EP3735461A1 - Nucleic acids for inhibiting expression of aldh2 in a cell - Google Patents
Nucleic acids for inhibiting expression of aldh2 in a cellInfo
- Publication number
- EP3735461A1 EP3735461A1 EP18803643.8A EP18803643A EP3735461A1 EP 3735461 A1 EP3735461 A1 EP 3735461A1 EP 18803643 A EP18803643 A EP 18803643A EP 3735461 A1 EP3735461 A1 EP 3735461A1
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- European Patent Office
- Prior art keywords
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- nucleic acid
- nucleotides
- modified
- rna
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- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C12Y—ENZYMES
- C12Y102/00—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2)
- C12Y102/01—Oxidoreductases acting on the aldehyde or oxo group of donors (1.2) with NAD+ or NADP+ as acceptor (1.2.1)
- C12Y102/01003—Aldehyde dehydrogenase (NAD+) (1.2.1.3)
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/317—Chemical structure of the backbone with an inverted bond, e.g. a cap structure
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/321—2'-O-R Modification
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/32—Chemical structure of the sugar
- C12N2310/322—2'-R Modification
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/35—Nature of the modification
- C12N2310/351—Conjugate
- C12N2310/3515—Lipophilic moiety, e.g. cholesterol
Definitions
- the present invention relates to products and compositions and their uses.
- the invention relates to nucleic acid products that interfere with or inhibit the aldehyde dehydrogenase-2 (ALDH2) gene expression and therapeutic uses of such repression such as for the treatment of alcohol use disorder and associated pathologies or syndromes.
- ADH2 aldehyde dehydrogenase-2
- Double-stranded RNA (dsRNA) able to complementarily bind expressed mRNA has been shown to be able to block gene expression (Fire et al., 1998, Nature. 1998 Feb
- RNA interference Short dsRNAs direct gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function. RNAi is mediated by the
- RNA-induced silencing complex a sequence-specific, multi-component nuclease that degrades messenger RNAs homologous to the silencing trigger loaded into the RISC complex.
- Interfering RNA such as siRNAs, antisense RNA, and micro RNA are oligonucleotides that prevent the formation of proteins by gene-silencing i.e. inhibiting gene translation of the protein through degradation of mRNA molecules.
- Gene-silencing agents are becoming increasingly important for therapeutic applications in medicine. According to Watts and Corey in the Journal of Pathology (2012; Vol 226, p 365-379) there are algorithms that can be used to design nucleic acid silencing triggers, but all of these have severe limitations.
- potent iRNAs it may take various experimental methods to identify potent iRNAs, as algorithms do not take into account factors such as tertiary structure of the target mRNA or the involvement of RNA binding proteins. Therefore, the discovery of a potent nucleic acid silencing trigger with minimal off-target effects is a complex process. For the pharmaceutical development of these highly charged molecules it is necessary that they can be synthesised economically, distributed to target tissues, enter cells and function within acceptable limits of toxicity.
- a first aspect of the invention relates to a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of a RNA transcribed from the ALDH2 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25, 27 or 55.
- the nucleic acid comprises in the first strand a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably at least 19 nucleotides of any one of the reference sequences SEQ ID NO: 1 1 , 17, 3, 1 , 5, 7, 9, 13, 15, 19, 21 , 23, 25 27 or 55.
- the number of single nucleotide mismatches in the first strand sequence relative to the portion of the reference sequence that is comprised in the first strand sequence is at most three, preferably at most two, more preferably at most one and most preferably zero
- the second strand may comprise a nucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.
- the first strand may comprise the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17 and/or the second strand may comprise the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:18.
- the first strand and/or the second strand may each be from 17-35 nucleotides in length and at least one duplex region may be from 10-25 nucleotides in length.
- the duplex may comprise two separate strands or it may comprise a single strand which comprises the first strand and the second strand.
- the nucleic acid may: a) be blunt ended at both ends; b) have an overhang at one end and a blunt end at the other; or c) have an overhang at both ends.
- One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides.
- One or more of the odd numbered nucleotides of the first strand may be modified.
- One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd nucleotides.
- At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.
- a plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention.
- a plurality of even numbered nucleotides in the first strand may be modified by a second modification.
- the first strand may comprise adjacent nucleotides that are modified by a common modification.
- the first strand may also comprise adjacent nucleotides that are modified by a second different modification.
- One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and/or one or more of the even numbered nucleotides of the second strand may be modified by the same modification of the odd numbered nucleotides of the first strand.
- At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides.
- a plurality of odd numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first strand odd numbered nucleotides.
- a plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.
- the second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.
- a common modification which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.
- each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with a second different modification.
- the nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.
- the modification and / or modifications may each and individually be selected from the group consisting of 3'-terminal deoxy-thymine, 2'-0-methyl, a 2'-deoxy-modification, a 2'-amino-modification, a 2'-alkyl-modification, a morpholino modification, a
- the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide.
- At least one modification may be 2'-0-methyl and/or at least one modification may be 2'-F.
- the invention further provides, as a second aspect, a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially
- first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, wherein the nucleotides of first strand are modified by a first modification on the odd numbered nucleotides, and modified by a second modification on the even numbered nucleotides, and nucleotides of the second strand are modified by a third modification on the even numbered nucleotides and modified by a fourth modification on the odd numbered nucleotides, wherein at least the first modification is different to the second modification and the third modification is different to the fourth modification.
- the second strand may comprise a nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.
- the third modification and the first modification may be the same and/or the second modification and the fourth modification may be the same.
- the first modification may be 2'OMe and the second modification may be 2'F.
- the first strand may comprise the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17 and the second strand may comprise the nucleotide sequence of SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:18.
- a nucleic acid of the invention may comprise a phosphorothioate linkage between the terminal one, two or three 3' nucleotides and/or one, two or three 5' nucleotides of the first and/or the second strand. It may comprise two phosphorothioate linkages between each of the three terminal 3' and between each of the three terminal 5' nucleotides on the first strand, and two phosphorothioate linkages between the three terminal nucleotides of the 3' end of the second strand.
- Such a nucleic acid may be conjugated to a ligand.
- the invention further provides, as a third aspect, a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially
- said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, and wherein the nucleic acid is conjugated to a ligand.
- the ligand may comprise (i) one or more N-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moieties to a sequence as defined in any preceding aspects.
- the linker may be a bivalent or trivalent or tetravalent branched structure.
- the nucleotides may be modified as defined herein.
- the ligand may comprise the formula I:
- S represents a saccharide, wherein the saccharide is N-acetyl galactosamine
- X 1 represents C3-C6 alkylene or (-CH2-CH2-0) m (-CH2)2- wherein m is 1 , 2, or 3;
- P is a phosphate or modified phosphate (preferably a thiophosphate);
- A is a branching unit
- X 3 represents a bridging unit
- nucleic acid according to the present invention is conjugated to X 3 via a phosphate or modified phosphate (preferably a thiophosphate).
- nucleic acid as defined herein before.
- a nucleic acid according to the present invention may be conjugated to a ligand of the following structure
- the present invention also relates to a conjugate for inhibiting expression of a ALDH2 gene in a cell, said conjugate comprising a nucleic acid portion, comprising the nucleic acid of any aspect of the invention, and ligand portions, said nucleic acid portion comprising at least one duplex region that comprises at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said ALDH2 gene, said ligand portions comprising a linker moiety, preferably a serinol-derived linker moiety, and a targeting ligand for in vivo targeting of cells and being conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, wherein:
- the second RNA strand is conjugated at the 5' end to the targeting ligand, and wherein (a) the second RNA strand is also conjugated at the 3' end to the targeting ligand and the 3' end of the first RNA strand is not conjugated; or (b) the first RNA strand is conjugated at the 3' end to the targeting ligand and the 3' end of the second RNA strand is not conjugated; or (c) both the second RNA strand and the first RNA strand are also conjugated at the 3' ends to the targeting ligand; or
- both the second RNA strand and the first RNA strand are conjugated at the 3' ends to the targeting ligand and the 5' end of the second RNA strand is not conjugated, or the present invention relates to a conjugate for inhibiting expression of a ALDH2 gene in a cell, said conjugate comprising a nucleic acid portion and ligand portions, said nucleic acid portion comprising at least one duplex region that comprises at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said ALDH2 gene, said ligand portions comprising a linker moiety and a targeting ligand for in vivo targeting of cells and being conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, wherein:
- the second RNA strand is conjugated at the 5' end to the targeting ligand, and wherein (a) the second RNA strand is also conjugated at the 3' end to the targeting ligand and the 3' end of the first RNA strand is not conjugated; or (b) the first RNA strand is conjugated at the 3' end to the targeting ligand and the 3' end of the second RNA strand is not conjugated; or (c) both the second RNA strand and the first RNA strand are also conjugated at the 3' ends to the targeting ligand; or (ii) both the second RNA strand and the first RNA strand are conjugated at the 3' ends to the targeting ligand and the 5' end of the second RNA strand is not conjugated.
- the linker moiety may for example be a serinol-derived linker moiety or one of the other linker types described herein.
- the invention also provides a composition comprising the nucleic acid or conjugated nucleic acid of any aspect of the invention, and a physiologically acceptable excipient.
- nucleic acid or conjugated nucleic acid for use in the treatment of a disease, disorder or syndrome and/or in the manufacture of a medicament for treating a disease, disorder, or syndrome.
- the invention provides a method of treating or preventing a disease, disorder or syndrome comprising administration of a composition comprising a nucleic acid or conjugated nucleic acid according to any aspect of the invention to an individual in need of treatment.
- the nucleic acid or conjugated nucleic acid may be administered to the subject subcutaneously, intravenously or using any other application routes such as oral, rectal or intraperitoneal.
- the invention may be delivered in a tissue specific manner to liver (hepatocytes) and target specifically ALDH2, in order to reduce unwanted side effects and achieve a lower therapeutic dose necessary to achieve the desired effect.
- the disease, disorder or syndrome may be alcohol use disorder which includes acute alcohol sensitivity (hangover), alcoholic neuropathy (alcohol-related polyneuropathy) alcohol dependence, alcohol abuse (alcoholism) or foetal alcohol syndrome (alcohol- related neurodevelopmental disorder) or any other pathology associated to acute or prolonged excessive alcohol consumption.
- alcohol use disorder which includes acute alcohol sensitivity (hangover), alcoholic neuropathy (alcohol-related polyneuropathy) alcohol dependence, alcohol abuse (alcoholism) or foetal alcohol syndrome (alcohol- related neurodevelopmental disorder) or any other pathology associated to acute or prolonged excessive alcohol consumption.
- the present invention relates to a nucleic acid which is double stranded and directed to an expressed RNA transcript of ALDH2 and compositions thereof.
- These nucleic acids or conjugated nucleic acids can be used in the treatment and prevention of a variety of diseases, disorders and syndromes where reduced expression of ALDH2 gene product is desirable.
- a first aspect of the invention relates to a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of a RNA transcribed from the ALDH2 gene, wherein said first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27.
- a related aspect of the invention is a nucleic acid for inhibiting expression of ALDH2 in a cell, wherein the nucleic acid comprises at least one duplex region that comprises a first strand and a second strand, wherein said second strand is at least partially complementary to the first strand, wherein said first strand comprises a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably at least 19 nucleotides of any one of the reference sequences SEQ ID NO: 1 1 , 17, 3, 1 , 5, 7, 9, 13, 15, 19, 21 , 23, 25, 27 or 55, and wherein the number of single nucleotide mismatches and/or deletions and/or insertions in the first strand sequence relative to the portion of the reference sequence that is comprised in the first strand sequence is at most three, preferably at most two, more preferably at most one and most preferably zero.
