Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
  • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid

Definitions

• the present disclosure relates to oligonucleotides conjugated to acyl chains having a terminal carboxyl group.
• the disclosure provides methods of making oligonucleotides conjugated to acyl chains having a terminal carboxyl group.
• Oligonucleotide based therapies such as antisense therapies, gene therapies and CRISPR gene editing therapies - are thought to hold promise for treatment of various conditions.
• delivery of oligonucleotides to specific tissues in the body has been a challenge for oligonucleotide-based therapies.
• One strategy for delivering oligonucleotides to specific tissues is to deliver the oligonucleotides packaged into lipid nanoparticles or polymer nanoparticles. Because oligonucleotides, unless specially modified, have a polyanionic backbone, cationic lipids or polymers are used in forming nanoparticles. The electrostatic interaction between the anionic oligonucleotides and cationic lipids or polymers causes the nanoparticles to form.
• lipid nanoparticles While a wide variety of lipid nanoparticles has been developed, almost all of these nanoparticles have limitations for use in therapeutics.
• One limitation is the amount of carrier, i.e., lipid, material that must be delivered to support the administration of oligonucleotides.
• siRNAs small interfering RNAs
• the siRNA makes up only a few percent of the total mass of the deliverable, with the remaining mass being lipids.
• Juliano Nucleic Acids Research 2016, 44(14):6518-6548.
• the delivery of large amount of cationic lipids can be problematic as many biological macromolecules are anionic and thus electrostatically associate with the cationic lipids leading to toxicity issues.
• lipid nanoparticles typically only accumulate in tissues having a fenestrated (e.g., porous) endothelium: liver, spleen and some tumor tissues. Osborn el al., Nucleic Acid Therapeutics 2018, 28(3): 128-136. This makes the use of lipid nanoparticles impractical for therapies where the oligonucleotide needs to be delivered to other tissues.
• Lipid conjugated oligonucleotides are being developed to improve the delivery of oligonucleotides.
• the oligonucleotide is chemically linked to the lipid, an electrostatic interaction between the two is not needed and potentially toxic cationic lipids are not required.
• the amount of lipid carrier material is substantially reduced.
• side effects with lipid conjugated oligonucleotides should be reduced.
• the lipids used for conjugation do not have to be suitable for lipid nanoparticle formation, a larger variety of lipids can be used. By varying the lipid portion of the lipid conjugated oligonucleotide, it is possible to target a wider range of tissues for uptake of the conjugates.
• the present disclosure is directed to acid acyl conjugated oligonucleotides comprising: a) a carboxy acyl group; b) an oligonucleotide; and c) a linker connecting the carboxy acyl group to the oligonucleotide.
• the carboxy acyl group is C4 to C32 and may be saturated, for example, monounsaturated or unsaturated, for instance polyunsaturated.
• the acid acyl conjugated oligonucleotides comprise a linker bound to the 5' end of the oligonucleotide. In some embodiments, the acid acyl conjugated oligonucleotides comprise a linker comprising an amino terminus connected to the carboxy acyl group. In some embodiments, the acid acyl conjugated oligonucleotides comprise a linker comprising a phosphate terminus connected to the oligonucleotide.
• the acid acyl conjugated oligonucleotides comprise a DNA oligonucleotide. In some embodiments, the acid acyl conjugated oligonucleotides comprise a RNA oligonucleotide. In some embodiments, the acid acyl conjugated oligonucleotides comprise an antisense oligonucleotide. In some embodiments, the oligonucleotide is a phosphorothioate oligonucleotide.
• the acid acyl conjugated oligonucleotides of the present disclosure may be chosen from compounds of formula (I): wherein:
• X is C 4 to C 32 alkyl or alkenyl
• A is a conjugation group
• L is a linker
• Y is an oligonucleotide
• X is C 10 to C 26 alkyl
• L is a linker -NH-C 6 H 12 -O-PO 2 -;
• Y is DNA, wherein the linker is attached to the DNA via a phosphate linkage at the 5' end of the DNA.
• the present disclosure further comprises methods to improve the reduction in cardiac cell expression compared comprising administering an acid acyl conjugated oligonucleotide disclosed herein to a mammal, wherein the acid acyl conjugated oligonucleotide improves reduction in cardiac cell expression compared to a non-acyl conjugated oligonucleotide.
• the present disclosure further provides methods of delivering an oligonucleotide to cardiac tissue in a subject comprising: a) providing acid acyl conjugated oligonucleotide according to the present disclosure, and b) administering the acid acyl conjugated oligonucleotide to the subject.
• FIG. 1 shows the dissociation constants of 5’ lipid conjugated Malat-1 ASOs from Human (HSA) and rat (RSA) serum albumin measured by surface plasmon resonance (SPR) as exemplified in Example 25.
• FIG. 2 shows the concentration dependent knock down of Malat-1 gene expression in human THP-1 monocytes after treatment with FA-ASO conjugates as exemplified in Example 26.
• FIGS. 3A-D shows the results of Example 27, that the lipidated CamK2D ASOs of Examples 12 (FIG. 3 A), Example 13 (FIG. 3B), Example 14 (FIG. 3C), and Example 15 (FIG. 3D) maintained functional activity in vitro.
• FIGS. 4A-B shows that conjugation to a saturated C 22 acid or C 18 (9Z) monounsaturated fatty diacid led to similar or increased knock down in the heart but also to an attenuation of the knock down measured in the liver and kidney compared to the parent ASO with FIG. 4A exemplifying Examples 12 and 15 MALAT-1 gene expression in the heart and FIG. 4B exemplifying Examples 7 and 10 MALAT-1 gene expression in the liver and kidney, ns: Analysis was carried out via two-way ANOVA followed by Bonferroni multiple comparisons. ***: p ⁇ 0.001, one way ANOVA followed by Dunnett multiple comparisons.
• FIG. 5 shows CamK2D gene expression level in the heart, kidney, and liver and shows that the saturated C22 acid chain conjugation improved knock down in the heart and tend to attenuate the knock-down in sink organs like kidney and liver in comparison to the naked parent ASOs as exemplified in Example 29. ** p ⁇ 0.01. *** p ⁇ 0.001, two way ANOVA followed by Bonferroni multiple comparisons.
• the present disclosure provides lipid conjugated oligonucleotides where the lipid comprises an acyl group and a free terminal carboxylic acid group.
• the present disclosure includes methods of making lipid conjugated oligonucleotides where the lipid comprises and acyl group and a free terminal carboxylic acid group.
• the present disclosure includes methods of delivering the lipid conjugate oligonucleotides disclosed herein to a subject.
• the present disclosure also includes methods of administering the lipid conjugate oligonucleotides disclosed herein to treat disease in a subject.
• nucleic acid means a polymeric compound including covalently linked nucleotides.
• a nucleotide includes a nucleoside linked to a phosphate group.
• a nucleoside includes a nucleobase and sugar moiety. The nucleobase may be naturally occurring or synthetic. The nucleobase and sugar base may each, independently, be modified or unmodified.
• Modified nucleoside means a nucleoside comprising a modified nucleobase and/or a modified sugar moiety.
• Modified nucleosides can include abasic nucleosides, which lack a nucleobase. Polynucleotides or oligonucleotides may be modified or unmodified and may contain one or more modified nucleosides. "Modified polynucleotide” or “modified oligonucleotide” means a polynucleotide or oligonucleotide, wherein at least one sugar, nucleobase, or intemucleoside linkage is modified. In some embodiments, the modified polynucleotide or modified oligonucleotide is oligonucleotide is a phosphorothioate polynucleotide.
