNA describe herein is administered to a cell in vitro or is administered to a subject in order for the SNA to come into contact with a cell of the subject in vivo.
  • a cell contemplated herein include a fibroblast, epithelial, endothelial, neuronal, adipose, cardiac, skeletal muscle, immune cells, hepatic, splenic, lung, circulating blood, gastrointestinal, renal, bone marrow, or pancreatic cell.
  • the differentiated cell can be a primary cell isolated from any somatic tissue including, but not limited to brain, liver, lung, gut, stomach, intestine, fat, muscle, uterus, skin, spleen, endocrine organ, bone, etc.
  • the terms“treat,”“treatment,”“treating,” or“amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder.
  • the term“treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with SMA.
  • Treatment is generally“effective” if one or more symptoms or clinical markers are reduced.
  • treatment is "effective” if the progression of a disease is reduced or halted. That is,“treatment” includes not just the improvement of symptoms or markers, but can also include a cessation or at least slowing of progress or worsening of symptoms that would be expected in absence of treatment.
  • Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s) of a malignant disease, diminishment of extent of a malignant disease, stabilized (i.e., not worsening) state of a malignant disease, delay or slowing of progression of a malignant disease, amelioration or palliation of the malignant disease state, and remission (whether partial or total), whether detectable or undetectable.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • the terms“significantly different than,”“statistically significant,” and similar phrases refer to comparisons between data or other measurements, wherein the differences between two compared individuals or groups are evidently or reasonably different to the trained observer, or statistically significant (if the phrase includes the term“statistically” or if there is some indication of statistical test, such as a p-value, or if the data, when analyzed, produce a statistical difference by standard statistical tests known in the art).
  • Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. , for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dosage can vary depending upon the dosage form employed and the route of administration utilized.
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i. e. , the concentration of the active ingredient, which achieves a half- maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography.
  • the effects of any particular dosage can be monitored by a suitable bioassay, e.g., synaptic function. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • “ameliorates symptoms and/or defects” is improving any defect or symptom associated with SMA. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique.
  • Non-limiting examples of SMA symptom management include (1) Orthopaedic treatment. Weak spine muscles may lead to development of kyphosis, scoliosis and other orthopaedic problems. Spine fusion is sometimes performed in people with SMA1 and SMA2 once they reach the age of 8-10 to relieve the pressure of a deformed spine on the lungs. People with SMA might also benefit greatly from various forms of physiotherapy and occupational therapy.
  • Orthotic devices can be used to support the body and to aid walking. For example, orthotics such as AFO's (ankle foot orthosis) are used to stabilize the foot and to aid gait, TLSO's (thoracic lumbar sacral orthosis) are used to stabilize the torso.
  • Assistive technologies may help in managing movement and daily activity, and greatly increase the quality of life.
  • Respiratory care and treatment Respiratory system requires utmost attention in SMA as once weakened it never fully recovers. Weakened pulmonary muscles in people with SMA1 and SMA2 can make breathing more difficult and pose a risk of hypoxiation, especially in sleep when muscles are more relaxed. Impaired cough reflex poses a constant risk of respiratory infection and pneumonia. Non-invasive ventilation (BiPAP) is frequently used and tracheostomy may be sometimes performed in more severe cases; both methods of ventilation prolong survival in a comparable degree, although tracheostomy prevents speech development. (4) Nutritional therapy.
  • kits typically defines a package or an assembly including one or more of the compositions of the invention, and/or other compositions associated with the invention, for example, as previously described.
  • Each of the compositions of the kit if present, may be provided in liquid form (e.g., in solution), or in solid form (e.g., a dried powder).
  • some of the compositions may be constitutable or otherwise processable (e.g., to an active form), for example, by the addition of a suitable solvent or other species, which may or may not be provided with the kit.
  • a kit associated with the invention includes one or more lipid cores.
  • a kit can also include one or more oligonucleotides.
  • a kit can also include one or more anchors or linkers.
  • a kit of the invention may, in some cases, include instructions in any form that are provided in connection with the compositions of the invention in such a manner that one of ordinary skill in the art would recognize that the instructions are to be associated with the compositions of the invention.
  • the instructions may include instructions for the use, modification, mixing, diluting, preserving, administering, assembly, storage, packaging, and/or preparation of the compositions and/or other compositions associated with the kit.
  • the instructions may also include instructions for the use of the compositions, for example, for a particular use, e.g., to a sample.