- the first strand of the nucleic acid comprises a sequence of at least 18 nucleotides of any one of the reference sequences, preferably of any one of the reference sequences SEQ ID NO: 1 1 , 17 and 3, and wherein the number of single-nucleotide mismatches and/or deletions and/or insertions in the first strand sequence relative to the portion of the reference sequence that is comprised in the first strand sequence is at most one, and preferably zero.
- the first strand of the nucleic acid comprises a sequence of at least 19 nucleotides of any of the reference sequences SEQ ID NO: 1 1 , 17 and 3.
- a certain number of mismatches, deletions or insertions between the first (antisense) strand and the target sequence, or between the first strand and the second (sense) strand can be tolerated in the context of siRNA and even have the potential in certain cases to increase activity.
- nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner.
- the nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand.
- the first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which 'folds' back to form a double stranded molecule.
- the nucleic acid may be an siRNA molecule.
- the nucleic acid may comprise ribonucleotides, modified ribonucleotides,
- the nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand).
- the duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive. By duplex region it is meant the region in two complementary or substantially
- oligonucleotide strand having 21 nucleotide units can base pair with another
- oligonucleotide of 21 nucleotide units yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the "duplex region" consists of 19 base pairs.
- the remaining base pairs may exist as 5' and 3' overhangs, or as single stranded regions.
- 100% complementarity is not required; substantial complementarity is allowable within a duplex region.
- complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another.
- the portion of the first strand and second strand that form at least one duplex region may be fully complementary and is at least partially complementary to each other.
- first and second strands must be able to hybridise under physiological conditions.
- the complementarity between the first strand and second strand in the at least one duplex region may be perfect in that there are no nucleotide mismatches or additional/deleted nucleotides in either strand. Alternatively, the complementarity may not be perfect. The complementarity may be from about 70% to about 100%. More specifically, the
- complementarity may be at least 70%, 75%, 80%, 85%, 90% or 95% and intermediate values.
- a portion of as for example in “one duplex region that comprises at least a portion of a first strand” should be understood to mean that the duplex region comprises at least 10, preferably at least 12, more preferably at least 14, yet more preferably at least 16, even more preferably at least 18 and most preferably all of the nucleotides of a given reference strand sequence.
- the portion of the reference sequence in the dublex region is at least 70%, preferably at least 80%, more preferably at least 90%, yet more preferably at least 95% and most preferably 100% identical to the corresponding portion of the reference sequence.
- the number of single nucleotide mismatches relative to the portion of the reference sequence is at most three, preferably at most two, more preferably at most one and most preferably zero.
- the first strand and the second strand may each comprise a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3
- the nucleic acid may comprise a second sequence comprising a nucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.
- the nucleic acid may comprise a first strand that comprises a nucleotide sequence of SEQ ID NO: 1 1 , and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 12; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 17, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 18; or a first strand that comprises a nucleotide sequence of SEQ ID NO: 3, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 4; or a first strand that comprises a nucleotide sequence of SEQ ID NO:1 , and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO: 2; or a first strand that comprises a nucleotide sequence of SEQ ID NO:5, and optionally wherein the second strand comprises a nucleotide sequence of SEQ ID NO:
- a nucleic acid involves the formation of a duplex region between all or a portion of the first strand and a portion of a target nucleic acid.
- the portion of the target nucleic acid that forms a duplex region with the first strand defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence, inclusive, is the target nucleic acid sequence or simply, target sequence.
- the duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a duplex structure with both the second strand and the target sequence, at least under physiological conditions.
- the complementarity between the first strand and the target sequence may be perfect (no nucleotide mismatches or additional/deleted nucleotides in either nucleic acid).
- the complementarity between the first strand and the target sequence may not be perfect.
- the complementarity may be from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 80%, 85%, 90% or 95% and intermediate values.
- the identity between the first strand and the complementary sequence of the target sequence may range from about 75% to about 100%. More specifically, the
- complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid is capable of reducing or inhibiting the expression of ALDH2.
- a nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of ALDH2 to the same level as a nucleic acid having perfect complementarity between the first strand and target sequence. Alternatively, it may be able to reduce expression of ALDH2 to a level that is 15% - 100% of the level of reduction achieved by the nucleic acid with perfect complementarity.
- the nucleic acid may comprise a first strand and a second strand that are each from 19-25 nucleotides in length.
- the first strand and the second strand may be of different lengths.
- the nucleic acid may be 15-25 nucleotide pairs in length.
- the nucleic acid may be 17-23 nucleotide pairs in length.
- the nucleic acid may be 17-25 nucleotide pairs in length.
- the nucleic acid may be 23-24 nucleotide pairs in length.
- the nucleic acid may be 19-21 nucleotide pairs in length.
- the nucleic acid may be 21-23 nucleotide pairs in length.
- the nucleic acid may comprise a duplex region that consists of 19-25 nucleotide base pairs.
- the duplex region may consist of 17, 18, 19, 20, 21 , 22, 23, 24 or 25 base pairs, which may be contiguous.
- the nucleic acid may comprise a first strand sequence of SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17.
- the nucleic acid may comprise a second strand sequence of SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:18.
- the nucleic acid mediates RNA interference.
- the nucleic acid for inhibiting expression of ALDH2 in a cell comprises at least one duplex region that comprises a first strand and a second strand that is at least partially complementary to the first strand, wherein said first strand comprises a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably at least 19 nucleotides with a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, yet more preferably at least 95% and most preferably 100% of any of sequences SEQ ID NOs: 1 1 , 17, 3, 1 , 5, 7, 9, 13, 15, 19, 21 , 23, 25, 27 or 55.
- SEQ ID NO: 55 is a 32-nucleotide sequence that corresponds to the RNA reverse complement of nucleotides 1 186 to 1217 of the ALDH2 reference sequence
- NM_000690.3 (SEQ ID NO: 55 - AUGUAGCCGAGGAUCUUCUUAAACUGAGUUUC).
- nucleic acid or conjugated nucleic acid as described may reduce the expression of ALDH2 by at least 15% compared to the expression observed in the absence of the nucleic acid or conjugated nucleic acid. All preferred features of any of the previous aspects also apply to this aspect.
- the expression of ALDH2 may be reduced to at least the following given % or less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15% or less, and intermediate values, than that observed in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.
- the nucleic acid may be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends.
- an "overhang” as used herein has its normal and customary meaning in the art, i.e. a single stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double strand nucleic acid.
- the term "blunt end” includes double stranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base-paired.
- the terminal nucleotide of a first strand and a second strand at a blunt end may be base paired.
- the terminal nucleotide of a first strand and a second strand at a blunt end may not be paired.
- the terminal two nucleotides of a first strand and a second strand at a blunt end may be base- paired.
- the terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.
- the nucleic acid may have an overhang at one end and a blunt end at the other.
- the nucleic acid may have an overhang at both ends.
- the nucleic acid may be blunt ended at both ends.
- the nucleic acid may be blunt ended at the end with the 5'-end of the first strand and the 3'-end of the second strand or at the 3'-end of the first strand and the 5'-end of the second strand.
- the nucleic acid may comprise an overhang at a 3'- or 5'-end.
- the nucleic acid may have a 3'-overhang on the first strand.
- the nucleic acid may have a 3'-overhang on the second strand.
- the nucleic acid may have a 5'-overhang on the first strand.
- the nucleic acid may have a 5'-overhang on the second strand.
- the nucleic acid may have an overhang at both the 5'-end and 3'-end of the first strand.
- the nucleic acid may have an overhang at both the 5'-end and 3'-end of the second strand.
- the nucleic acid may have a 5' overhang on the first strand and a 3' overhang on the second strand.
- the nucleic acid may have a 3' overhang on the first strand and a 5' overhang on the second strand.
- the nucleic acid may have a 3' overhang on the first strand and a 3' overhang on the second strand.
- the nucleic acid may have a 5' overhang on the first strand and a 5' overhang on the second strand.
- An overhang at the 3'-end or 5' end of the second strand or the first strand may be selected from consisting of 1 , 2, 3, 4 and 5 nucleotides in length.
- an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.
- Unmodified polynucleotides may be prone to degradation by cellular nucleases, and, as such, modifications/ modified nucleotides may be included in the nucleic acid of the invention. Such modifications may help to stabilise the nucleic acid by making them more resistant against nucleases. This improved resistance allows nucleic acids to be active in mediating RNA interference for longer time periods and is especially desirable when the nucleic acids are to be used for treatment.
- nucleic acid on the second and/or first strand of the nucleic acid of the invention may be modified. Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules.
- the nucleic acid according to the invention may be modified by chemical modifications. Modified nucleic acids can also minimise the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of a nucleic acid to a target cell.
- the modified nucleic acid of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand.
- a ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties.
- the ribonucleic acid may be modified by substitution or insertion with analogues of nucleic acids or bases.
- nucleic acid of the present invention may be modified.
- the nucleic acid may comprise at least one modified nucleotide.
- the modified nucleotide may be in the first strand.
- the modified nucleotide may be in the second strand.
- the modified nucleotide may be in the duplex region.
- the modified nucleotide may be outside the duplex region, i.e., in a single stranded region.
- the modified nucleotide may be on the first strand and may be outside the duplex region.
- the modified nucleotide may be on the second strand and may be outside the duplex region.
- the 3'-terminal nucleotide of the first strand may be a modified nucleotide.
- the 3'-terminal nucleotide of the second strand may be a modified nucleotide.
- the 5'-terminal nucleotide of the first strand may be a modified nucleotide.
- the 5'-terminal nucleotide of the second strand may be a modified nucleotide.
- a nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, 24-26 modified nucleotides or about 26-28 modified nucleotides.
- the nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides or vice versa.
- the nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate values of its activity as compared to the same nucleic acid but without said modified nucleotides, or may have more than 100% of the activity of the same nucleic acid without said modified nucleotides.
- the modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified.
- the modified nucleotides may be selected from the group consisting of a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-0-methyl modified nucleotide, a 2' modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a
- dT deoxy-thymine
- nucleotide 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a nucleotide comprising a 5' phosphate or 5' phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.
- the nucleic acid may comprise a nucleotide comprising a modified nucleotide, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine,inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5- alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6- alkylpyrimidine (e.g.
- 6-methyluridine 6-methyluridine
- propyne quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5'- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D- galactosylqueosine, 1 -methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3- methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7- methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6- isopentenyladenosine, beta-D-mannosylqueo
- Nucleic acids discussed herein include unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates.
- Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, for example as occur naturally in the human body.
- Modified nucleotide as used herein refers to a nucleotide in which one or more of the components of the nucleotides, namely sugars, bases, and phosphate moieties, are different from those which occur in nature.
- modified nucleotides While they are referred to as modified nucleotides they will of course, because of the modification, the term also includes molecules which are not nucleotides, for example a polynucleotide molecule in which the ribophosphate backbone is replaced with a non- ribophosphate construct that allows hybridisation between strands i.e. the modified nucleotides mimic the ribophosphate backbone.
- a modification may only occur at a 3' or 5' terminal position, may only occur in a terminal regions, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand.
- a modification may occur in a double strand region, a single strand region, or in both.
- a modification may occur only in the double strand region of a nucleic acid of the invention or may only occur in a single strand region of a nucleic acid of the invention.
- a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and/or in single strand regions, particularly at termini.
- the 5' end or 3' ends may be phosphorylated.