• Unmodified polynucleotide means a polynucleotide that does not comprise any sugar, nucleobase, or intemucleoside modification.
• nucleic acid includes ribonucleic acid (RNA) and deoxyribonucleic acid (DNA), both of which may be single- or double-stranded.
• DNA includes, but is not limited to, complementary DNA (cDNA), genomic DNA, plasmid or vector DNA, and synthetic DNA.
• cDNA complementary DNA
• genomic DNA genomic DNA
• plasmid or vector DNA and synthetic DNA.
• the polynucleotide or oligonucleotide is double stranded DNA.
• polynucleotide or oligonucleotide is single stranded DNA.
• the polynucleotide or oligonucleotide is double stranded RNA. In some embodiments the polynucleotide or oligonucleotide is single stranded RNA. In some embodiments the polynucleotide or oligonucleotide is an antisense RNA. Nucleic acids of the present disclosure may be any length. In some embodiments, a nucleic acid provided herein is 8 to 80 nucleotides in length.
• a nucleic acid provided herein is 10 to 70 nucleotides in length, 12 to 60 nucleotides in length, 15 to 50 nucleotides in length, 15 to 45 nucleotides in length, 16 to 40 nucleotides in length, 17 to 35 nucleotides in length, 18 to 30 nucleotides in length, 19 to 29 nucleotides in length or 20 to 28 nucleotides in length.
• the term "antisense molecule” means an oligomeric molecule that is capable of undergoing hybridization to a target nucleic acid, e.g., through hydrogen bonding.
• antisense molecules include single-stranded and double-stranded nucleic acids, such as, e.g., antisense oligonucleotides (ASO), small interfering RNAs (siRNA), short hairpin RNAs (shRNA), small nucleolar RNAs (snoRNA), microRNAs (miRNA), and meroduplexes (mdRNA), and satellite repeat sequences.
• ASO antisense oligonucleotides
• siRNA small interfering RNAs
• shRNA short hairpin RNAs
• snoRNA small nucleolar RNAs
• miRNA microRNAs
• mdRNA meroduplexes
• an "antisense oligonucleotide” or “ASO” refers to a polynucleotide comprising a sequence that is complementary to a target nucleic acid or region or segment thereof.
• an ASO is specifically hybridizable to a target nucleic acid or region or segment thereof.
• ASOs are capable of influencing RNA processing and/or modulating protein expression.
• an ASO is a single-stranded oligonucleotide that binds to single-stranded RNA to inactivate the RNA.
• an ASO binds to messenger RNA (mRNA) for a gene, thereby inactivating the gene.
• mRNA messenger RNA
• an ASO binds to a non-coding mRNA, thereby disrupting the function of the non-coding mRNA. In some embodiments, an ASO binds to a transcription initiation site, a translation initiation site, 5 '-untranslated sequence, 3 '-untranslated sequence, coding sequence, a pre-mRNA sequence, an mRNA splice site, and/or an intron/exon junction of an mRNA encoding a gene, thereby inactivating the gene. In some embodiments, the ASO includes DNA, RNA, or combination thereof.
• ASOs are further described in, e.g., Goodchild, Methods Mol Biol 764:1- 15, 2011; Smith et al., Ann Rev Pharmacol Toxicol 59:605-630, 2019; and Stein et al., Mol Ther 25(5): 1069-1075, 2017.
• the oligonucleotide can be unmodified DNA, RNA or may be modified.
• Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase and/or at least one modified intemucleoside linkage).
• the oligonucleotide can be selected from any of the oligonucleotides described herein.
• the oligonucleotide is an antisense oligonucleotide.
• the antisense oligonucleotide contains at least one phosphorothioate intemucleoside linkage.
• the antisense oligonucleotide contains at least one modified sugar moiety, for example, a bicyclic sugar moiety (e.g., comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure), such as a furanosyl moiety.
• the antisense oligonucleotide contains at least one modified nucleobase.
• the present disclosure provides an acyl acid conjugated oligonucleotide comprising a carboxy acyl group connected to an oligonucleotide.
• the carboxy acyl group is connected to the oligonucleotide by a linker.
• the carboxy acyl group of the conjugate is a fatty acid chain having a free terminal carboxylic acid group.
• the acyl group has a length of from C 4 to C 32 .
• the acyl group has a length of from C 6 to C 30 .
• the acyl group has a length of from C 8 to C 28 .
• the acyl group has a length of from C 10 to C 26 .
• the acyl group has a length of from C 12 to C 26 .
• the acyl group has a length of from C 14 to C 24 .
• the acyl group has a length of from C 16 to C 22 .
• the acyl group has a length of Ci6.
• the acyl group has a length of C 18 .
• the acyl group has a length of C 22 .
• the carboxy terminal on the acyl group provides improved uptake of the conjugated oligonucleotide in specific tissues and/or organs upon administration. In embodiments, the carboxy terminal on the acyl group provides improved uptake in cardiac tissue or liver tissue. In embodiments, the carboxyl terminal on the acyl group provides improved uptake in the heart or liver. In embodiments, the carboxyl terminal on the acyl group provides improved uptake in cardiac tissue. In embodiments, the carboxyl terminal on the acyl group provides improved uptake in the heart.
• the acyl group is saturated, i.e., having only single carbon-carbon bonds. In embodiments, the acyl group is unsaturated, i.e., having one or more double carbon- carbon bonds. In embodiments, the acyl group is monounsaturated. In embodiments, the acyl group is polyunsaturated, e.g., having 2-15 double bond, e.g., having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 double bonds.
• the lipid conjugated oligonucleotides can include a single carboxy acyl group attached to the oligonucleotide. In embodiments, the lipid conjugated oligonucleotides can include a two carboxy acyl groups attached to the oligonucleotide. In embodiments, the lipid conjugated oligonucleotides can include a three or more carboxy acyl groups attached to the oligonucleotide. In embodiments, the lipid conjugated oligonucleotides can include a single carboxy acyl group attached to the oligonucleotide along with one, two or more other acyl groups that do not have a terminal carboxy. In embodiments, the lipid conjugated oligonucleotides can include two carboxy acyl groups attached to the oligonucleotide along with one, two or more other acyl groups that do not have a terminal carboxy.
• the carboxyl acyl group is attached to the 5' end of the oligonucleotide. In embodiments, the carboxyl acyl group is attached to the 3' end of the oligonucleotide. In embodiments, the carboxy acyl group is attached to the oligonucleotide at a modified base in the oligonucleotide instead of the 5' or 3' ends of the oligonucleotide.
• the carboxyl acyl group is connected to the oligonucleotide by a linker.
• the linker is bound to the 5' end of the oligonucleotide.
• the linker forms an amide bond with the carboxy acyl group.
• the linker comprises a phosphate linkage connected to the oligonucleotide.
• the linker is attached to the 3' end of the oligonucleotide.
• the linker is attached to the oligonucleotide at a modified base in the oligonucleotide.
• the linker may be any chemical moiety that allows for chemical attachment of the carboxy acyl group at one end of the linker and the oligonucleotide at another end of the linker.
• the linker forms an amide bond with the carboxy acyl group.
• the linker comprises a phosphate linkage connected to the oligonucleotide.
• the phosphate linkage of the linker can be a 5' phosphate on the oligonucleotide.
• the linker comprises an acyl chain between termini.
• the linker comprises an acyl chain of C 1 to C 10 in length.
• the acyl chain can be saturated.