  • the instructions may be provided in any form recognizable by one of ordinary skill in the art as a suitable vehicle for containing such instructions, for example, written or published, verbal, audible (e.g., telephonic), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communications (including Internet or web-based communications), provided in any manner.
  • verbal e.g., telephonic
  • digital e.g., optical, visual
  • visual e.g., videotape, DVD, etc.
  • electronic communications including Internet or web-based communications
  • the present invention is directed to methods of promoting one or more embodiments of the invention as discussed herein.
  • “promoting” includes all methods of doing business including, but not limited to, methods of selling, advertising, assigning, licensing, contracting, instructing, educating, researching, importing, exporting, negotiating, financing, loaning, trading, vending, reselling, distributing, repairing, replacing, insuring, suing, patenting, or the like that are associated with the systems, devices, apparatuses, articles, methods, compositions, kits, etc. of the invention as discussed herein.
  • Methods of promotion can be performed by any party including, but not limited to, personal parties, businesses (public or private), partnerships, corporations, trusts, contractual or sub- contractual agencies, educational institutions such as colleges and universities, research institutions, hospitals or other clinical institutions, governmental agencies, etc.
  • Promotional activities may include communications of any form (e.g., written, oral, and/or electronic communications, such as, but not limited to, e-mail, telephonic, Internet, Web-based, etc.) that are clearly associated with the invention.
  • the method of promotion may involve one or more instructions.
  • instructions can define a component of instructional utility (e.g., directions, guides, warnings, labels, notes, FAQs or“frequently asked questions,” etc.), and typically involve written instructions on or associated with the invention and/or with the packaging of the invention. Instructions can also include instructional communications in any form (e.g., oral, electronic, audible, digital, optical, visual, etc.), provided in any manner such that a user will clearly recognize that the instructions are to be associated with the invention, e.g., as discussed herein.
  • instructional utility e.g., directions, guides, warnings, labels, notes, FAQs or“frequently asked questions,” etc.
  • Instructions can also include instructional communications in any form (e.g., oral, electronic, audible, digital, optical, visual, etc.), provided in any manner such that a user will clearly recognize that the instructions are to be associated with the invention, e.g., as discussed herein.
  • genomic nucleic acid sequence, pre-mRNA nucleic acid sequence, mRNA nucleic acid sequence and amino acid sequence of SMN2 are well known to one of ordinary skill in the art. Non-limiting examples include:
  • CAAGACTCCG TCTCAAATAA ATAAATAAAT AAATAAATAA ATAATAAAAA
  • TCACGCCTGT AATCCCAGCA CTTTGGGAGA CTGAGGTGGG TGGATTACCT GAGGTCAGGA
  • CTGTAATCCC AGCACTTTGG GAGGCCGAGT TGGGCAGATC ACAAGGTCAG GAGTTCGAGA
  • AAAAAAAAAA AAAAAAAAAAAG TGGGAGGATC AATGTACTGC CAGTCCTAAT GAAGTGGAAT
  • CTCTCAAGTA GCTGGAACTA CAGGTGCTGA CCACCATGCC TGGCTACTTT TTGTCAGGAT
  • CAGCCTCCAC CTCCTGCGCT CAGTCTTCTT GTCTCAGCCT TCTGAGTAGC TGAAATTACG
  • GGCATGTCCA CCATGGTGGC TCCACCTCCC CTTATTTAGC ACATGCACAA TAGGAAAGAG