- Stability of a nucleic acid of the invention may be increased by including particular bases in overhangs, or to include modified nucleotides, in single strand overhangs, e.g., in a 5' or 3' overhang, or in both.
- Purine nucleotides may be included in overhangs. All or some of the bases in a 3' or 5' overhang may be modified. Modifications can include the use of modifications at the 2' OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as
- Overhangs need not be homologous with the target sequence.
- Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render
- Modified nucleic acids can include one or more of:
- alteration e.g., replacement, of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens (referred to as linking even if at the 5' and 3' terminus of the nucleic acid of the invention);
- modification of the 3' end or 5' end of the RNA e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g., a
- RNA fluorescently labelled moiety, to either the 3' or 5' end of RNA.
- modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates,
- Phosphorodithioates have both non-linking oxygens replaced by sulphur.
- One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).
- the phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
- a modified nucleotide can include modification of the sugar groups.
- the 2' hydroxyl group (OH) can be modified or replaced with a number of different "oxy" or "deoxy" substituents.
- dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino),— NHC(0)R (R alkyl, cycloalkyi, aryl, aralkyl, heteroaryl or sugar), cyano;
- a modified nucleotides may contain a sugar such as arabinose.
- Modified nucleotides can also include "abasic" sugars, which lack a nucleobase at C— V. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.
- the 2' modifications may be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate).
- phosphate linker modifications e.g., phosphorothioate
- the phosphate groups can individually be replaced by non-phosphorus containing connectors.
- moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate,
- replacements may include the methylenecarbonylamino and
- the phosphate linker and ribose sugar may be replaced by nuclease resistant
- Examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.
- PNA peptide nucleic acid
- the 3' and 5' ends of an oligonucleotide can be modified. Such modifications can be at the 3' end or the 5' end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group.
- an oligonucleotide can be conjugated to other functional molecular entities such as labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
- labelling moieties e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester).
- the functional molecular entities can be attached to the sugar through a phosphate group and/or a linker.
- the terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3' or C-5' O, N, S or C group of the sugar.
- the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs).
- a nucleotide surrogate e.g., PNAs
- the 3' end can be an— OH group.
- Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation.
- Terminal modifications useful for modulating activity include modification of the 5' end with phosphate or phosphate analogues.
- Nucleic acids of the invention, on the first or second strand, may be 5' phosphorylated or include a phosphoryl analogue at the 5' prime terminus.
- 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing.
- Suitable modifications include: 5'-monophosphate ((HO)2(0)P— 0-5'); 5'-diphosphate ((HO) 2 (0)P— O— P(HO)(0)— 0-5'); 5'-triphosphate ((HO) 2 (0)P— O— (HO)(0)P— O— P(HO)(0)— 0-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-0-5 - (HO)(0)P— O— (HO)(0)P— O— P(HO)(0)— 0-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N— 0-5'-(H0)(0)P— O— (HO)(0)P— O— P(HO)(0)— 0-5'); 5'-monothiophosphate (phosphorothioate; (HO) 2 (S)P— 0-5'); 5'- monodithiophosphate (phosphorodithio
- the nucleic acid of the present invention may include one or more phosphorothioate modifications on one or more of the terminal ends of the first and/or the second strand.
- each or either end of the first strand may comprise one or two or three phosphorothioate modified nucleotides.
- each or either end of the second strand may comprise one or two or three phosphorothioate modified nucleotides.
- Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye.
- Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.
- Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNAs having improved properties.
- nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.
- substituted or modified analogues of any of the above bases and "universal bases" can be employed.
- Examples include 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5- halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines
- non-pairing nucleotide analogue means a nucleotide analogue which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, N3-Me dC.
- the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.
- terminal functional group includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, ether groups.
- moieties may be linked to the 5' terminus of the first strand or the second strand. These include abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2' O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5'OMe nucleotide; and nucleotide analogues including 4',5'-methylene nucleotide; 1 -(3-D-erythrofuranosyl)nucleotide; 4'-thio nucleotide, carbocyclic nucleotide; 5'-amino-alkyl phosphate; 1 ,3-diamino-2-propyl phosphate, 3- aminoprop
- hydroxypropyl phosphate 1 ,5-anhydrohexitol nucleotide; alpha-nucleotide; threo- pentofuranosyl nucleotide; acyclic 3',4'-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted abasic moiety; 1 ,4-butanediol phosphate; 5 - amino; and bridging or non-bridging methylphosphonate and 5'-mercapto moieties.
- the nucleic acids of the invention may include one or more inverted nucleotides, for example inverted thymidine or inverted adenine (for example see Takei, et al., 2002. JBC 277 (26):23800-06).
- the term “inhibit”, “down-regulate”, or “reduce” with respect to gene expression means the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or activity of one or more proteins or protein subunits, is reduced below that observed in the absence of the nucleic acid or conjugated nucleic acid of the invention or in reference to an siRNA molecule with no known homology to human transcripts (herein termed non- silencing control).
- control may be conjugated and modified in an analogous manner to the molecule of the invention and delivered into the target cell by the same route; for example the expression may be reduced to 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, or to intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid or in the presence of a non-silencing control.
- the nucleic acid of the present invention may comprise an abasic nucleotide.
- abasic refers to moieties lacking a base or having other chemical groups in place of a base at the V position, for example a 3',3'-linked or 5',5'-linked deoxyabasic ribose derivative.
- the nucleic acid may comprise one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides. Alternating as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.
- One or more of the odd numbered nucleotides of the first strand of the nucleic acid of the invention may be modified wherein the first strand is numbered 5' to 3', the 5'-most nucleotide being nucleotide number 1 of the first strand.
- the term "odd numbered” as described herein means a number not divisible by two. Examples of odd numbers are 1 , 3, 5, 7, 9, 1 1 and so on.
- One or more of the even numbered nucleotides of the first strand of the nucleic acid of the invention may be modified, wherein the first strand is numbered 5' to 3'.
- the term "even numbered” as described herein means a number which is evenly divisible by two.
- Examples of even numbers are 2, 4, 6, 8, 10, 12, 14 and so on.
- One or more of the odd numbered nucleotides of the second strand of the nucleic acid of the invention may be modified wherein the second strand is numbered 3' to 5', the 3'-most nucleotide being nucleotide number 1 of the second strand.
- One or more of the even numbered nucleotides of the second strand of the nucleic acid of the invention may be modified, wherein the second strand is numbered 3' to 5'.
- One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides.
- One or more of the odd numbered nucleotides of the first strand may be modified.
- One or more of the even numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd nucleotides. At least one of the one or more modified even numbered nucleotides may be adjacent to at least one of the one or more modified odd numbered nucleotides.
- a plurality of odd numbered nucleotides in the first strand may be modified in the nucleic acid of the invention.
- a plurality of even numbered nucleotides in the first strand may be modified by a second modification.
- the first strand may comprise adjacent nucleotides that are modified by a common modification.
- the first strand may also comprise adjacent nucleotides that are modified by a second different modification.
- One or more of the odd numbered nucleotides of the second strand may be modified by a modification that is different to the modification of the odd numbered nucleotides on the first strand and/or one or more of the even numbered nucleotides of the second strand may be modified by the same modification of the odd numbered nucleotides of the first strand. At least one of the one or more modified even numbered nucleotides of the second strand may be adjacent to the one or more modified odd numbered nucleotides.
- a plurality of odd numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even numbered nucleotides may be modified by the same modification that is present on the first stand odd numbered nucleotides.
- a plurality of odd numbered nucleotides on the second strand may be modified by a second modification, wherein the second modification is different from the modification of the first strand odd numbered nucleotides.
- the second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a second modification that is different from the modification of the odd numbered nucleotides of the first strand.
- each of the odd numbered nucleotides in the first strand and each of the even numbered nucleotides in the second strand may be modified with a common modification and, each of the even numbered nucleotides may be modified in the first strand with a second modification and each of the odd numbered nucleotides may be modified in the second strand with the second modification.
- the nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.
- One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand.
- One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
- One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the even numbered nucleotides may be modified in the second strand.
- One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
- One or more or each of the odd numbered nucleotides may be modified in the first strand and one or more of the odd numbered nucleotides may be modified in the second strand by a common modification.
- One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
- One or more or each of the even numbered nucleotides may be modified in the first strand and one or more or each of the odd numbered nucleotides may be modified in the second strand by a common modification.
- One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.
- the nucleic acid of the invention may comprise single or double stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions can comprise up to about 12 nucleotides but preferably comprise from about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acid of the invention.
- the nucleic acid may comprise from 4 to about 10 nucleotides of alternating nucleotides at each termini (3' and 5') and these regions may be separated by from about 5 to about 12 contiguous unmodified or differently or commonly modified nucleotides.
- the odd numbered nucleotides of the first strand may be modified and the even numbered nucleotides may be modified with a second modification.
- the second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as the modification of the odd numbered nucleotides of the first strand.
- One or more nucleotides of second strand may also be modified with the second modification.
- One or more nucleotides with the second modification may be adjacent to each other and to nucleotides having a modification that is the same as the modification of the odd numbered nucleotides of the first strand.
- the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3' end and at the 5' end.
- the second strand may comprise a phosphorothioate linkage between the two
- the second strand may also be conjugated to a ligand at the 5' end.
- the nucleic acid of the invention may comprise a first strand comprising adjacent nucleotides that are modified with a common modification.
- One or more of such nucleotides may be adjacent to one or more nucleotides which may be modified with a second modification.
- One or more nucleotides with the second modification may be adjacent.
- the second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand.
- One or more nucleotides of second strand may also be modified with the second modification.
- One or more nucleotides with the second modification may be adjacent.
- the first strand may also comprise phosphorothioate linkages between the two nucleotides at the 5' end and at the 3' end.
- the second strand may comprise a phosphorothioate linkage between the two nucleotides at the 3' end.
- the second strand may also be conjugated to a ligand at the 5' end.
- the nucleotides numbered from 5' to 3' on the first strand and 3' to 5' on the second strand, 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23 and 25 may be modified by a modification on the first strand.
- the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand.
- the nucleotides numbered 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23 may be modified by a modification on the second strand.
- the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.
- Nucleotides are numbered for the sake of the nucleic acid of the present invention from 5' to 3' on the first strand and 3' to 5' on the second strand
- the nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a modification on the first strand.
- 17, 19, 21 , 23 may be modified by a second modification on the first strand.
- the nucleotides numbered 1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23 may be modified by a modification on the second strand.
- 18, 20, 22 and 24 may be modified by a second modification on the second strand.
- first and/or the second strand are shorter than 25 nucleotides in length, such as 19 nucleotides in length, there are no nucleotides numbered 20, 21 , 22, 23, 24 and 25 to be modified.
- nucleotides numbered 20, 21 , 22, 23, 24 and 25 there are no nucleotides numbered 20, 21 , 22, 23, 24 and 25 to be modified. The skilled person understands the description above to apply to shorter strands, accordingly.
- One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification.
- One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification.
- One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand.
- One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand.
- the alternating nucleotides can be aligned on the two strands such as, for example, all the modifications in the alternating regions of the second strand are paired with identical modifications in the first strand or alternatively the modifications can be offset by one nucleotide with the common modifications in the alternating regions of one strand pairing with dissimilar modifications (i.e. a second or further modification) in the other strand.
- dissimilar modifications i.e. a second or further modification
- the modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand, such that common modified nucleotides are not paired with each other.