• the acyl chain can be monounsaturated or polyunsaturated.
• the linker forms an amide bond with the carboxy acyl group, a phosphate linkage with the oligonucleotide, and an acyl chain of C 1 to C 10 in length connecting the amide bond with the phosphate terminal.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (I) wherein: X is C4 to C32 alkyl or alkenyl; A is a conjugation group; L is a linker; and Y is an oligonucleotide.
• X is C 6 to C 30 alkyl or alkenyl. In embodiments, X is C 8 to C 28 alkyl or alkenyl. In embodiments, X is C 10 to C 26 alkyl or alkenyl. In embodiments, X is C 12 to C 26 alkyl or alkenyl. In embodiments, X is C 14 to C 24 alkyl or alkenyl. In embodiments, X is C 14 alkyl or alkenyl. In embodiments, X is C 16 alkyl or alkenyl. In embodiments, X is C 20 alkyl or alkenyl.
• X is C 6 to C 30 alkyl. In embodiments, X is C 8 to C 28 alkyl. In embodiments, X is C 10 to C 26 alkyl. In embodiments, X is C 12 to C 26 alkyl. In embodiments, X is C 14 to C 24 alkyl. In embodiments, X is C 14 alkyl. In embodiments, X is C 16 alkyl. In embodiments, X is C 20 alkyl.
• X is C 6 to C 30 monounsaturated alkenyl. In embodiments, X is C 8 to C 28 monounsaturated alkenyl. In embodiments, X is C 10 to C 26 monounsaturated alkenyl. In embodiments, X is C12 to C26 monounsaturated alkenyl. In embodiments, X is C 14 to C 24 monounsaturated alkenyl. In embodiments, X is C 14 monounsaturated alkenyl. In embodiments, X is C 16 monounsaturated alkenyl. In embodiments, X is C 20 monounsaturated alkenyl.
• X is C 6 to C 30 polyunsaturated alkenyl. In embodiments, X is C 8 to C 28 polyunsaturated alkenyl. In embodiments, X is C 10 to C 26 polyunsaturated alkenyl. In embodiments, X is C 12 to C 26 polyunsaturated alkenyl. In embodiments, X is C 14 to C 24 polyunsaturated alkenyl. In embodiments, X is C 14 polyunsaturated alkenyl. In embodiments, X is C 16 polyunsaturated alkenyl. In embodiments, X is C 20 polyunsaturated alkenyl.
• the conjugation group A contains at least one spacer between the conjugation group A and X in the formula (I) above (e.g., a polyethylene glycol (PEG) chain (e.g., molecular weight ranging from 100 to 2000 Da) and/or at least one amino acid (e.g., cysteine, glutamic acid, lysine, glycine, etc.).
• a polyethylene glycol (PEG) chain e.g., molecular weight ranging from 100 to 2000 Da
• amino acid e.g., cysteine, glutamic acid, lysine, glycine, etc.
• the conjugation group A contains at least one other fatty acid chain other than the fatty acid chain of formula (I).
• the conjugation group A contains two fatty acid chains such as illustrated below: wherein in each instance, X is C 4 to C 32 alkyl or alkenyl.
• L is a linker: where Z and W are defined below.
• Z is O or S.
• W is C 1 to C 10 alkyl or alkenyl, or in some embodiments, W is
• W is C 2 to C 9 alkyl, such as C 3 to C8 a lkyl, for example, W is C 4 to C 8 alkyl. In some embodiments, W is C 5 to C 7 alkyl.
• W is C 2 to C 9 monounsaturated alkenyl, for instance W is C 3 to C 8 monounsaturated alkenyl. In some embodiments, W is C 4 to C 8 monounsaturated alkenyl, such as W is C 5 to C 7 monounsaturated alkenyl. [0052] In embodiments, W is C 2 to C 9 polyunsaturated alkenyl, for example W is C 3 to C 8 polyunsaturated alkenyl. In some embodiments, W is C 4 to C 8 polyunsaturated alkenyl, such as W is C 5 to C 7 polyunsaturated alkenyl.
• W is C 1 alkyl. In embodiments, W is C 2 alkyl. In embodiments, W is C 3 alkyl. In embodiments, W is C 4 alkyl. In embodiments, W is C 5 alkyl. In embodiments, W is C 6 alkyl. In embodiments, W is C 7 alkyl. In embodiments, W is C 8 alkyl. In embodiments, W is C 9 alkyl. In embodiments, W is C 10 alkyl.
• W is C 2 monounsaturated alkenyl. In embodiments, W is C 3 monounsaturated alkenyl. In embodiments, W is C 4 monounsaturated alkenyl. In embodiments, W is C 5 monounsaturated alkenyl. In embodiments, W is C 6 monounsaturated alkenyl. In embodiments, W is C 7 monounsaturated alkenyl. In embodiments, W is C 8 monounsaturated alkenyl. In embodiments, W is C 9 monounsaturated alkenyl. In embodiments, W is C 10 monounsaturated alkenyl.
• W is C 2 polyunsaturated alkenyl. In embodiments, W is C 3 polyunsaturated alkenyl. In embodiments, W is C 4 polyunsaturated alkenyl. In embodiments, W is C 5 polyunsaturated alkenyl. In embodiments, W is C 6 polyunsaturated alkenyl. In embodiments, W is C 7 polyunsaturated alkenyl. In embodiments, W is C 8 polyunsaturated alkenyl. In embodiments, W is C 9 polyunsaturated alkenyl. In embodiments, W is C 10 polyunsaturated alkenyl.
• L is -NH-CH 2 -O-PO2-. In embodiments, L is -NH-C 2 H 4 -O-PO 2 -. In embodiments, L is -NH-C 3 H6-O-PO 2 -. In embodiments, L is -NH-C 4 H 8 -O-PO 2 -. In embodiments, L is -NH-C 5 H 10 -O-PO 2 -. In embodiments, L is -NH-C 6 H 12 -O-PO 2 -. In embodiments, L is -NH-C 7 H 14 -O-PO 2 -. In embodiments, L is -NH-C 8 H 16 -O-PO 2 -. In embodiments, L is -NH-C 9 H 18 -O-PO 2 -. In embodiments, L is -NH-C 10 H 20 -O-PO 2 -.
• L is attached to the 5' end of the oligonucleotide Y.
• the phosphate group in L is a 5' phosphate group on the oligonucleotide.
• L is attached to the 3' end of the oligonucleotide Y.
• L is attached to the oligonucleotide at a modified base in the oligonucleotide Y.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (I) wherein: X is C 10 to C 26 alkyl; A is a conjugation group chosen from the groups described above; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (I) wherein: X is chosen from C 14 alkyl, C 16 alkyl, and C 20 alkyl; A is a conjugation group chosen from the groups described above; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• formula (I) wherein: X is chosen from C 14 alkyl, C 16 alkyl, and C 20 alkyl; A is a conjugation group chosen from the groups described above; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (la) wherein: X is C 14 alkyl; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (la) wherein: X is C 16 alkyl; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• the present disclosure provides an acid acyl conjugated oligonucleotide of formula (la) wherein: X is C 20 alkyl; L is -NH-C 6 H 12 -O-PO 2 -; and Y is an oligonucleotide as described herein attached to L at the 5' end of Y.
• the oligonucleotide Y can be any oligonucleotide described herein.
• the oligonucleotide (Y) of the compounds described herein can comprise DNA, RNA or nucleic acids having unnatural backbones.
• the oligonucleotide can comprise the natural DNA and RNA nucleobases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), and may also comprise non-natural and modified nucleobases.