  • AACTTTGGCA AAATAAACTT TCTAAATTGA TTGAAACCTG TCTTAGCTAC TTCTGGTTTA
  • CAGTCTTAAA GTTAGATAAT GTAAATTGTC CAGCTTTGGT TTATTTTTGT CCTTAGTAGT
  • CAAGTTTCCC TGGTCATATC TTGGTTGGAT GAAGCGTATT TTCAATAGAT TACCCTGGAA
  • CTAATCACTA GACCACCAGG AAGATTGTTT GTTTTGTTTT GTTTTTATTC TTCAGGGACC
  • GCCTCCCCCC TACCCCCCTT TTTTTCTTTC GAGACAGAGA TTATAGGTGT GAGCCACTGG
  • GACTATAGGC ATGTGCCACC ATGCCCAGCT AAATTTGGTT TTTTTGTTTG TTTGTTTTTG
  • AAATTGGTAC CAGGAAAGCA GGAAAGGGAA ATGGAAGTAA AAAAATAATAATAATAATAATAA
  • AAATCTACTC ATGGTATGTG GATAGGAATT
  • AAATCAGGTG ATATCCTCCT TTAGAAGTTG ATAGCCTATA TATGTCATCC TTTGTGGAGG
  • CAACTTCAAA AACAACTATT AAATTTTCTG TTATTTAGGA ACATGCATAT TAGTCATGAA
  • CTCCACCACC CCCCATGCCA GGGCCAAGAC TGGGACCAGG AAAGGTAAAC CTTCTATGAA
  • AATAAGAACA CATTATTTAC ATCTAATATA GATAAAATCC TGAGGCGCTC TCAGATTGTT
  • AAATCAACTC T AAAAA GATT TTTATTATAG GTTAGATTAT GTCATGGAAC CTTAAGGCTT
  • CTGTAATCCC GGCATTTTAG AAGGCTGAGG CAGGAGGATC ACTTGAGCTC AGGAGTTTGA GACCAGTCTG GGCAACATAG CAAGACCTCG TCTTTGTTTA G G G G AAAAAA AAGAAATTTA AGTAGGAGAT TATATAAGCA AAAATACAAT TAATTTCCAG CATTCACTAT ATAATATAAA TCTCCAGACT TTACTTTTTT GTTTACTGGA TATAAACAAT ATCTTTTTCT GTCTCCAGAT AATTCCCCCA CCACCTCCCA TATGTCCAGA TTCTCTTGAT GATGCTGATG CTTTGGGAAG TATGTTAATT TCATGGTACA TGAGTGGCTA TCATACTGGC TATTATATGG TAAGTAATCA CTCAGCATCT TTTCCTGACA ATTTTTTTGT AGTTATGTGA CTTTGTTTTG TAAATTTATA AAATACTACT TGCTTCTCTC TTTATATTAC TAAAAAATAA AAA T AAAAAA ATACAACTGT CTGAGG
  • GAAGAGAGAC AGAGGACATT TGGGCTAGAT CTGACAAGAA AAACAAATGT TTTAGTATTA
  • GTAGGCATGA GCCACTGCAA GAAAACCTTA ACTGCAGCCT AATAATTGTT TTCTTTGGGA
  • GCAAAAUGUU ACAGAAUCUA ACUGGUGGAC AUGGCUGUUC AUUGUACUGU UUUUUUCUAU
  • GCCATCTGCA CAGAGCGTGA ATCTCTGGCA TCCCCCACCC CAACCTTTTA TTATGCAGGC
  • SMS pre-mRNA refers to an RNA sequence, including all exons, introns, and untranslated regions, transcribed from DNA encoding human SMN2.
  • “intronic splicing silencer Nl” or“ISS-N1” refers to an intronic splice silencing domain in intron 7 of the SMN2 gene or pre-mRNA (see e.g., Singh et ah, Mol Cell Biol (2006) 26(4): 1333-46). Splicing of a critical exon of human Survival Motor Neuron is regulated by a unique silencer element located in the last intron.
  • ISS-N1 comprises the nucleic acid sequence:
  • the SMN2 pre-mRNA is targeted with one or more of the exemplary oligonucleotides disclosed in Tables 2-6 below in one or more SNAs. Unless indicated otherwise, the sequences contain phosphodiester internucleotide linkages.
  • percent identical refers to sequence identity between two amino acid sequences or between two nucleotide sequences. Identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position.
  • Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. Expression as a percentage of homology, similarity, or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • Various alignment algorithms and/or programs may be used, including FASTA, BLAST, or ENTREZ. FASTA and BLAST are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default settings.
  • the percent identity of two sequences can be determined by the GCG program with a gap weight of 1, e.g., each amino acid gap is weighted as if it were a single amino acid or nucleotide mismatch between the two sequences.
  • SMN2-targeted SNA Increases Expression of SMN2 mRNA and Protein for
  • SNAs have been developed targeting mRNA for down regulation of gene expression and TLR9 protein to activate the immune system.
  • Antisense SNAs for dermal diseases and TLR9 agonist SNAs for immuno -oncology applications are in clinical development.