- the modification and/or modifications may each and individually be selected from the group consisting of 3'-terminal deoxy-thymine, 2'-0-methyl, a 2'-deoxy-modification, a 2'-amino-modification, a 2'-alkyl-modification, a morpholino modification, a
- the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide. At least one modification may be 2'-0-methyl and/or at least one modification may be 2'-F. Further modifications as described herein may be present on the first and/or second strand.
- the nucleic acid of the invention may comprise an inverted RNA nucleotide at one or several of the strand ends.
- the nucleic acid comprises at least an inverted nucleotide at one or several of the 3' end of at least one of the strands and/or at the 5' end of the of the second strand. More preferably, the nucleic acid comprises an inverted nucleotde at the 3' end of the second strand. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3' end of the second strand and this nucleotide is preferably an inverted A.
- the inverted nucleotide is preferably present at an end of a strand not as an overhang but opposite a corresponding nucleotide in the other strand. A nucleic acid with such a modification is stable and easy to synthesise.
- “same or common modification” means the same modification to any nucleotide, be that A, G, C or U modified with a group such as a methyl group or a fluoro group. Is it not taken to mean the same addition on the same nucleotide.
- 2 ' F-dU, 2 ' F-dA, 2 ' F-dC, 2 ' F-dG are all considered to be the same or common modification, as are 2'-OMe-rU, 2'-OMe-rA; 2'-OMe-rC; 2'-OMe-rG.
- a 2'F modification is a different modification to a 2'OMe modification.
- the nucleic acid may comprise a modification and a second or further modification which are each and individually selected from the group comprising 2'-0- methyl modification and 2'-F modification.
- the nucleic acid may comprise a modification that is 2'-0-methyl (2'OMe) that may be a first modification, and a second modification that is 2'-F.
- the nucleic acid of the invention may also include a phosphorothioate modification and/or a deoxy modification which may be present in or between the terminal 1 , 2 or 3 nucleotides of each or any end of each or both strands.
- the invention provides as a further aspect, a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising a nucleotide sequence of SEQ ID NO:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, wherein the nucleotides of first strand are modified by a first modification on the odd numbered nucleotides, and modified by a second modification on the even numbered nucleotides, and nucleotides of the second strand are modified by a third modification on the even numbered nucleotides and modified by a fourth modification the odd numbered nucleotides, wherein at least the first modification is different to the second modification and the third modification is different to the fourth modification.
- the third and first modifications may be the same or different, the second and fourth modifications may be the same or different.
- the first and second modifications may be different to each other and the third and fourth modifications may be different to each other.
- the second strand may comprise a nucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.
- the nucleotides of the first strand may be modified by a first modification on the odd numbered nucleotides, and modified with a second modification on the even numbered nucleotides, and the second strand may be modified on the odd numbered nucleotides with the second modification and modified with the first modification on the even numbered nucleotides.
- the first modification may be 2'OMe and the second modification may be 2' F.
- the first strand may comprise the nucleotide sequence of SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17 and/or the second strand may comprise the nucleotide sequence of SEQ ID NO:4, SEQ ID NO: 12 or SEQ ID NO:18.
- the modifications may be those as set out in Table 1.
- the nucleic acid of the invention may be conjugated to a ligand.
- Efficient delivery of oligonucleotides, in particular double stranded nucleic acids of the invention, to cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins.
- One method of achieving specific targeting is to conjugate a ligand to the nucleic acid.
- the ligand helps in targeting the nucleic acid to the required target site.
- conjugate appropriate ligands for the desired receptor molecules in order for the conjugated molecules to be taken up by the target cells by mechanisms such as different receptor-mediated endocytosis pathways or functionally analogous processes.
- ASGP-R asialoglycoprotein receptor complex
- ASGP-R asialoglycoprotein receptor complex
- GalNAc N- Acetyl-D-Galactosylamine
- the asialoglycoprotein receptor complex (ASGP-R), which recognizes specifically terminal ⁇ -galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, P.H. et. al., Biochim. Biophys. Acta. 2002 Sep 19;1572(2-3):341-63) can be used for delivering a drug to the liver's hepatocytes expressing the receptor complex by covalent coupling of galactose or galactosamine to the drug substance (lshibashi,S.; et. al., J Biol. Chem. 1994 Nov 1 1 ;269(45):27803-6). Furthermore the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting moiety (Biessen EA, et al., J Med Chem. 1995 Apr 28;38(9):1538-46).
- the ASGP-R complex is a mediator for an active uptake of terminal ⁇ -galactosyl containing glycoproteins to the cell's endosomes.
- the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells (Akinc et al., Mol Ther. 2010 Jul; 18(7): 1357-64).
- the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell.
- the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).
- the saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose.
- the saccharide may be N-acetyl galactosamine (GalNAc).
- a ligand for use in the present invention may therefore comprise (i) one or more N-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moieties to a sequence as defined in any preceding aspects.
- the linker may be a bivalent or trivalent or tetravalent branched structure.
- the nucleotides may be modified as defined herein.
- GalNAc refers to 2-(Acetylamino)-2-deoxy-D- galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to “GalNAc” or “N-acetyl
- galactosamine includes both the ⁇ - form: 2-(Acetylamino)-2-deoxy-3 -D-galactopyranose and the oform: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose.
- Both the ⁇ -form: 2- (Acetylarnino)-2-deoxy-3-D-galactopyranose and oform: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose may be used interchangeably.
- the compounds of the invention comprise the ⁇ -form, 2-(Acetylarnino)-2-deoxy-3-D-galactopyranose.
- the ligand may therefore comprise GalNAc.
- the ligand may comprise a compound of formula I :
- S represents a saccharide, wherein the saccharide is N-acetyl galactosamine
- X 1 represents C3-C6 alkylene or (-CH2-CH2-0) m (-CH2)2- wherein m is 1 , 2, or 3;
- P is a phosphate or modified phosphate (preferably a thiophosphate);
- A is a branching unit
- X 3 represents a bridging unit
- nucleic acid according to the present invention is conjugated to X 3 via a phosphate or modified phosphate (preferably a thiophosphate).
- branching unit "A" branches into three in order to accommodate the three saccharide ligands.
- the branching unit is covalently attached to the remaining tethered portions of the ligand and the nucleic acid.
- the branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups.
- the branching unit may comprise groups selected from alkyl and ether groups.
- the branching unit A may have a structure selected from:
- each n independently represents an integer from 1 to 20.
- the branchin unit may have a structure selected from:
- each n independently represents an integer from 1 to 20.
- the branching unit may have the structure:
- the branching unit may have the structure:
- the branching unit may have the structure:
- the branching unit consists of only a carbon atom.
- the "X 3 " portion is a bridging unit.
- the bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.
- X 3 may be selected from -C1-C20 alkylene-, -C2-C20 alkenylene-, an alkylene ether of formula -(C1-C20 alkylene)-O-(Ci-C 20 alkylene)-, -C(0)-Ci-C 2 o alkylene-, -C0-C4 alkylene(Cy)C 0 -C 4 alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C1-C4 alkylene-NHC(0)-Ci- C 4 alkylene-, -C1-C4 alkylene-C(0)NH-Ci-C 4 alkylene-, -C1-C4 alkylene-SC(0)-Ci-C 4 alkylene-, -C1-C4 alkylene-C(0)S-Ci-C 4 alkylene-, -C1-
- X 3 may be an alkylene ether of formula -(C1-C20 alkylene)-0-(Ci-C2o alkylene)-.
- X 3 may be an alkylene ether of formula -(C1-C20 alkylene)-0-(C 4 -C2o alkylene)-, wherein said (C4-C20 alkylene) is linked to Z.
- X 3 may be selected from the group consisting of -CH2-O-C3H6-, - CH2-O-C4H8-, -CH 2 -0-C 6 Hi2- and -CH 2 -0-C 8 Hi6-, especially -CH2-O-C4H8-, -CH 2 -0-C 6 Hi2- and -CH2-O-C8H16-, wherein in each case the -CH 2 - group is linked to A.
- the ligand may comprise a compound of formula (II):
- S represents a saccharide
- X 1 represents C3-C6 alkylene or (-CH2-CH2-0) m (-CH2)2- wherein m is 1 , 2, or 3;
- P is a phosphate or modified phosphate (preferably a thiophosphate);
- X 2 is Ci-Ce alkylene
- A is a branching unit selected from:
- X 3 is a bridging unit
- nucleic acid according to the present invention is conjugated to X 3 via a phosphate or modified phosphate (preferably a thiophosphate)
- Branching unit A may have the structure: .
- Branching unit A may have the structure: , wherein X 3 is attached to the nitrogen atom.
- X 3 may be C1-C20 alkylene.
- X 3 is selected from the group consisting of -C3H6-, - C4H8-, -C6H12- and -C8H16-, especially -C4H8-, -C6H12- and - ⁇ -.
- the ligand may comprise a compound of formula (III):
- S represents a saccharide
- X 1 represents C3-C6 alkylene or (-CH2-CH2-0) m (-CH2)2- wherein m is 1 , 2, or 3;
- P is a phosphate or modified phosphate (preferably a thiophosphate);
- X 2 is an alkylene ether of formula -C3H6-O-CH2-;
- A is a branching unit
- X 3 is an alkylene ether of formula selected from the group consisting of -CH2-O-CH2-, - CH2-O-C2H4-, -CH2-O-C3H6-, -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O-C6H12-, -CH2-O- C7H14-, and -CH2-O-C8H16-, wherein in each case the -CH2- group is linked to A, and wherein X 3 is conjugated to a nucleic acid according to the present invention by a phosphate or modified phosphate (preferably a thiophosphate).
- a phosphate or modified phosphate preferably a thiophosphate
- the branching unit may comprise carbon.
- the branching unit is carbon.
- X 3 may be selected from the group consisting of -CH2-O-C4H8-, -CH2-O-C5H10-, -CH2-O- C6H12-, -CH2-O-C7H14-, and -CH2-O-C8H16-.
- X 3 is selected from the group consisting of -CH2-O-C4H8-, -CH 2 -0-C 6 Hi2- and -CH 2 -0-C 8 Hi 6 .
- P when P represents a modified phosphate group, P can be represented by:
- modified phosphate it is meant a phosphate group wherein one or more of the non- linking oxygens is replaced.
- modified phosphate groups include
- Phosphorodithioates have both non-linking oxygens replaced by sulphur.
- One, each or both non-linking oxygens in the phosphate group can be
- R is alkyl or aryl
- the phosphate can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates).
- the replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.
- the modified phosphate group is a thiophosphate group.
- P is a monothiophosphate. The inventors have found that conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo.
- the saccharide may be selected to have an affinity for at least one type of receptor on a target cell.
- the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).
- the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose.
- a ligand to be used in the present invention may include N-acetyl galactosamine (GalNAc).
- GalNAc N-acetyl galactosamine
- the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.
- GalNAc refers to 2-(Acetylamino)-2-deoxy-D- galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to “GalNAc” or “N-acetyl
- galactosamine includes both the ⁇ - form: 2-(Acetylamino)-2-deoxy-3 -D-galactopyranose and the oform: 2-(Acetylamino)-2-deoxy-a-D- galactopyranose.
- both the ⁇ -form: 2-(Acetylarnino)-2-deoxy-3-D-galactopyranose and oform: 2- (Acetylamino)-2-deoxy-a-D-galactopyranose may be used interchangeably.
- the compounds of the invention comprise the ⁇ -form, 2-(Acetylarnino)-2-deoxy-3-D- galactopyranose.
- X 1 may be (-CH2-CH2-0)(-CH2)2-.
- X 1 may be (-CH2-CH 2 -0)2(-CH 2 )2-.