• the oligonucleotide can have a non-natural backbone such as a phosphorothioate backbone.
• the oligonucleotide is single stranded. In some embodiments, the oligonucleotide is double stranded.
• the oligonucleotide comprises an antisense oligonucleotide.
• the oligonucleotide comprises a sequence that can be expressed in a cell, e.g., a coding sequence such as a gene or a messenger RNA (mRNA).
• the oligonucleotide comprises a sequence that does not encode a protein but has another function in the cell, e.g., a non-coding RNA, a transfer RNA (tRNA), a ribosomal RNA (rRNA) or a small-nucleolar RNA (snRNA).
• the oligonucleotide is a CRISPR guide RNA (gRNA).
• the oligonucleotide is an antisense oligonucleotide targeting MALAT1, apolipoprotein B, apolipoprotein C III, endothelial lipase, p53, clusterin, signal transducer and activator of transcription 3 (STAT3), Mothers against decapentaplegic homolog 7 (SMAD7), intercellular adhesion molecule 1 (CD54), dystrophin (for example, myotonic dystrophy protein kinase (DMPK), transthyretin (TTR), huntingtin (HTT), microRNA-122 (miR-122).
• DMPK myotonic dystrophy protein kinase
• TTR transthyretin
• HTT huntingtin
• microRNA-122 miR-122
• the oligonucleotide is an antisense oligonucleotide that interferes with splicing of dystrophin mRNA such as eteplirsen, golodirsen, casimersen, drisapersen and viltolarsen).
• the oligonucleotide is an antisense oligonucleotide that reduces expression of any of the above targets.
• the oligonucleotide is an antisense oligonucleotide that reduces mRNA splicing of any of the above targets.
• the oligonucleotide is an antisense oligonucleotide that targets a non-coding sequence in order to reduce expression of any of the above targets, e.g., an exon, a 5' non-coding sequence or a 3' non-coding sequence.
• the oligonucleotide comprises a sequence that can be expressed in a cell, such as a protein expressed by a gene
• the lipid conjugated oligonucleotide can be used in methods of treating subjects that lack sufficient expression of the protein, e.g., in gene therapy type methods of treatment.
• the lipid conjugated oligonucleotide can be used in methods of treating subjects that lack the function provided by the oligonucleotide.
• the lipid conjugated oligonucleotide can be used in methods of treating subjects having disorders relating to increased levels of expression of the target.
• the oligonucleotide reduces expression of the target in cells by about 1% to about 100%.
• the oligonucleotide reduces expression of the target in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the oligonucleotide reduces expression of the target in cells by about 1% to about 100%, by about 5% to about 50%, by about 10% to about 50%, by about 30%, by about 10% to about 30%, or by about 15% to about 25%.
• the oligonucleotide is an antisense oligonucleotide targeting a condition effecting the heart, as the carboxy acyl conjugate can be used to preferentially target to the oligonucleotide to cardiac tissue.
• the oligonucleotide reduces expression of the target in cardiac cells by about 1% to about 100%.
• the oligonucleotide reduces expression of the target in cardiac cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the oligonucleotide reduces expression of the target in cardiac cells by about 1% to about 100%, by about 5% to about 50%, by about 10% to about 50%, by about 30%, by about 10% to about 30%, or by about 15% to about 25%.
• the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1). In embodiments, the oligonucleotide is an antisense oligonucleotide targeting MALAT1, variant 1 (SEQ ID NO:1). In embodiments, the oligonucleotide is an antisense oligonucleotide targeting MALAT1, variant 2 (SEQ ID NO:2). In embodiments, the oligonucleotide is an antisense oligonucleotide targeting MALAT1, variant 3 (SEQ ID NO:3).
• the antisense oligonucleotide targets MALAT1 and has the sequence: tcagcattctaatagcagc (SEQ ID NO:4). In embodiments, the antisense oligonucleotide targets MALAT1 and has the sequence: tm5cagm5cattm5ctaatagm5cagm5c, where m5c is 5 -methylcytidine (SEQ ID NO:5). In embodiments, the antisense oligonucleotide targets MALAT1 and has the sequence: gcattctaatagcagc (SEQ ID NO: 6).
• the antisense oligonucleotide targets MALAT1 and has the sequence: gm5cattm5ctaatagm5cagm5c, where m5c is 5-methylcytidine (SEQ ID NO: 7).
• the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1% to about 100%.
• the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the oligonucleotide is an antisense oligonucleotide targeting metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) comprising at least one nucleic acid with a locked sugar modified moiety (“LNA”).
• MALAT1 metastasis-associated lung adenocarcinoma transcript 1
• LNA locked sugar modified moiety
• the antisense oligonucleotide targets MALAT1 and has the sequence: GM5CAttm5ctaatagm5cAGM5C, where m5c is 5-methylcytidine and capital letters are LNA nucleosides (SEQ ID NO: 8).
• the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1% to about 100%. In embodiments, the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the oligonucleotide is an antisense oligonucleotide targeting Calcium/Calmodulin Dependent Protein Kinase II Delta (CAMK2D).
• the antisense oligonucleotide targeting CAMK2D comprises at least one nucleic acid with a locked sugar modified moiety (“LNA”).
• LNA locked sugar modified moiety
• the antisense oligonucleotide targets CAMK2D and has the sequence: GTGtm5caam5caam5cm5caTTT, where m5c is 5 -methylcytidine and capital letters are LNA nucleosides (SEQ ID NO: 10).
• the antisense oligonucleotide targets CAMK2D and has the sequence: M5CAM5CAaatttattaaM5CTM5CT, where m5c is 5-methylcytidine and capital letters are LNA nucleosides (SEQ ID NO: 11).
• the antisense oligonucleotide targets CAMK2D and has the sequence: M5CTGttm5cttm5caAtaATG, where m5c is 5- methylcytidine and capital letters are LNA nucleosides (SEQ ID NO: 12).
• the antisense oligonucleotide targets CAMK2D and has the sequence: AM5CM5Catgagm5ctataM5CTT, where m5c is 5-methylcytidine and capital letters are LNA nucleosides (SEQ ID NO: 13).
• the oligonucleotide is an antisense oligonucleotide that targets CAMK2D and reduces expression of CAMK2D in cells by about 1% to about 100%. In embodiments, the oligonucleotide is an antisense oligonucleotide that targets CAMK2D and reduces expression of CAMK2D in cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cardiac cells by about 1% to about 100%. In embodiments, the oligonucleotide is an antisense oligonucleotide that targets MALAT1 and reduces expression of MALAT1 in cardiac cells by about 1%, by about 10%, by about 20%, by about 30%, by about 40%, by about 50%, by about 60%, by about 70%, by about 80%, by about 90%, by about 95% or by about 100%.
• the lipid conjugated oligonucleotide can be used in a method of treatment of a cardiac disease in a subject. In embodiments where the oligonucleotide is an antisense oligonucleotide that targets MALAT1, the lipid conjugated oligonucleotide can be used in a method of treatment of a myocardial infarction in a subject.
• the lipid conjugated oligonucleotide can be used in a method of preventing a myocardial infarction in a subject.
• the oligonucleotide is administered to a subject at risk for a myocardial infarction.
• the present disclosure provides methods of making the lipid conjugated oligonucleotides described herein.
• the present disclosure provides a method of making an acid acyl oligonucleotide of formula (la): wherein: X is C 4 to C 32 alkyl or alkenyl; L is a linker; and Y is an oligonucleotide.