  • a linear and a SNA version of Spinraza were compared for their effect on the inclusion of exon 7 in SMN2 mRNA in SMA patient-derived fibroblasts. The results show that in patient-derived fibroblasts, SNA version of Spinraza yields greater expression of exon 7 included SMN2 mRNA and protein compared with the linear version of Spinraza currently used to treat SMA patients.
  • Linear oligonucleotides linear ASO
  • oligonucleotides via two hexaethyleneglycol (spacer 18) moieties for SNA were synthesized with 2’-methoxyethyl (2’-MOE) and phosphorothioate (PS) backbone modification.
  • the oligonucleotide sequence is the same as that of Spinraza.
  • SNAs (SNA-ASO) were prepared by loading 3’-cholesterol attached oligonucleotides onto DOPC liposomes at a ratio of 30 oligonucleotide molecules per 20 nm liposome.
  • Oligo sequence 5 ' - TCA CTT TCA TAA TGC TGG - ( Spacer 18 ) 2 - 3 ChoiTEG ( SEQ ID NO : 1 )
  • SMA patient fibroblast cells (GM03813C, GM09677C and GM00232D) were obtained from Coriell Institute for Medical Research. Cells were cultured in DMEM medium containing 10% FBS and 100 U/ml penicillin and 100 pg/ml streptomycin. Linear and SNA ASOs were added to the cell cultures without transfecting agents and incubated for 48 hours or 72 hours. Then the cells were collected at 48 hours for mRNA extraction and at 72 hours for protein isolation. The levels of SMN2 mRNA, SMNA7 mRNA, and total SMN mRNAs were measured by qPCR using the following set of probes and primers.
  • SMN2 mRNA and SMNA7 mRNA primers were obtained from IDT and the probes were from Thermo Fisher Scientific, and the commercially available primers and probes for total SMN mRNA were purchased from Life Technologies (cat # Hs00l65806_ml).
  • SMN2 mRNA forward primer
  • SMNA7 mRNA reverse primer 5 ' - ATGCCAGCATTT CCATATAATAGCC-3 ' ( SEQ ID NO : 6 ) and SMNA7 mRNA probe: 5 ' - 6 FAM-TACATGAGTGGCTATCATACT-MGBNFQ-3 ' ( SEQ ID NO : 7 ) .
  • the levels of SMN2 protein were measured by Western blotting using SMN antibody obtained from BD Biosciences (cat # 610646) and the control GRP94 protein by the Grp94 (9G10) antibody obtained from Enzo Lifesciences (cat # ADI-SPA-850).
  • the fold increase of SMN2 mRNA over SMNA7 mRNA was calculated by dividing the values of % SMN2 mRNA expression with % SMNA7 mRNA expression.
  • ASO-SNAs and Linear ASOs targeting ISS-N1 site of the SMN2 mRNA were tested at various concentrations in three different SMA patient-derived fibroblasts.
  • phenylbutyrate (PBA, a known small molecule compound, positive control) and negative controls (control SNA and control linear) were included in the assays for comparison.
  • Fig. 1A Full-length SMN mRNA
  • 1B D7 SMN mRNA
  • pPBA not shown
  • the results showed that ASO-SNA treatment led to greater inclusion of exon 7 in SMN2 mRNA compared with linear ASO.
  • ASO-SNA treatment resulted in up to 45-fold increase in the inclusion of exon 7 over SMNA7 mRNA depending on the source of fibroblasts.
  • linear ASO resulted in about 2.5-fold higher inclusion of exon 7 over SMNA7 mRNA.
  • SMN2 protein upregulation was measured by ASO-SNA and linear ASO at 72 hours by Western blotting.
  • GM09677C were treated with SNAs for 72 hours and, then assessed by western blot and qRT-PCR.
  • ASO-SNA treatment resulted in greater expression of SMN2 protein compared with linear ASO in GM09677C (Fig. 2A and 2B), which is consistent with the results from the mRNA levels from above.
  • Fig. 2A Western blot showing total SMN protein and loading control GRP94.
  • GRP94 protein loading control was detected with ADI-SPA-850-F (Enzo Life Sciences).
  • SMN was detected with VMA00249 (Bio-Rad).
  • 2B is a densitometric quantification of SMN western blot (solid bars) and qRT-PCR of full-length SMN mRNA (hashed bars) from identically treated wells.
  • SMN qRT-PCR was performed on SMA patient fibroblasts (GM09677C) that were plated in 96-well plates and treated in triplicate with SNAs in complete media. After cell lysis, cDNA was derived from extracted RNA and assessed by qRT-PCR with technical duplicates for each sample. Full- length SMN2 was measured relative to GAPDH.