- X 1 may be (-CH 2 -CH2-0)3(-CH 2 )2-.
- X 1 is (-CH 2 -CH 2 - 0)2(-CH2)2-.
- X 1 represents C3-C6 alkylene.
- X 1 may be propylene.
- X 1 may be butylene.
- X 1 may be pentylene.
- X 1 may be hexylene.
- the alkyl is a linear alkylene. In particular, X 1 may be butylene.
- X 2 represents an alkylene ether of formula -C3H6-O-CH2- i.e. C 3 alkoxy methylene, or -Ch ChbCI-bOCI-b-.
- Z is a nucleic acid as defined herein before.
- a ligand of formula (I), (II) or (III) can be attached at the 3'-end of the first (antisense) strand and/or at any of the 3'- and/or 5'-end of the second (sense) strand.
- the nucleic acid can comprise more than one ligand of formula (I), (II), or (III). However, a single ligand of formula (I), (II) or (III) is preferred because a single such ligand is sufficient for efficient targeting of the nucleic acid to the target cells.
- the 5'-end of the first (antisense) strand is not attached to a ligand of formula (I), (II) or (III), since a ligand in this position can potentially interfere with the biological activity of the nucleic acid.
- a nucleic acid with a single ligand of formula (I), (II) or (III) at the 5'-end of a strand is easier and therefore cheaper to synthesis than the same nucleic acid with the same ligand at the 3'-end.
- a single ligand of any of formulae (I), (II) or (III) is covalently attached to (conjugated with) the 5'-end of the second strand of the nucleic acid.
- the nucleic acid is conjugated to a ligand that comprises a lipid, and more preferably a ligand that comprises a cholesterol.
- a conjugate of the invention can comprise any nucleic acid as disclosed herein
- the present invention also relates to a conjugate for inhibiting expression of a ALDH2 gene in a cell, said conjugate comprising a nucleic acid portion, comprising the nucleic acid of any aspect of the invention, and at least one ligand portion, said nucleic acid portion comprising at least one duplex region that comprises at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said ALDH2 gene, said at least one ligand portion comprising a linker moiety, preferably a serinol-derived linker moiety, and a targeting ligand for in vivo targeting of cells and being conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, wherein: (i) the second RNA strand is conjugated
- both the second RNA strand and the first RNA strand are conjugated at the 3' ends to the targeting ligand and the 5' end of the second RNA strand is not conjugated.
- the second RNA strand i.e. the sense strand
- the first RNA strand i.e. the antisense strand
- the 3' end of the second RNA strand i.e. the sense strand
- the second RNA strand (i.e. the sense strand) is conjugated at the 5' end to the targeting ligand
- the second RNA strand (i.e. the sense strand) is also conjugated at the 3' end to the targeting ligand and the 3' end of the first RNA strand (i.e. the antisense strand) is not conjugated, such that a conjugate with the schematic structure as shown in Figure 59B is formed.
- both the second RNA strand i.e. the sense strand
- the first RNA strand i.e.
- the antisense strand are conjugated at the 3' ends to the targeting ligand and the 5' end of the second RNA strand (i.e. the sense strand) is not conjugated , such that a conjugate with the schematic structure as shown in Figure 59C is formed.
- the second RNA strand (i.e. the sense strand) is conjugated at the 5' end to the targeting ligand and both the second RNA strand (i.e. the sense strand) and the first RNA strand (i.e. the antisense strand) are also conjugated at the 3' ends to the targeting ligand, such that a conjugate with the schematic structure as shown in Figure 59D is formed.
- « ⁇ w indicates the linker which conjugates the ligand to the ends of the nucleic acid portion; the ligand may be a GalNAc moiety such as GalNAc; and the schematic structure as shown in Figure 59E represents the nucleic acid portion.
- the ligands may be monomeric or multimeric (e.g. dimeric, trimeric, etc.).
- the ligands are monomeric, thus containing a single targeting ligand moiety, e.g. a single GalNAc moiety.
- the ligands may be dimeric ligands wherein the ligand portions comprise two linker moieties, such as serinol-derived linker moieties or non-serinol linker moieties, each linked to a single targeting ligand moiety.
- the ligands may be trimeric ligands wherein the ligand portions comprise three linker moieties, such as serinol-derived linker moieties or non-serinol linker moieties, each linked to a single targeting ligand moiety.
- the two or three serinol-derived linker moieties may be linked in series e.g. as shown below: wherein n is 1 or 2 and Y is S or O.
- the ligands are monomeric.
- the conjugated RNA strands are conjugated to a targeting ligand via a linker moiety including a further linker wherein the further linker is or comprises a saturated, unbranched or branched CMS alkyl chain, wherein optionally one or more carbons (for example 1 , 2 or 3 carbons, suitably 1 or 2, in particular 1 ) is/are replaced by a heteroatom selected from O, N, S(0)p, wherein p is 0, 1 or 2 (for example a CH2 group is replaced with O, or with NH, or with S, or with SO2 or a -CH 3 group at the terminus of the chain or on a branch is replaced with OH or with NH 2 ) wherein said chain is optionally substituted by one or more oxo groups (for example 1 to 3, such as 1 group).
- the linker moiety is a serinol-derived linker moiety.
- the further linker comprises a saturated, unbranched CMS alkyl chain wherein one or more carbons (for example 1 , 2 or 3 carbons, suitably 1 or 2, in particular 1 ) is/are replaced by an oxygen atom.
- the further linker comprises a PEG-chain.
- the further linker comprises a saturated, unbranched C1-15 alkyl chain. More suitably, the further linker comprises a saturated, unbranched C1-6 alkyl chain.
- the further linker comprises a saturated, unbranched C 4 or C6 alkyl chain, e.g. a C 4 alkyl chain.
- > ⁇ / is a linking moiety of formula (V): wherein n, Y and Li are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- the targeting ligand portion is a linking moiety of formula (VI):
- n, Y and Li are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- > ⁇ ⁇ / is a linking moiety of formula (XIV):
- L is connected to the targeting ligand e.g. GalNAc and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- the targeting ligand portion is a linking moiety of formula (IV):
- n, Y, Ri and L are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- > ⁇ ⁇ / is a linking moiety of formula (VII):
- n, Y and l_2 are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- the targeting ligand portion is a linking moiety of formula (VIII):
- n, Y and L 2 are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- vr w is a linking moiety of formula (IX):
- F, Y and L are defined below and the O of the phosphoro-group is attached to the terminal oligonucleoside of the RNA strands.
- the targeting ligand portion is a linking moiety of formula (IXa):
- L is:
- the ligands are selected from GalNAc and galactose moieties, especially GalNAc moieties.
- GalNac may be replaced by another targeting ligand, e.g. a saccharide.
- the second RNA strand is a compound of formula (XI):
- c and d are independently 0 or 1 ;
- Zi and 2.2 are the RNA portions of the first and second RNA strands respectively;
- Y is O or S
- n 0, 1 , 2 or 3;
- Li is a linker to which a ligand is attached
- the first RNA strand is a compound of formula (XV):
- the second RNA strand is a compound of formula (XVI):
- c and d are independently 0 or 1 ;
- Zi and Z 2 are the RNA portions of the first and second RNA strands respectively;
- Y is O or S
- Ri is H or methyl
- n 0, 1 , 2 or 3;
- L is the same or different in formulae (XV) and (XVI) and is selected from the group consisting of:
- the first RNA strand is a compound of formula (XII):
- the second RNA strand is a compound of formula (XIII):
- c and d are independently 0 or 1 ;
- Zi and Z 2 are the RNA portions of the first and second RNA strands respectively;
- Y is O or S
- n 0, 1 , 2 or 3;
- L 2 is the same or different in formulae (XII) and (XIII) and is the same or different in moieties bracketed by b, c and d, and is selected from the group consisting of:
- F is a saturated branched or unbranched (such as unbranched) Ci-ealkyl (e.g. Ci- 6alkyl) chain wherein one of the carbon atoms is optionally replaced with an oxygen atom provided that said oxygen atom is separated from another heteroatom (e.g. an O or N atom) by at least 2 carbon atoms;
- L is the same or different in formulae (XII) and (XIII) and is selected from the group consisting of:
- the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein.
- b is 0, c is 1 and d is 1 ; b is 1 , c is 0 and d is 1 ; b is 1 , c is 1 and d is 0; or b is 1 , c is 1 and d is 1 .
- b is 0, c is 1 and d is 1 ; b is 1 , c is 0 and d is 1 ; or b is 1 , c is 1 and d is 1.
- b is 0, c is 1 and d is 1.
- Y is O. In another embodiment, Y is S.
- Ri is H or methyl. In one embodiment, Ri is H. In another embodiment, Ri is methyl.
- n is 0, 1 , 2 or 3.
- n is 0.
- L is selected from the group consisting of:
- L is:
- Example F moieties include (CH 2 )i-6 e.g. (CH 2 )i-4 e.g. CH 2 , (CH 2 )4, (CH 2 )5 or (CH 2 ) 6 , CH 2 0(CH 2 ) 2 -3, e.g. CH 2 0(CH 2 )CH 3 .
- L 2 is:
- L 2 is:
- L 2 is:
- L 2 is:
- n 0 and L 2 is:
- l_2 is typically the same. Between moieties bracketed by b, c and d, l_2 may be the same or different. In an embodiment, l_2 in the moiety bracketed by c is the same as the l_2 in the moiety bracketed by d. In an
- l_2 in the moiety bracketed by c is not the same as l_2 in the moiety bracketed by d.
- the l_2 in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.
- Serinol derived linker moieties may be based on serinol in any stereochemistry i.e.
- the serinol-GalNAc moiety (SerGN) has the following stereochemistry:
- the targeted cells are hepatocytes.
- Example compounds can be synthesised according to methods described below and known to the person skilled in the art. Whilst the schemes illustrate the synthesis of particular conjugates, it will be understood that other claimed conjugates may be prepared by analogous methods. Assembly of the oligonucleotide chain and linker building blocks may, for example, be performed by solid phase synthesis applying phosphoramidite methodology. Solid phase synthesis may start from a base or modified building block loaded lcaa CPG.
- Phosphoramidite synthesis coupling cycle consists of 1 ) DMT-removal, 2) chain elongation using the required DMT-masked phosphoramidite and an activator, which may be benzylthiotetrazole (BTT), 3) capping of non-elongated oligonucleotide chains, followed by oxidation of the P(lll) to P(V) either by Iodine (if phosphodiester linkage is desired) or EDITH (if phosphorothioate linkage is desired) and again capping (Cap/Ox/Cap or Cap/Thio/Cap).
- BTT benzylthiotetrazole
- GalNAc conjugation may be achieved by peptide bond formation of a GalNAc-carboxylic acid building block to the prior assembled and purified oligonucleotide having the necessary number of amino modified linker building blocks attached.
- the necessary building blocks are either commercially available or synthesis is described below. All final single stranded products were analysed by AEX-HPLC to prove their purity. Purity is given in %FLP (% full length product) which is the percentage of the UV-area under the assigned product signal in the UV-trace of the AEX-HPLC analysis of the final product. Identity of the respective single stranded products was proved by LC-MS analysis.
- ethyl trifluoroacetate NEt 3 , MeOH, 0°C, 16h, 2: 86% 5: 90%, ii) DMTCI, pyridine, 0°C, 16h, 74%, iii) LiBH4, EtOH/THF (1/1 , v/v), 0°C, 1 h, 76%, iv) 2-cyanoethyl-N,N- diisopropylchloro phosphoramidite, EtN/Pr 2 , CH2CI2, 56%, v) succinic anhydride, DMAP, pyridine, RT, 16h, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), RT, 18h, 29% (26 umol/g loading).