• the method of making formula (I) comprises the following steps:
• the activating group A* is an activated ester amine.
• the activating group A* in step B) may be chosen from:
• the reaction in step B) requires reacting a carboxylic acid with an amine to form an activated ester amine.
• the reaction in step B) of the method of making requires reacting a carboxylic acid with N- hydroxysuccinimide (NHS) to form an NHS ester as an activated ester amine.
• NHS N- hydroxysuccinimide
• the reaction in step B) of the method of making is performed in an organic solvent.
• the reaction in step B) of the method of making is performed in a solvent comprising one or more of ethyl acetate, dioxane, tetrahydro furan, dimethylfuran and dichloromethane and mixtures thereof.
• the reaction in step B) of the method of making is performed in the presence of a coupling reagent.
• the coupling reagent is a diimine.
• the coupling reagent is dicyclohexylmethanediimine.
• the reaction in step C) requires coupling between the activating group A* attached to the fatty acyl compound and a primary amine on the linker (L).
• the compound of formula (IV) is dissolved in an aqueous buffer.
• the compoud of formula (IV) is dissolved in a phosphate buffer, a borate buffer, a carbonate buffer, an acetate buffer, a Tris buffer, a HEPES buffer, a MOPS buffer or a PIPES buffer, or is dissolved in pure water with a base such as triethylamine or DIPEA.
• the compound of formula (III) is dissolved in a polar aprotic solvent. In embodiments of the method of making formula (I), the compound of formula (III) is dissolved in acetonitrile. In other embodiments of the method of making formula (I), the compound of formula (III) is dissolved in dimethyl sulfoxide. In other embodiments of the method of making formula (I), the compound of formula (III) is dissolved in a mixture of acetonitrile and dimethyl sulfoxide.
• the method of making formula (I) comprises the following steps:
• the resultant product of either step B) and /or step C) can be purified.
• the resultant product of either step B) and/or step C) is purified by chromatography.
• the resultant product of either step B) and/or step C) is purified by flash chromatography.
• the length of the acyl group, X is C 6 to C 30 alkyl or alkenyl.
• X is C 8 to C 28 alkyl or alkenyl.
• X is C 10 to C 26 alkyl or alkenyl.
• X is C 12 to C 26 alkyl or alkenyl.
• X is C 14 to C 24 alkyl or alkenyl.
• X is C 14 alkyl or alkenyl.
• X is C 16 alkyl or alkenyl.
• X is C 20 alkyl or alkenyl.
• the length of the acyl group, X is C 6 to C 30 alkyl.
• X is C 8 to C 28 alkyl.
• X is C 10 to C 26 alkyl.
• X is C 12 to C 26 alkyl.
• X is C 14 to C 24 alkyl.
• X is C 14 alkyl.
• X is C 16 alkyl.
• X is C 20 alkyl.
• X is C 6 to C 30 monounsaturated alkenyl. In embodiments, X is C 8 to C 28 monounsaturated alkenyl. In embodiments, X is C 10 to C 26 monounsaturated alkenyl. In embodiments, X is C 12 to C 26 monounsaturated alkenyl. In embodiments, X is C 14 to C 24 monounsaturated alkenyl. In embodiments, X is C 14 monounsaturated alkenyl. In embodiments, X is C 16 monounsaturated alkenyl. In embodiments, X is C 20 monounsaturated alkenyl.
• X is C 6 to C 30 polyunsaturated alkenyl. In embodiments, X is C 8 to C 28 polyunsaturated alkenyl. In embodiments, X is C 10 to C 26 polyunsaturated alkenyl. In embodiments, X is C 12 to C 26 polyunsaturated alkenyl. In embodiments, X is C 14 to C 24 polyunsaturated alkenyl. In embodiments, X is C 14 polyunsaturated alkenyl. In embodiments, X is C 16 polyunsaturated alkenyl. In embodiments, X is C 20 polyunsaturated alkenyl.
• the linker (L) comprises a C 3 -C 10 amine. In embodiments of the method of making formula (I), the linker (L) comprises a C 4 -C 9 amine. In embodiments of the method of making formula (I), the linker (L) comprises a C 5 -C 8 amine. In embodiments of the method of making formula (I), the linker (L) comprises a C 6 or C 7 amine. In embodiments of the method of making formula (I), the linker (L) comprises a C 6 amine (hexylamine).
• the linker (L) forms an amide bond with the carboxy acyl group.
• the linker (L) comprises a phosphate terminal connected to the oligonucleotide.
• the linker (L) is wherein W is C 1 to C 10 alkyl or alkenyl.
• W is C 2 to C 9 alkyl. In embodiments, W is C 3 to C 8 alkyl. In embodiments, W is C 4 to C 8 alkyl. In embodiments, W is C 5 to C 7 alkyl.
• W is C 2 to C 9 monounsaturated alkenyl. In embodiments, W is C 3 to C 8 monounsaturated alkenyl. In embodiments, W is C 4 to C 8 monounsaturated alkenyl. In embodiments, W is C 5 to C 7 monounsaturated alkenyl.
• W is C 2 to C 9 polyunsaturated alkenyl. In embodiments, W is C 3 to C 8 polyunsaturated alkenyl. In embodiments, W is C 4 to C 8 polyunsaturated alkenyl. In embodiments, W is C 5 to C 7 polyunsaturated alkenyl. [0096] In embodiments, W is C 1 alkyl. In embodiments, W is C 2 alkyl. In embodiments, W is C 3 alkyl. In embodiments, W is C 4 alkyl. In embodiments, W is C 5 alkyl. In embodiments, W is C 6 alkyl. In embodiments, W is C 7 alkyl. In embodiments, W is C 8 alkyl. In embodiments, W is C 9 alkyl. In embodiments, W is C 10 alkyl.
• W is C 2 monounsaturated alkenyl. In embodiments, W is C 3 monounsaturated alkenyl. In embodiments, W is C 4 monounsaturated alkenyl. In embodiments, W is C 5 monounsaturated alkenyl. In embodiments, W is C 6 monounsaturated alkenyl. In embodiments, W is C 7 monounsaturated alkenyl. In embodiments, W is C 8 monounsaturated alkenyl. In embodiments, W is C 9 monounsaturated alkenyl. In embodiments, W is C 10 monounsaturated alkenyl.
• W is C 2 polyunsaturated alkenyl. In embodiments, W is C 3 polyunsaturated alkenyl. In embodiments, W is C 4 polyunsaturated alkenyl. In embodiments, W is C 5 polyunsaturated alkenyl. In embodiments, W is C 6 polyunsaturated alkenyl. In embodiments, W is C 7 polyunsaturated alkenyl. In embodiments, W is C 8 polyunsaturated alkenyl. In embodiments, W is C 9 polyunsaturated alkenyl. In embodiments, W is C 10 polyunsaturated alkenyl.
• the linker (L) is -NH-CH 2 -O-PO 2 -.
• L is -NH-C 3 H 6 - O-PO2-.
• L is -NH-C4H8-O-PO2-.
• L is -NH-C 5 H 10 -O-PO 2 -.
• L is -NH-C 6 H 12 -O-PO 2 -.
• L is -NH-C 7 H 14 -O-PO 2 -.
• L is -NH-C 8 H 16 -O-PO 2 -.
• L is -NH-C 9 H 18 -O-PO 2 -.
• L is -NH-C 10 H 20 -O-PO 2 -.
• L is attached to the 5' end of the oligonucleotide Y. In embodiments, L is attached to the 3' end of the oligonucleotide Y. In embodiments, L is attached to the oligonucleotide at a modified base in the oligonucleotide Y.