  • ASO-SNA treatment of SMA patient-derived fibroblasts facilitates increased of exon 7 inclusion and SMN2 protein expression compared with the same sequence of linear ASO (Spinraza).
  • SNA linear ASO
  • Previous studies have shown that oligonucleotides in SNA format are taken up by cells to a greater extent than linear oligonucleotides and function as potent antisense agents at mRNA level in the cytoplasm to down regulate gene expression.
  • the current results are the first demonstration of SNAs interacting with pre-mRNA in the nucleus facilitating exon 7 inclusion in SMN2 mRNA in SMA patient-derived fibroblasts.
  • SNA Spherical Nucleic Acid
  • Example 1 Based on the data presented in Example 1, the constructs were tested in vivo in a mouse model to evaluate the potency of SMN2-targeted SNA in comparison with a linear MOE-ASO. Tolerability of SNA compounds can be evaluated by intrathecal (IT) or intracerebroventricular injection (ICY). Spinraza is administered to patients using IT administration so one ideal comparison will involve IT administration in mouse models. It would be a great improvement to be able to deliver the therapeutic SNA into central nervous system using other administration modalities, such subcutaneous, intramuscular, intravenous, oral, ophthalmic, topical delivery in the ear, such as ear drops or similar forms, transtympanic administration, etc.
  • IT intrathecal
  • ICY intracerebroventricular injection
  • Passini et al used a single dose of MOE-ASO via intracerebral ventricular (ICV) injection up to 8 pg and obtained a survival improvement from 14 days to 23 days, Hua et al used a ICV dose of 20 mg with no adverse effect and in a different SMA animal model had an increased survival from 10 days to 16 days. Hua et al also obtained further improvement by giving the ASO into the periphery. This contrasts with the morpholino data that showed survival beyond 100 days in the delta7 SMA mice whereas mice without treatment lived for 13 days.
  • Linear oligonucleotides linear ASO
  • oligonucleotides via two hexaethyleneglycol (spacer 18) moieties for SNA were synthesized with 2’-methoxyethyl (2’-MOE) and phosphorothioate (PS) backbone modification.
  • the oligonucleotide sequence is the same as that of Spinraza.
  • SNAs (SNA-ASO) were prepared by loading 3’-cholesterol attached oligonucleotides onto DOPC liposomes at a ratio of 30 oligonucleotide molecules per 20 nm liposome particle.
  • mice were administered to mice by intracerebro- ventricular injections as described previously (P. N. Porensky, et al, Hum. Mol. Genet. 21, 1625-1638, 2012). Briefly, P0 pup was cryo- anesthetized and hand-mounted over a back-light to visualize the intersection of the coronal and sagittal cranial sutures (bregma). A fine-drawn capillary needle with injection assembly was inserted 1 mm lateral and 1 mm posterior to bregma, and then tunneled 1 mm deep to the skin edge
  • mice in each group The pharmacodynamic activity of the compounds is followed by survival of mice in each group compared with untreated mice.
  • morpholino ASO prolonged the Smn _/ SMN2 D7 mice survival over 100 days, which serves as a reference for the current study.
  • the EMGs will be recorded for muscle action potential (CMAP) as well as motor unit number estimation. Both these parameters are reduced in SMA at 6 days and beyond.
  • CMAP muscle action potential
  • SMN levels are corrected due to the action of the test compounds, these values recover and when mice live out can reach normal levels. This is an important measure as it shows that the motor neuron has recovered and the muscle is innervated correctly. It is one of the only measures that can be made in man and mouse and is altered in human SMA.
  • SMN protein and RNA give a measure of the increased
  • a single dose of SNA- ASO or linear ASO was injected to mice on P0 at 10, 20 or 30 mg.
  • the Kaplan-Meier survival plots of SMA mice treated with SNA-ASO and linear ASO and untreated mice are shown in Fig. 3 A and 3B.
  • Mice were genotyped at P0 (day of birth) and injected via Intracerebroventricular injection (ICV) on P0. The recorder of events was blinded to genotype and treatment. Control untreated mice died within 18 days with a median survival of about 14 days.
  • Mice treated with linear ASO showed a median survival of 16, 17 and 2 days at 10, 20 and 30 mg doses, respectively with a maximal survival prolongation of about 28 days.