- (S)-DMT-Serinol(TFA)-phosphoramidite 7 can be synthesised from (L)-serine methyl ester derivative 1 according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).
- (S)-DMT-Serinol(TFA)-succinate 6 can be made by conversion of intermediate 5 with succinic anhydride in presence of a catalyst such as DMAP.
- Loading of 6 to a solid support such as a controlled pore glass (CPG) support may be achieved by peptide bond formation to a solid support such as an amino modified native CPG support (500A) using a coupling reagent such as HBTU.
- a coupling reagent such as HBTU.
- the (S)-DMT-Serinol(TFA)- succinate 6 and a coupling reagent such as HBTU is dissolved in a solvent such as CH3CN .
- a base, such as diisopropylethylamine, is added to the solution, and the reaction mixture is stirred for 2 min.
- a solid support such as a native amino-lcaa-CPG support (500 A, 3 g, amine content: 136 umol/g) is added to the reaction mixture and a suspension forms.
- the suspension is gently shaken at room temperature on a wrist-action shaker for 16h then filtered, and washed with solvent such as DCM and EtOH.
- the support is dried under vacuum for 2 h.
- the unreacted amines on the support can be capped by stirring with acetic anhydride/lutidine/N-methylimidazole at room temperature. Washing of the support may be repeated as above.
- the solid support is dried under vacuum to yield solid support 10.
- GalNAc synthon 9 can be prepared according to methods as described in Nair et al. (2014), starting from commercially available per-acetylated galactose amine 8.
- Oligonucleotide synthesis of 3' mono-GalNAc conjugated oligonucleotides is outlined in Figure 25 and summarised in Scheme 3. Synthesis is commenced using (S)-DMT-Serinol(TFA) -succinate-lcaa-CPG 10 as in example compound A0264. In case additional serinol building blocks are needed the (S)-DMT- serinol(TFA) amidite (7) is used in the appropriate solid phase synthesis cycle. For example, to make compound A0329, the chain assembly is finished with an additional serinol amidite coupling after the base sequence is fully assembled.
- oligonucleotide synthesis of 5' mono-GalNAc conjugated oligonucleotides may be commenced from a solid support loaded with the appropriate nucleoside of its respected sequence. In example compound A0220 this may be 2'fA.
- the oligonucleotide chain is assembled according to its sequence and as appropriate, the building block (S)-DMT-serinol(TFA)-amidite (7) is used.
- the protective DMT group of the last coupled amidite building block is removed, as in step 1 ) of the phosphoramidite synthesis cycle.
- the single strands can be cleaved off the solid support by treatment with an amine such as 40% aq. methylamine treatment. Any remaining protecting groups are also removed in this step and methylamine treatment also liberates the serinol amino function.
- the crude products were then purified each by AEX-HPLC and SEC to yield the precursor oligonucleotide for further GalNAc conjugation.
- GalNAc-conjugation was achieved by pre-activation of the GalN(Ac4)-C4-acid (9) by a peptide coupling reagent such as HBTU.
- the pre-activated acid 9 was then reacted with the amino-groups in 11 (e.g. A0264) to form the intermediate GalN(Ac4)-conjugates.
- the acetyl groups protecting the hydroxyl groups in the GalNAc- moieties were cleaved off by methylamine treatment to yield the desired example compounds 12 (e.g. A0268), which were further purified by AEX-HPLC and SEC.
- Amino modified building blocks other than serinol are commercially available from various suppliers and can be used instead of serinol to provide reactive amino-groups that allow for GalNAc conjugation.
- the commercially available building blocks shown in Table 6 below can be used to provide non-serinol-derived amino modified precursor oligonucleotides 14 (Scheme 5A) by using amino-modifier loaded CPG such as 10-1 to 10- 3 followed by sequence assembly as described above and finally coupling of amino-modifier phosophoramidites such as 13-1 , 13-2 or 13-4.
- GlyC3Am-CPG (10-2) was used in combination with GlyC3Am-Amidite 13-2.
- Structurally differing modifiers can be used to make 14, for example for A0651 C7Am-CPG was used in combination with C6Am-Amidite as second amino modification.
- commercially available amino-modifier loaded CPG 10-5 and amino-modified phosphoramidite 13-5 can be used to synthesise amino-modified precursor molecules 14 (A0655).
- C3Am-CPG (10-1 ) is: GlyC3Am-CPG (10-2) is: NHTFA
- C7Am-CPG (10-3) is:
- PipAm-CPG (10-5) is:
- C6Am-Phos (13-4) is: PipAm-Phos (13-5) is:
- the resulting precursor oligonucleotides 14 can then be conjugated with GalN(Ac4)-C4- acid (9) to yield the desired example compounds 15 (Scheme 6).
- Oligonucleotides synthesis of tri-antennary GalNAc-cluster conjugated siRNA is outlined in Figure 26. Oligonucleotide chain assembly is commenced using base loaded support e.g. 5'DMT-2'FdA(bz)-succinate-lcaa-CPG as in example compound A0006.
- Phosphoramidite synthesis coupling cycle consisting of 1 ) DMT-removal, 2) chain elongation using the required DMT-masked phosphoramidite, 3) capping of non-elongated oligonucleotide chains, followed by oxidation of the P(lll) to P(V) either by Iodine or EDITH (if phosphorothioate linkage is desired) and again capping (Cap/Ox/Cap or Cap/Thio/Cap) is repeated until full length of the product is reached.
- individual single strands are dissolved in a concentration of 60 OD/mL in H2O. Both individual oligonucleotide solutions can be added together to a reaction vessel. For reaction monitoring a titration can be performed. The first strand is added in 25% excess over the second strand as determined by UV-absorption at 260nm. The reaction mixture is heated e.g. to 80°C for 5min and then slowly cooled to RT. Double strand formation may be monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand can be calculated and added to the reaction mixture. The reaction is heated e.g. to 80°C again and slowly cooled to RT. This procedure can be repeated until less than 10% of residual single strand is detected.
- Example compounds can be synthesised according to methods described below and known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks may, for example, be performed by solid phase synthesis applying phosphoramidite methodology. GalNAc conjugation may be achieved by peptide bond formation of a GalNAc-carboxylic acid building block to the prior assembled and purified oligonucleotide having the necessary number of amino modified linker building blocks attached.
- Scheme 1 Synthesis of DMT-serinol(TFA) linker svnthons
- ethyl trifluoroacetate NEt 3 , MeOH, 0°C, 16h, 2: 86% 5: 90%, ii) DMTCI, pyridine, 0°C, 16h, 74%, iii) LiBH4, EtOH/THF (1/1 , v/v), 0°C, 1 h, 76%, iv) 2-cyanoethyl-N,N- diisopropylchloro phosphoramidite, EtN/Pr 2 , CH2CI2, 56%, v) succinic anhydride, DMAP, pyridine, RT, 16h, 38%, vi) HBTU, DIEA, amino-lcaa CPG (500A), RT, 18h, 29%.
- DMT-Serinol(TFA)-phosphoramidite 7 can be synthesised from serinol derivative 1 according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135).
- DMT-Serinol(TFA)-succinate 6 can be made by conversion of intermediate 5 with succinic anhydride in presence of a catalyst such as DMAP.
- Loading of 6 to a solid support such as a CPG support may be achieved by peptide bond formation to a solid support such as an amino modified native CPG support (500A) using a coupling reagent such as HBTU.
- a coupling reagent such as HBTU.
- the DMT-Serinol(TFA)-succinate 6 and a coupling reagent such as HBTU is dissolved in a solvent such as CH 3 CN.
- a base such as diisopropylethylamine, is added to the solution, and the reaction mixture is stirred for 2 min.
- a solid support such as a native amino-lcaa-CPG support (500 A, 3 g, amine content: 136 micromol/g) is added to the reaction mixture and a suspension forms.
- the suspension is gently shaken at room temperature on a wrist-action shaker for 16h then filtered and washed with solvent such as DCM and EtOH.
- solvent such as DCM and EtOH.
- the support is dried under vacuum for 2 h.
- the unreacted amines on the support can be capped by stirring with acetic anhydride/lutidine/N- methylimidazole at room temperature. Washing of the support may be repeated as above.
- the solid support is dried under vacuum to yield solid support 10.
- GalNAc synthon 9 can be prepared according to methods as described in Nair et al. J. Am. Chem. Soc, 2014, 136 (49), pp 16958-16961 , starting from commercially available per-acetylated galactose amine 8.
- Oligonucleotides can be synthesized on an AKTA oligopilot 10 synthesizer using standard phosphoramidite chemistry which is described in detail below.
- Oligonucleotide synthesis of 3' and 5' GalNAc conjugated oligonucleotides precursors is outlined in Figure 29 and summarised in Scheme 3. Synthesis is commenced using DMT-Serinol(TFA)-succinate-lcaa-CPG 10. A phosphoramidite synthesis cycle is applied until full length of the product was reached. Upon completion of chain elongation, the protective DMT group of the last coupled amidite building block can be removed in the same manner as in every individual synthesis cycle.
- example compound X0385B-prec which has a serinol-derived linker moiety at the 3' and 5' ends of the second strand
- the chain assembly was finished with an additional serinol amidite coupling after the base sequence was fully assembled.
- the single strands can be cleaved off the solid support by treatment with an amine such as 40% aq. methylamine. Any remaining protecting groups are also removed in this step and methylamine treatment also liberates the serinol amino function.
- the resulting crude oligonucleotide can be purified by ion exchange chromatography (Resource Q, 6ml_, GE Healthcare) on a AKTA Pure HPLC System using a gradient such as a sodium chloride gradient. Excess salt from I EX purification can be removed by SEC to yield the amino modified precursor oligonucleotide 11. Product containing fractions are pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.
- Conjugation of the GalNAc synthon 9, as described above, can be achieved by coupling 9 to the serinol-amino function of the respective oligonucleotide strand 11 using standard peptide coupling conditions known to the skilled person.
- the respective amino- modified precursor molecule 11 is dissolved in H2O and a polar solvent such as DMSO (e.g. DMSO/H2O, 2/1 , v/v) is added, followed by a base such as DIPEA (e.g. 2.5% of total volume).
- a polar solvent such as DMSO (e.g. DMSO/H2O, 2/1 , v/v)
- DIPEA e.g. 2.5% of total volume.
- pre-activation of the GalNAc synthon 9 can be performed by reacting 2 eq.
- acetyl hydroxy-protecting groups are removed under basic conditions, such as 40% MeNH 2 (1 ml_ per 500 OD). After 15 min at RT H 2 0 (1 :10 v/v) is added and compound 12 (such as X0385B shown in Figure 7) are isolated, purified again by anion exchange and size exclusion chromatography and then lyophilised.
- Individual single strands are dissolved in a concentration of 60 OD/mL in H2O. Both individual oligonucleotide solutions can be added together to a reaction vessel. For reaction monitoring a titration can be performed. The first strand is added in 25% excess over the second strand as determined by UV-absorption at 260nm. The reaction mixture is heated e.g. to 80°C for 5min and then slowly cooled to RT. Double strand formation may be monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand can be calculated and added to the reaction mixture. The reaction is heated e.g. to 80°C again and slowly cooled to RT. This procedure can be repeated until less than 10% of residual single strand is detected.
- the above process may be easily adapted to replace GalNac with another targeting ligand e.g. a saccharide.