• the oligonucleotide can be unmodified DNA, RNA or may be modified.
• Modified oligonucleotides comprise at least one modification relative to unmodified RNA or DNA (i.e., comprise at least one modified nucleoside (comprising a modified sugar moiety and/or a modified nucleobase and/or at least one modified intemucleoside linkage).
• the oligonucleotide can be selected from any of the oligonucleotides described herein.
• the oligonucleotide is an antisense oligonucleotide.
• the antisense oligonucleotide contains at least one phosphorothioate intemucleoside linkage.
• the antisense oligonucleotide contains at least one modified sugar moiety, for example, a bicyclic sugar moiety (e.g., comprising two rings, wherein the second ring is formed via a bridge connecting two of the atoms in the first ring thereby forming a bicyclic structure).
• the antisense oligonucleotide contains at least one modified nucleobase.
• the lipid conjugated oligonucleotides are preferentially delivered to a specific tissue in the body upon administration.
• the lipid conjugated oligonucleotides are, for instance, delivered to cardiac tissue upon administration.
• the lipid conjugated oligonucleotides are, for example, delivered to liver tissue upon administration.
• the lipid conjugated oligonucleotides are, for instance, delivered to spleen tissue upon administration.
• the lipid conjugated oligonucleotides are, for example, delivered to kidney tissue upon administration.
• the lipid conjugated oligonucleotides are, for example, delivered to skin tissue upon administration.
• the lipid conjugated oligonucleotides are, for example, delivered to muscle tissue upon administration. In embodiments, the lipid conjugated oligonucleotides are, for example, delivered to lung tissue upon administration. In embodiments, the lipid conjugated oligonucleotides are, for example, delivered to adipose tissue upon administration.
• the present disclosure provides a method of delivering an oligonucleotide to cardiac tissue in a subject comprising: a) providing an acid acyl conjugated oligonucleotide as described herein, and b) administering the acid acyl conjugated oligonucleotide to the subject.
• the present disclosure provides a method of reducing expression of a gene of interest in the cardiac cells of a subject, comprising a) providing an acid acyl conjugated oligonucleotide as described herein, and b) administering the acid acyl conjugated oligonucleotide to the subject.
• the gene of interest is any antisense target described herein.
• the gene of interest is MALATl.
• the oligonucleotide can be DNA, RNA or a modified oligonucleotide. In embodiments, the oligonucleotide can be selected from any of the oligonucleotides described herein. In embodiments, the oligonucleotide is an antisense oligonucleotide. In embodiments, the oligonucleotide has a phosphorothioate backbone.
• the subject is a mammal.
• the mammalian subject is an animal such as an agricultural animal (e.g., cattle, sheep, swine), research animal (e.g., mice, rats, monkeys, chimpanzees) or companion animal (e.g., dogs, cats and rabbits).
• the mammalian subject is a human.
• the oligonucleotide is administered to treat a cardiac disease. In embodiments of the methods herein, the oligonucleotide is administered to treat a myocardial infarction. In embodiments of the methods herein, the oligonucleotide is administered to prevent a myocardial infarction. In embodiments of the method of delivering an oligonucleotide to cardiac tissue in a subject, the oligonucleotide is administered to a subject at risk for a myocardial infarction.
• the lipid conjugated oligonucleotides described herein are formulated into a pharmaceutically acceptable formulation.
• the pharmaceutical formulation further comprises a pharmaceutically acceptable excipient, e.g., tonicity adjusting agent, preservative, solubilizing agent, complexing agent, dispersing agent, buffering agent, or combination thereof.
• the pharmaceutical formulation is suitable for administration to a patient.
• the pharmaceutical formulation is suitable for intramuscular, subcutaneous, intravenous, intraperitoneal or oral administration to a patient.
• the compound was made according to Intermediate 1 starting from hexadecanedioic acid (0.3g, 1.05 mmol). After crystallization, the residue was purified with flash chromatography on silica using ethyl acetate/ Heptane 1/1 as eluent. The pure fractions was evaporated to give the desired product (16-((2,5-dioxopyrrolidin-l-yl)oxy)-16-oxohexadecanoic acid) Yield: 120 mg (30%).
• the compound was made according to Intermediate 1 starting from heptadecanedioic acid (0.3g, 1.0 mmol). After crystallization, the residue was purified with flash chromatography on silica using ethyl acetate/ Heptane 1/1 as eluent. The pure fraction was evaporated to give of the desired product (17-((2,5-dioxopyrrolidin-l-yl)oxy)-17-oxoheptadecanoic acid) Yield: 99 mg, 25%.
• the compound was made according to Intermediate 1 starting from octadecanedioic acid (0.3g, 0.95 mmol). After crystallization, the residue was purified with flash chromatography on silica using ethyl acetate/ Heptane 2/1 as eluent. The pure fraction was evaporated to give of the desired product (18-((2,5-dioxopyrrolidin-l-yl)oxy)-18-oxooctadecanoic acid) Yield: 93 mg, 24%.
• the compound (20-((2,5-dioxopyrrolidin-l-yl)oxy)-20-oxoicosanoic acid was made according to Intermediate 1 starting from icosanedioic acid (0.3g, 0.88 mmol). Yield: 229 mg, 59%.
• the compound was made according to Intermediate 1 starting from henicosanedioic acid (0.3g, 1.05 mmol). After crystallization, the residue was purified with flash chromatography on silica using ethyl acetate/ Heptane 1/1 as eluent. The pure fraction was evaporated to give of the desired product (21-((2,5-dioxopyrrolidin-l-yl)oxy)-21-oxohenicosanoic acid) Yield: 21 mg, (6%).
• the compound was made according to Intermediate 1 starting from tetracosanedioic acid (0.3g, 0.75 mmol). After crystallization, the residue was purified with flash chromatography on silica using ethyl acetate/ Heptane 1/1 as eluent. The pure fraction was evaporated to give of the desired product (24-((2,5-dioxopyrrolidin-l-yl)oxy)-24-oxotetracosanoic acid) Yield: 44 mg, 12%.
• the compound was purified by preparative HPLC on a XBridge C18 column (10 pm 250x50 ID mm) using a gradient of 20-75% acetonitrile in ammonia(0.2%) buffer over 20minutes with a flow of 100 mL/min.
• the product was detected by LS-MS analyses.
• Product fractions were concentrated to give the desired compound (tert-butyl (1- azido- 15- ⁇ 2-[(tert-butoxycarbonyl)amino]ethyl ⁇ - 11 -oxo-3,6,9-trioxa- 12, 15-diazaheptadecan- 17-yl)carbamate). Yield 131mg (58.1%)
• MALATl-LNA-hexylamine was dissolved in Phosphate buffer (0.1M pH 7.34, 660 ⁇ l) and 2,5-dioxopyrrolidin-l-yl 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoate (9.76 mg, 0.04 mmol) dissolved in acetonitrile (330 ⁇ L) was added. The clear solution was stirred for 1.5h at rt. Sodium acetate (3M, pH 5.2, 100 ⁇ l) was added and then the oligo was precipitated by addition of ethanol (4mL), vortexed briefly and left standing at -20C for 30 min.
• the mixture was centrifuged at 0 °C for 10 mins at 3500 rpm and the clear supernatant was removed.