  • SNA- ASO treatment lead to increased survival of SMA mice at all dose levels compared with linear ASO.
  • the median survival of SNA- ASO treated mice was 26, 69 and 70 days at 10, 20, and 30 mg doses, respectively.
  • the survival of SMA mice was prolonged up to about 117 days in 20 mg SNA- ASO dose group and the mice in 30 mg dose group have not reached end point.
  • Fig. 3A shows A7SMA mice treated with the 30pg dose Nusinersen-SNA had increased survival to a maximum of 82 days while scramble SNA has no effect on survival.
  • Fig. 3B shows that linear Nusinersen improved survival of D7 SMA mice to a maximum of
  • Phenotypic changes, including weight changes, on the treated mice were assessed. Weight curves to 21 days of age in treated and untreated control mice are shown in Fig. 4A and 4B. Mice were weighed each day. Fig. 4A shows that weights are similar in A7SMA mice treated with linear or Nusinersen-SNA treated mice. Fig. 4B shows that weights are similar in A7SMA mice treated with morpholino to ISS-N1 or Nusinersen-SNA . The scramble-SNA did not alter the weight of the A7SMA mice.
  • treatment of SMA mice with a single ICV dose of ASO-SNA increased exon 7 inclusion.
  • treatment of SMA mice with ASO-SNA resulted in increased median survival of up to 69/70 days with a prolongation of survival beyond 100 days compared with linear ASO.
  • SNA-ASOs are safe and well tolerated in SMA mice compared with linear ASO.
  • SNAs increased uptake of MOE Nusinersen in cell models lacking SMN 1 but containing SMN2, resulting in increased amounts of full-length mRNA and SMN protein from SMN2. Additionally, SNAs when delivered to CSF in the A7SMA mouse model allow increased dosing of Nusinersen and increased efficacy with prolonged survival of SMA mice. SNAs when delivered to CSF in the A7SMA mouse model also have increased full- length SMN mRNA levels in spinal cord tissue. In view of these data demonstrating the enhanced use of SNA relative to Nusinersen, the therapeutic utility of the SNA is substantial.
  • Additional experiments for further analysis include: Performing EMG, compound muscle action potential (CMAP) and motor unit number estimation (MUNE) to assess the extent of motor neuron correction and determining Nusinersen-SNA bio-distribution and SMN levels in all treatment groups using ELISA and Western blot.
  • CMAP compound muscle action potential
  • MUNE motor unit number estimation

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L'invention concerne des compositions associées à des acides nucléiques sphériques (SNA) présentant des oligonucléotides antisens et des procédés de traitement de maladies et de troubles.
PCT/US2018/045685 2018-02-28 2018-08-07 Constructions liposomales d'acides nucléiques sphériques (sna) pour des inhibiteurs de survie de neurones moteurs (smn) WO2019168558A1 (fr)

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* Cited by examiner, † Cited by third party
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US11633503B2 (en) 2009-01-08 2023-04-25 Northwestern University Delivery of oligonucleotide-functionalized nanoparticles
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10894963B2 (en) 2013-07-25 2021-01-19 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11957788B2 (en) 2014-06-04 2024-04-16 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US12013403B2 (en) 2014-09-12 2024-06-18 Biogen Ma Inc. Compositions and methods for detection of SMN protein in a subject and treatment of a subject
US10760080B2 (en) 2014-10-06 2020-09-01 Exicure, Inc. Anti-TNF compounds
US10704043B2 (en) 2015-01-14 2020-07-07 Exicure, Inc. Nucleic acid nanostructures with core motifs
US11866700B2 (en) 2016-05-06 2024-01-09 Exicure Operating Company Liposomal spherical nucleic acid (SNA) constructs presenting antisense oligonucleotides (ASO) for specific knockdown of interleukin 17 receptor mRNA
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
WO2021046254A1 (fr) * 2019-09-04 2021-03-11 Exicure, Inc. Constructions liposomales d'acides nucléiques sphériques (sna) pour la modulation de l'épissage
US11299737B1 (en) 2020-02-28 2022-04-12 Ionis Pharmaceuticals, Inc. Compounds and methods for modulating SMN2
WO2022150706A3 (fr) * 2021-01-08 2022-08-18 The General Hospital Corporation Approches d'édition de génome pour traiter une amyotrophie spinale

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