- the present invention relates to a conjugate for inhibiting expression of a ALDH2 gene in a cell, said conjugate comprising a nucleic acid portion and ligand portions, said nucleic acid portion comprising at least one duplex region that comprises at least a portion of a first RNA strand and at least a portion of a second RNA strand that is at least partially complementary to the first strand, wherein said first strand is at least partially complementary to at least a portion of RNA transcribed from said ALDH2 gene, said ligand portions comprising a linker moiety and a targeting ligand for in vivo targeting of cells and being conjugated exclusively to the 3' and/or 5' ends of one or both RNA strands, wherein the 5' end of the first RNA strand is not conjugated, wherein: (i) the second RNA strand is conjugated at the 5' end to the targeting ligand, and wherein (a) the second RNA strand is also conjugated at the 3' end to the targeting ligand
- both the second RNA strand and the first RNA strand are conjugated at the 3' ends and the 5' end of the second RNA strand is not conjugated.
- the linker may be a serinol-derived linker moiety.
- the invention provides, as another aspect, a nucleic acid for inhibiting expression of ALDH2 in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially
- the first strand comprises a nucleotide sequence selected from the following sequences: SEQ ID NOs:1 , 3, 5, 7, 9, 1 1 , 13, 15, 17, 19, 21 , 23, 25 or 27, wherein the nucleic acid is conjugated to a ligand.
- the second strand may comprise a nucleotide sequence of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 or 28.
- the nucleotides of the first and/or second strand may be modified, as herein described.
- the nucleic acid comprises SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17 and SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:18 conjugated to a ligand of formula I (as set out above), wherein the ligand is conjugated to the nucleic acid as described and wherein the first strand is modified with a 2'OMe modification on the odd numbered nucleotides, and modified with a 2'F on the even numbered nucleotides, and the second strand is modified with a 2'OMe on the even numbered nucleotides and modified with a 2'F on the odd numbered nucleotides.
- the nucleic acid comprises SEQ ID NO:3, SEQ ID NO:1 1 or SEQ ID NO:17 and SEQ ID NO:4, SEQ ID NO:12 or SEQ ID NO:18, wherein the nucleic acid is conjugated to a ligand of formula I (as set out above), and furthermore wherein the nucleic acid has a modification pattern as shown below which is an extract of Table 1 as herein provided.
- the ligand may comprise GalNAc and Figure 10A or Figure 10B further illustrate examples of the present invention.
- the present invention also provides pharmaceutical compositions comprising the nucleic acid or conjugated nucleic acid of the invention.
- the pharmaceutical compositions may be used as medicaments or as diagnostic agents, alone or in combination with other agents.
- one or more nucleic acid conjugates of the invention can be combined with a delivery vehicle (e.g., liposomes) and/or excipients, such as carriers, diluents.
- a delivery vehicle e.g., liposomes
- excipients such as carriers, diluents.
- Other agents such as preservatives and stabilizers can also be added.
- the nucleic acid or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose.
- the invention also includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids according to the present invention in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like. Dosage levels for the medicament and pharmaceutical compositions of the invention can be determined by those skilled in the art by routine experimentation.
- a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid or conjugated nucleic acid.
- the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight.
- Dosage levels may also be calculated via other parameters such as, e.g., body surface area.
- the pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilized form.
- compositions and medicaments of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose.
- the mammal may be selected from a human, a non-human primate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, a goat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig, or other species of relevance.
- the wording "ALDH2" or "ALDH2” as used herein denotes nucleic acid or protein in any of the above mentioned species, if expressed therein naturally or artificially, but preferably this wording denotes human nucleic acids or proteins.
- a further aspect of the invention relates to a nucleic acid or conjugated nucleic acid of the invention or the pharmaceutical composition comprising the nucleic acid or conjugated nucleic acid of the invention for use in the treatment of a disease, disorder or syndrome.
- the disease, disorder or syndrome may be alcohol use disorder which includes acute alcohol sensitivity (hangover), alcoholic neuropathy (alcohol-related polyneuropathy), alcohol dependence, alcohol abuse (alcoholism), or foetal alcohol syndrome (alcohol- related neurodevelopmental disorder) or any other pathology associated to acute or prolonged excessive alcohol consumption.
- the invention includes a pharmaceutical composition comprising one or more nucleic acids or conjugated nucleic acids according to the present invention in a physiologically/ pharmaceutically acceptable excipient, such as a stabiliser, preservative, diluent, buffer and the like.
- the pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilised form or adhered, absorbed or included to or into any other suitable galenic carrier substance such as pellets, tablets, capsules, nanoparticles, gels, tablets, beads or similar structures.
- the nucleic acid described herein may be capable of inhibiting the expression of ALDH2.
- the nucleic acid described herein may be capable of partially inhibiting the expression of ALDH2.
- Inhibition may be complete, i.e. 0% compared of the expression level of ALDH2 in the absence of the nucleic acid of the invention.
- Inhibition of ALDH2 expression may be partial, i.e. it may be 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or intermediate values of ALDH2 expression in the absence of a nucleic acid of the invention.
- a nucleic acid or conjugated nucleic acid of the invention, or compositions including the same may be for use in a regimen comprising treatments once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks, or in regimens with varying dosing frequency such as combinations of the before- mentioned intervals.
- the nucleic acid may be for use subcutaneously, intravenously or using any other application routes such as oral, rectal or intraperitoneal.
- the ALDH2 expression may be inhibited compared to untreated cells and/or subjects by a range from 15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or intermediate values.
- the level of inhibition may allow treatment of a disease associated with ALDH2 expression or overexpression, or may serve to further investigate the functions and physiological roles of the ALDH2 gene product.
- a further aspect of the invention relates to a nucleic acid or conjugated nucleic acid of the invention in the manufacture of a medicament for treating a disease, disorder or syndromes, such as those as listed above or additional pathologies associated with acute or prolonged excessive alcohol consumption, or additional therapeutic approaches where inhibition of ALDH2 expression is desired.
- a method of treating or preventing a disease, disorder or syndrome, such as those listed above comprising administration of a pharmaceutical composition comprising a nucleic acid or conjugated nucleic acid as described herein, to an individual in need of treatment (to improve such pathologies).
- the nucleic acid composition may be administered in a regimen comprising treatments twice every week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight weeks or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals.
- the nucleic acid or conjugated nucleic acid may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.
- nucleic acid or conjugated nucleic acid of the present invention can also be administered for use in combination with other therapeutic compounds, either
- a molecular conjugation to other biologically active molecular entities such as peptides, cellular or artificial ligands or small and large molecules is also possible.
- the nucleic acid or conjugated nucleic acid of the present invention can be produced using routine methods in the art including chemical synthesis or expressing the nucleic acid either in vitro (e.g., run off transcription) or in vivo.
- chemical synthesis or expressing the nucleic acid either in vitro (e.g., run off transcription) or in vivo.
- solid phase chemical synthesis or using a nucleic acid-based expression vector including viral derivates or partially or completely synthetic expression systems can be used to produce the nucleic acid of the invention in vitro, within an intermediate host organism or cell type, within an intermediate or the final organism or within the desired target cell.
- Methods for the production (synthesis or enzymatic transcription) of the nucleic acid described herein are known to persons skilled in the art.
- the invention consists of chemical molecular entities that mediate ALDH2 mRNA degradation by binding to the ALDH2 gene transcripts through cellular RNA interference mechanisms.
- the molecular compounds invented may be used as conjugates with, but are not limited to an N-acetylgalactosamin (GalNAc) sugar moiety that ensures hepatocyte-specific cellular uptake, though specific binding to the asialoglycoprotein receptor complex (ASGPR).
- GalNAc N-acetylgalactosamin
- the invention may be linked to other different chemical structures conferring different properties as referred to in the following.
- the use of a chemical modification pattern of the nucleic acids confers nuclease stability in serum and makes for example subcutaneous application route feasible.
- the present invention aims at conferring assistance in alcohol dishabituation inducing alcohol aversion with a better safety profile than the currently available treatments.
- the invention is characterized by high specificity at the molecular and tissue-directed delivery level.
- the invention is characterized by high specificity at the molecular and tissue-directed delivery level, potentially conferring a better safety profile than the currently available treatments.
- the invention also provides a nucleic acid according to any aspect of the invention described herein, wherein the first RNA strand has a terminal 5' (E)-vinylphosphonate nucleotide, and the terminal 5' (E)-vinylphosphonate nucleotide is linked to the second nucleotide in the first strand by a phosphodiester linkage.
- the first strand may include more than 1 phosphodiester linkage.
- the first strand may comprise phosphodiester linkages between at least the terminal three 5' nucleotides.
- the first strand may comprise phosphodiester linkages between at least the terminal four 5' nucleotides.
- the first strand may comprise formula (XVII):
- n is from 1 to (the total number of nucleotides in the first strand - 2), preferably wherein n is from 1 to (the total number of nucleotides in the first strand -3), more preferably wherein n is from 1 to (the total number of nucleotides in the first strand -
- the first strand may include at least one phosphorothioate (ps) linkage.
- the first strand may further comprise a phosphorothioate linkage between the terminal two 3' nucleotides or phosphorothioate linkages between the terminal three 3' nucleotides.
- the linkages between the other nucleotides in the first strand are phosphodiester linkages.
- the first strand may include more than 1 phosphorothioate linkage.
- the second strand may comprise a phosphorothioate linkage between the terminal two 3' nucleotides or phosphorothioate linkages between the terminal three 3' nucleotides.
- the second strand may comprise a phosphorothioate linkage between the terminal two 5' nucleotides or phosphorothioate linkages between the terminal three 5' nucleotides.
- the terminal 5' (E)-vinylphosphonate nucleotide is an RNA nucleotide.
- 5' (E) vinylphosphonate is a 5' phosphate mimic.
- a biological mimic is a molecule that is capable of carrying out the same function as and is structurally very similar to the original molecule that is being mimicked.
- 5' (E) vinylphosphonate mimics the function of a normal 5' phosphate, e.g. enabling efficient RISC loading.
- 5' (E) vinylphosphonate is capable of stabilizing the 5'-end nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases.
- One aspect of the invention is a nucleic acid as disclosed herein for inhibiting expression of a target gene in a cell, comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said first strand is at least partially
- RNA transcribed from said target gene complementary to at least a portion of RNA transcribed from said target gene, wherein said first strand includes modified nucleotides or unmodified nucleotides at a plurality of positions in order to facilitate processing of the nucleic acid by RISC.
- RISC in one aspect "facilitate processing by RISC” means that the nucleic acid can be processed by RISC, for example any modification present will permit the nucleic acid to be processed by RISC, suitably such that siRNA activity can take place.
- a nucleotide on the second strand that "corresponds to" a position on the first strand is suitably the nucleotide that base pairs with that nucleotide on the first strand.
- the nucleotide on the second strand which corresponds to position 13 of the first strand is the nucleotide that forms a base pair with position 13 of the first strand.
- the nucleotide on the second strand which corresponds to position 1 1 of the first strand is the nucleotide that forms a base pair with position 1 1 of the first strand.
- the nucleotide on the second strand which corresponds to position 12 of the first strand is the nucleotide that forms a base pair with position 12 of the first strand.
- This nomenclature may be applied to other positions of the second strand. For example, in a 19-mer nucleic acid which is double stranded and blunt ended, position 13 of the first strand would pair with position 7 of the second strand. Position 1 1 of the first strand would pair with position 9 of the second strand. This nomenclature may be applied to other positions of the second strand.
- the nucleotide that corresponds to position 13 of the first strand is suitably position 13 of the second strand, counting from the 3' of the second strand, starting from the first nucleotide of the double stranded region.