• the oligo was washed once more by dissolving the pellet in ImL of water and Sodium acetate 3M, pH 5.2 (100 ⁇ l) (shaking necessary for full dissolution) followed by addition of 4 mL of ethanol, cooled for 30 mins at -20, then centrifuged at 0 °C for 10 mins at 3500 rpm. The supernatant was discarded, and the pellet was dried under a nitrogen flow. Yield 27mg (97 %)
• the resulting opaque reaction mixture was stirred for 24h at rt.
• the mixture was diluted with diethyl ether and washed with water, sodium bicarbonate (10%), potassium hydrogen sulphate (0.5M), water and brine.
• the organic phase was evaporated, and the residue was passed through a 2g silica plug with heptane and increasing diethyl ether.
• the product was detected by TLC with cerium ammonium molybdate stain.
• the resin was finally washed with DMF (4 x), methanol and DCM.
• the resin was filtered off and washed with TFA ( ⁇ 2 mL) which was combined with the filtrate.
• the product was precipitated in cold Et20.
• the precipitated product was centrifugated and the supernatant discarded.
• the solid material was washed three times by addition of cold Et20 and centrifugation.
• the resulting solid material was suspended in MeCH/H2O/TFA (50/50/0.1) and freeze dried.
• the compound was purified by preparative HPLC on a Kromasil C8 column (10 pm 250x50 ID mm) using a gradient of 35-80% acetonitrile in H2O/ACN/FA 95/5/0.2 buffer over 20 minutes with a flow of 100 mL/min. The compound was detected by UV at 220 nm. The product fractions were freeze dried to give the desired compound.
• the IvDde-group was then removed by treating the resin with 5%Hydrazine/DMF six times at rt for 2+5+5+5+5+5min.
• the same procedures used in the synthesis of intermediate 21 was then performed for the coupling of 22-(tert-butoxy)- 22-oxodocosanoic acid (137 mg, 0.32 mmol) and for the cleavage and purification of the final product.
• MALATl-LNA-hexylamine 34 mg, 6.15 pmol was dissolved in water (800 pl) and triethylamine (9.53 pl, 0.07 mmol) was added.
• 20-((2,5-dioxopyrrolidin-l-yl)oxy)-20- oxoicosanoic acid (4.06 mg, 9.23 pmol) (Intermediate 5) solved in a mixture of warm (60 °C) acetonitrile (200 pl) and DMSO (120 pl) was added to the solved oligonucleotide and the reaction mixture was stirred at RT for 60 min.
• EXAMPLE 2 SYNTHESIS OF C 16 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• Example 2 compound was made according to Example 1 (C20-acid-MALAT 1 - LNA) starting from MALATl-LNA-hexylamine (36 mg, 6.52 pmol) and 16-((2,5- dioxopyrrolidin-l-yl)oxy)-16-oxohexadecanoic acid (5.00 mg, 13.0 ⁇ mol (Intermediate 2).
• EXAMPLE 3 SYNTHESIS OF C 17 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• Example 3 compound was made according to Example 1 (C20-acid-MALAT 1 - LNA) starting from MALATl-LNA-hexylamine (34 mg, 6.15 ⁇ mol) and 17-((2,5- dioxopyrrolidin-l-yl)oxy)-17-oxoheptadecanoic acid (4.89 mg, 12.3 ⁇ mol) (Intermediate 3) Reaction time 10 min. The yield was 18 mg (48%).
• EXAMPLE 4 SYNTHESIS OF C 18 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• Example 4 compound was made according to Example 1 (C20-acid-MALAT 1 - LNA) starting from MALATl-LNA-hexylamine (38 mg, 6.88 ⁇ mol) and triethylamine (9.53 pl, 0.07 mmol) was added. 18-((2,5-dioxopyrrolidin-l-yl)oxy)-18-oxooctadecanoic acid (5.66 mg, 13.8 pmol) (Intermediate 4). Reaction time: 90 min. Purification on a XBridge C18, 5pm 19x150 mm column by using a gradient from 15-90% acetonitrile in NH4HCO3 (50 mM, pH8) at R.T. Yield: 24 mg (57%).
• EXAMPLE 5 SYNTHESIS OF C19 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the compound was made according to Example 1 (C20-acid-MALATl-LNA) starting from MALATl-LNA-hexylamine (34 mg, 6.15 ⁇ mol) and 19-((2,5-dioxopyrrolidin- l-yl)oxy)-19-oxononadecanoic acid (5.24 mg, 12.3 mmol) (Intermediate 10). Reaction time: 30 min. The yield was 21 mg (56%).
• EXAMPLE 7 SYNTHESIS OF C22 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• MALATl-LNA-hexylamine 100 mg, 0.02 mmol
• triethylamine 10.03 pl, 0.07 mmol
• 22-((2,5-dioxopyrrolidin-l-yl)oxy)-22-oxodocosanoic acid 21.16 mg, 0.05 mmol
• Intermediate 1 dissolved in warm acetonitrile (400 pl) and DMSO (200 pl) was added. Stirred at 40 °C for 24 h. Additional 2.5 eqv of the activated lipid was added and the mixture stirred additional 24 h. at 40 °C.
• EXAMPLE 8 SYNTHESIS OF C23 SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the compound was made according to Example 1 (C20-acid-MALATl-LNA) starting from MALATl-LNA-hexylamine (39 mg, 7.06 pmol) and 23-((2,5-dioxopyrrolidin- l-yl)oxy)-23-oxotricosanoic acid (8.50 mg, 15.2 ⁇ mo) (Intermediate 7). The yield was 17 mg (39%).
• the compound was made according to Example 1 C20-acid-MALATl-LNA starting from MALATl-LNA-hexylamine (41 mg, 7.42 ⁇ mol) and 24-((2,5-dioxopyrrolidin- l-yl)oxy)-24-oxotetracosanoic acid (7.36 mg, 0.01 mmol) (Intermediate 8). Reaction time: over night. The yield was 23 mg (50%).
• EXAMPLE 10 SYNTHESIS OF C 18 (9Z) SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• MALATl-LNA-hexylamine 180 mg, 0.03 mmol was dissolved in Borate buffer 0.1M pH 9.5 (4.8 ml).
• (Z)-18-((2,5-dioxopyrro lidin- l-yl)oxy)-18-oxooctadec-9-enoic acid (Intermediate 11) (26.7 mg, 0.07 mmol) dissolved in acetonitrile (1.2 mL) was added and the mixture was stirred at RT for 45 m. Additional 1 eqv of (Intermediate 11) was added and the mixture was stirred for additional 2 h. IM NaOH solution (1.4 ml) was added during 1 h.
• EXAMPLE 11 SYNTHESIS OF C22 CLICK SATURATED ACID CONJUGATE OF MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the mixture was centrifuged at 0 °C for 10 mins at 3500 rpm, the clear supernatant was removed. The pellet was dried under vacuum. The residue was purified on a XBridge C18, 5pm 19x150 mm column by using a gradient from 10-90% acetonitrile in NH4HCO3 (50 mM, pH8) at R.T. The pure fractions was freeze-dried twice. Yield: 5.0 mg.
• Example 1 C20-acid-MALATl-LNA
• CamK2D ASO -hexylamine 40 mg, 7.24 ⁇ mol
• 22-((2,5-dioxopyrrolidin-l- yl)oxy)-22-oxodocosanoic acid (Intermediate 1) (6.77 mg, 14.5 ⁇ mol).
• Reaction time 90 min.
• CAMK2D ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 12) [00165] The compound was made according to Example 2 C22-acid-MALATl-LNA starting from CamK2D ASO -hexylamine (35 mg, 5.68 ⁇ mol) and 22-((2,5-dioxopyrrolidin-l- yl)oxy)-22-oxodocosanoic acid (Intermediate 1) (5.31 mg, 11.4 ⁇ mol). Reaction time: 40 min.
• CAMK2D ANTISENSE OLIGONUCLEOTIDE SEQ ID NO: 13
• the compound was made according to Example 2 C22-acid-MALATl-LNA starting from CamK2D ASO -hexylamine (35 mg, 5.68 ⁇ mol) and 22-((2,5-dioxopyrrolidin-l- yl)oxy)-22-oxodocosanoic acid (Intermediate 1) (5.31 mg, 11.4 ⁇ mol). Reaction time: 40 min. Purified on a XBridge C18, 5 ⁇ m 19x150 mm column by using a gradient from 10-50% acetonitrile 1 Imin, 50-90% acetonitrile in NH 4 HCO 3 (50 mM, pH8) at R.T. The yield was 18.5 mg (53%).
• PEG-4 MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE SEQ ID NO: 8
• EXAMPLE 18 SYNTHESIS OF BILIPID C22 SATURATED ACID CONJUGATE OF PEG-4 MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the residue was purified on a XBridge Cl 8, 5 ⁇ m 19x150 mm column by using a gradient from 20-90% acetonitrile in NH 4 HCO 3 (50 mM, pH8) at R.T.
• the pure fractions were evaporated to 2/3 of the volume and freeze-dried twice. Yield: 3 mg (11%).
• the residue was purified on a XBridge C18, 5pm 19x150 mm column by using a gradient from 10-90% acetonitrile in NH4HCO3 (50 mM, pH8) at R.T. The pure fractions were freeze-dried twice. Yield: 13 mg (32.2%).
• EXAMPLE 20 SYNTHESIS OF C22 SATURATED ACID CONJUGATE OF CYSTEINE MALEIMIDE MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the oligo was precipitated by addition of ethanol (14mL), vortexed briefly and left standing at -20C for 30 min. The mixture was centrifuged at 0 °C for 10 mins at 3500 rpm and the clear supernatant was removed. The oligo was washed once more by dissolving the pellet in ImL of water and Sodium acetate 3M, pH 5.2 (100 ⁇ l) followed by addition of 14 mL of ethanol, cooled for 30 mins at -20, then centrifuged at 0 °C for 10 mins at 3500 rpm. The supernatant was discarded, and the product was dried in vacuum.
• the residue was purified on a XBridge Cl 8, 5pm 19x150 mm column by using a gradient from 5-90% acetonitrile in NH4HCO3 (50 mM, pH8) at R.T. The pure fractions were freeze-dried twice. Yield: 7.5 mg (26.4%).
• EXAMPLE 21 SYNTHESIS OF C22 SATURATED ACID CONJUGATE OF PEG CYSTEINE MALEIMIDE MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the mixture was extracted with diethylether/tetrahydofuran (0.5ml) to remove excess of Intermediate 21.
• To the water phase was then added ethanol (14mL) to precipitate the oligo.
• the mixture was vortexed briefly and left standing at -20C for 30 min, centrifuged at 0 °C for 10 mins at 3500 rpm and the clear supernatant was removed.
• the oligo was washed once more by dissolving the pellet in 1 mL of water and adding sodium acetate 3M, pH 5.2 ( 100 ⁇ l) followed by addition of 14 mL of ethanol, cooled for 30 mins at -20, then centrifuged at 0 °C for 10 mins at 3500 rpm.
• EXAMPLE 23 SYNTHESIS OF C22 SATURATED ACID CONJUGATE OF LYS-PEG SQUARIC AMIDE MALAT-1 LNA ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO: 8)
• the product was purified on a XBridge Cl 8, 5 ⁇ m 19x150 mm column by using a gradient from 10-90% acetonitrile in NH4HCO3 (50 mM, pH8) at R.T. The pure fractions were freeze-dried twice. Yield: 8.5 mg (25.7%).
• the mixture was stirred for 30 min at 45 °C.
• the product was purified on a XBridge Cl 8, 5 ⁇ m 19x150 mm column by using a gradient from 10-90% acetonitrile in NH 4 HCO 3 (50 mM, pH8) at R.T. The pure fractions were freeze-dried twice. Yield 12.5mg (32.3%)
• THP-1 cells (ATCC® TIB-202TM) were cultured according to standard procedures in RPMI 1640 with GlutaMax, 2g/L glucose, HEPES, MEM Non-Essential Amino Acids, ImM sodium pyruvate, 10% FBS and 50pM P-mercaptoethanol. Cells were collected by centrifugation, resuspended in serum free medium and plated at 70.000 cells per well in 96 well culture plates. FA-ASO conjugates were dosed into the medium at final concentrations 1, 0.3, 0.1, and 0.03 pM.
• LA-4 cells (ATCC CCL- 196 ® TM) were cultured according to standard procedures in Ham’s F12 nutrient mix media with GlutaMax, 1.8g/L glucose, 1% MEM Non-Essential Amino Acids and 15% FBS. Cells were trypsinised, resuspended in standard culture medium and plated at 6000 cells per well in 384 well culture plates. 16h later the media was removed from the cells and replaced with serum free media. 12 -point dilution series (concentration range 0.000085-15 ⁇ M) were prepared for both naked and C22 lipid conjugated ASOs. These were dosed into the medium and cells were incubated at 37°C, 5% CO2 for 24 hours.
• Real-Time PCR reactions were set up using 3pL cDNA, TaqManTM Fast Advanced Master Mix, and Malat-1 or RplpO TaqManTM Gene Expression Assays (Mm00499266_ml and Mm00725448_sl, all Applied Biosystems) in a total volume of lOpL. Amplifications were performed on a QuantStudioTM 7 Flex Real-Time PCR System (Applied Biosystems) and were conducted at 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
• Cq Quantification cycle
• mice Male B6NTac mice arrived at 8- 10 weeks of age, 2/cage-housed and placed on Chow Diet. Mice were allowed to acclimate for at least one week before subjected to the study. On day -1 , mice were weighed and randomized to appropriate drug treatment groups based on the body weight. Compounds were dosed by subcutaneous injection through tail vein at 5mg/Kg in PBS at pH7.4 at days 0, 2, 4, 7 and 21. Mice were euthanized via CO 2 inhalation at day 28. The hearts were removed and approximately 20 mg of apex were weighed out for RNA extraction. One piece of the liver left lobe was snap frozen in liquid nitrogen for RNA extraction. The right kidney were removed and snap frozen for RNA extraction. Knock-down effect was evaluated by RT-qPCR as described in example 26.
• FIGS. 4A-B show that conjugation to a saturated C 22 acid or C 18 (9Z) monounsaturated fatty diacid led to similar or increased knock down in the heart but also to an attenuation of the knock down measured in the liver and kidney compared to the parent ASO.
• mice Male B6NTac mice arrived at 8- 10 weeks of age, 2/cage-housed and placed on Chow Diet. Mice were allowed to acclimate for at least one week before subjected to the study. On day 0, mice were weighed and randomized to appropriate drug treatment groups based on the body weight. On dayl compounds were dosed by subcutaneous injection through tail vein at 5mg/Kg in PBS at pH7.4. Mice were euthanized via CO2 inhalation at day 4. The hearts were removed and approximately 20 mg of apex were weighed out for RNA extraction. One piece of the liver left lobe was snap frozen in liquid nitrogen for RNA extraction. The right kidney was removed and snap frozen for RNA extraction. Knock-down effect was evaluated as described in example 26.

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• 2021
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