- position 1 1 of the second strand is suitably the 1 1 th nucleotide from the 3' of the second strand, starting from the first nucleotide of the double stranded region. This nomenclature may be applied to other positions of the second strand.
- the nucleotide on the second strand that "corresponds to" a position on the first strand may not necessarily form a base pair if that position is the position in which there is a mismatch, but the principle of the nomenclature still applies.
- Preferred is a first and second strand that are fully complementary over the duplex region (ignoring any overhang regions) and there are no mismatches within the double stranded region of the nucleic acid.
- nucleotide on the second strand which corresponds to position 12 of the first strand is not modified with a 2' O-methyl modification.
- This limitation on the nucleic acid may be seen with any other limitation described herein.
- nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides on the second strand which corresponds to position 1 1-13 of the first strand are not modified with a 2' O-methyl modification.
- Suitable naturally occurring modifications include, as well as 2 O' methyl, other 2' sugar modifications, in particular a 2' H
- a nucleic acid as disclosed herein comprising no more than 20%, such as no more than 15% such as more than 10%, of nucleotides which have 2' modifications that are not 2' O methyl modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both the first and second strands.
- a nucleic acid as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2' fluoro modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both strands.
- nucleic acid as disclosed herein wherein all nucleotides are modified with a 2' O-methyl modification except positions 2 and 14 from the 5' end of the first strand and the nucleotides on the second strand which correspond to position 1 1 , or 13, or 1 1 and 13, or 1 1 -13 of the first strand.
- nucleotides that are not modified with 2' O-methyl are modified with fluoro at the 2' position.
- nucleic acid as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2' position of the sugar.
- these nucleotides are modified with a 2'- fluoro modification where the modification is not a 2' O-Methyl modification.
- Nucleic acids of the invention may comprise one or more nucleotides modified at the 2' position with a 2' H, and therefore having a DNA nucleotide within the nucleic acid.
- Nucleic acids of the invention may comprise DNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5' end of the first strand. Nucleic acids may comprise DNA nucleotides on the second strand which correspond to position 1 1 , or 13, or 1 1 and 13, or 1 1 -13 of the first strand. In one aspect there is no more than one DNA per nucleic acid of the invention. Nucleic acids of the invention may comprise one or more LNA nucleotides. Nucleic acids of the invention may comprise LNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5' end of the first strand. Nucleic acids may comprise LNA on the second strand which correspond to position 1 1 , or 13, or 1 1 and 13, or 1 1 -13 of the first strand.
- the nucleic acid is modified on the first strand with alternating 2-0 methyl modifications and 2 fluoro modifications, and positions 2 and 14 (starting from the 5' end) are modified with 2' fluoro.
- the second strand is modified with 2' fluoro modifications at nucleotides on the second strand which correspond to position 1 1 , or 13, or 1 1 and 13, or 1 1-13 of the first strand.
- the second strand is modified with 2' fluoro modifications at positions 1 1-13 counting from the 3' end starting at the first position of the complementary (double stranded) region, and the remaining modifications are naturally occurring modifications, preferably 2' O-methyl.
- nucleic acids of the invention comprise one or more inverted ribonucleotides, preferably an inverted adenine, using a 5'-5' linkage or a 3'-3' linkage, preferably a 3'-3' linkage at the 3' end of the second strand.
- nucleic acid comprises one or more phosphorodithioate linkages, such as 1 , 2, 3 or 4 phosphorodithioate linkages. Preferably there are up to 4
- nucleic acids can be combined with all other aspects of the invention disclosed herein.
- nucleic acids which are siRNA molecules wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleic acid comprises one or more or all of:
- the second strand nucleotide corresponding to position 1 1 or 13 of the first strand is not modified with a 2' O-methyl modification, preferably wherein one or both of these positions comprise a 2' fluoro modification;
- the nucleic acid comprises at least 80% of all nucleotides having a 2'-0-methly modification; (v) the nucleic acid comprises no more than 20% of nucleotides which have 2' fluoro modifications.
- nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand and the nucleotides at positions 7 and/or 9, or 7 - 9 from the 5' end of the second strand are modified with a 2' fluoro modification, and at least 90% of the remaining nucleotides are 2'-0 methyl modified or comprise another naturally occurring 2' modification.
- Specific preferred examples, for a blunt double stranded 19 base nucleic acid, with no overhang, are:
- nucleic acid as disclosed herein wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotide at position 9 from the 5' end of the second strand is not modified with a 2' O- methyl modification
- a nucleic acid as disclosed herein wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides at position 7 and 9 from the 5' end of the second strand are not modified with a 2' O-methyl modification.
- a nucleic acid as disclosed herein wherein the nucleotides at positions 2 and 14 from the 5' end of the first strand are not modified with a 2' O-methyl modification, and the nucleotides at positions 7 - 9 from the 5' end of the second strand are not modified with a 2' O-methyl modification.
- Suitable naturally occurring modifications include, as well as 2 O' methyl, other 2' sugar modifications, in particular a 2' H
- a nucleic acid as disclosed herein comprising no more than 20%, such as no more than 15% such as more than 10%, of nucleotides which have 2' modifications that are not 2' O methyl modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both the first and second strands.
- a nucleic acid as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2' fluoro modifications on the first and/or second strand, preferably as a percentage of the total nucleotides of both strands.
- nucleic acid as disclosed herein wherein all nucleotides are modified with a 2' O-methyl modification except positions 2 and 14 from the 5' end of the first strand and the nucleotides at positions 7 and/or 9 from the 5' end of the second strand.
- nucleotides that are not modified with 2' O-methyl are modified with fluoro at the 2' position.
- nucleic acid as disclosed herein wherein all nucleotides are modified with a 2' O-methyl modification except positions 2 and 14 from the 5' end of the first strand and the nucleotides at positions 7 - 9 from the 5' end of the second strand.
- nucleotides that are not modified with 2' O-methyl are modified with fluoro at the 2' position.
- the second strand preferably does not have a 2' O-methyl group at nucleotides 8 or 9 or 10 counting from the 5' end of the duplex corresponding to positions 13, 12, and 1 1 of the first strand respectively.
- the second strand preferably does not have a 2' O-methyl group at nucleotides 9 or 10 or 1 1 counting from the 5' end of the duplex corresponding to positions 13, 12, and 1 1 of the first strand respectively.
- the present invention also relates to the unmodified sequences of all modified sequences disclosed herein.
- Figure 1 shows the results of a unconjugated RNAi molecule screen for inhibition of ALDH2 expression in human Hep3B cells.
- Figure 2 shows the dose response of ALDH2 siRNA molecules in human HepG2 cells.
- Figure 3 shows mRNA and protein reduction by ALDH2-hcm-2 siRNA in human HepG2 cells as well as its effect on ALDH enzymatic activity in HepG2 total cell extracts.
- Figure 4 shows the dose-response for GalNAc-coupled siRNAs targeting Aldh2 mRNA in primary mouse hepatocytes.
- Figure 5 shows the dose response for GalNAc-coupled siRNAs against ALDH2 mRNA in primary human hepatocytes.
- Figure 6 shows the reduction of ALDH2 mRNA by 15nM of GalNAc-coupled ALDH2-hcm- 2 siRNA in 1 ° human, cynomolgus and mouse hepatocytes.
- Figure 7 shows the comparison in dose-response on ALDH2 mRNA knockdown by GalNAc-conjugated ALDH2 siRNAs hcm2, hcm6, hcm9 in conjugation to L1 linker vs. conjugation to the L6 GalNAc-linker in 1 ° human hepatocytes.
- Figure 8a show Aldh2 mRNA knockdown in mice in vivo 10 days post treatment, 8b the resulting reduction of ALDH2 protein and 8c the resulting hepatic acetaldehyde metabolite levels in the same mice.
- Figure 9 shows the dose-dependent duration of the Aldh2 mRNA knockdown up to 41 days upon a single dose application in mice using as an example GalNAc-ALDH2-hcm6 and hcm9 conjugated to the L6 linked GalNAc moiety.
- Figures 10a and 10b show the structure of the GalNAc L1 and L6 ligands, respectively, to which the example oligonucleotides were conjugated.
- Figure 1 1 shows different siRNA duplexes containing inverted RNA nucleotides at both 3'- ends tested for serum stability.
- Figure 12 shows the influence on ALDH2 mRNA knockdown of inverted A, U, C and G RNA nucleotides at 3'-overhang positions using derivatives of the siRNA ALDH2-hcm2 (see Table 3).
- Figure 13 shows the influence on ALDH2 mRNA knockdown of inverted A, U, C and G RNA nucleotides at terminal 3' positions using derivatives of the siRNA ALDH2-hcm2 (see Table 3).
- Figure 14 shows different GalNAc-L6-linker conjugates of ALDH2-hcm2 containing inverted RNA nucleotides (see Table 4) tested for serum stability.
- Figure 15 shows the dose response of inverted RNA nucleotides at terminal 3' positions on the efficacy of ALDH2 mRNA knockdown using GalNAc-L6-conjugated derivatives of the siRNA ALDH2-hcm2 (see Table 4) by ASGPR receptor complex-mediated uptake in mouse primary hepatocytes.
- Figure 16 shows the results of an in vivo study in mice testing the influence of ivA at the first strand's 3'-end in GalNAc-L6 derivatives of ALDH2-hcm6 and ALDH2-hcm9 siRNA conjugates on the efficacy of ALDH2 mRNA knockdown.
- Figure 17 shows the effect of different modified derivatives of the GalNAc-siRNA conjugates of ALDH2-hcm6 and ALDH2-hcm9 (Table 5) on knockdown efficacy on Aldh2 mRNA expression levels in vivo in mice.
- FIG. 18 depicts Conjugate 1.
- FIG. 19 depicts Conjugate 2.
- FIG. 20 depicts Conjugate 3.
- Figure 21 depicts Reference Conjugate 1.
- Figure 22 depicts Reference Conjugate 2.
- Figure 23 depicts Reference Conjugate 3.
- Figure 24 depicts Reference Conjugate 4.
- the top strand is the antisense strand and the bottom strand is the sense strand.
- the nucleotide at the end of the respective conjugated strands is drawn in full.
- Figure 25 shows the synthesis of A0268, which is a 3' mono-GalNAc conjugated single stranded oligonucleotide and is the starting material in the synthesis of Conjugate 1 and Conjugate 3.
- (ps) denotes phosphorothioate linkage.
- Figure 26 shows the synthesis of A0006, which is a 5' tri-antennary GalNAc conjugated single stranded oligonucleotide used for the synthesis of Reference Conjugate 4.
- ps denotes phosphorothioate linkage.
- Figure 27 illustrates the in vitro determination of TTR knockdown.
- Figure 27A shows the in vitro determination of TTR knockdown by Reference Conjugates (RC) 1 and 3 as well as the untreated control "UT”
- Figure 27B shows the in vitro determination of TTR knockdown by Reference Conjugates (RC) 2 and 3, as well as the untreated control "UT”
- Figure 27C shows the in vitro determination of TTR knockdown by Conjugates 1 , 2 and 3, as well as by RC3 and untreated control "UT”.
- Reference Conjugates 1 and 2 represent comparator conjugates.
- Reference Conjugate 3 represents a non-targeting GalNAc siRNA and "untreated" ("UT") represents untreated cells. Both RC3 and UT are negative controls. mRNA levels were normalised against Ptenll.
- Figure 29 shows oligonucleotide synthesis of 3' and 5' GalNAc conjugated oligonucleotides precursors (such as compound X0385B-prec).
- Figure 30 discloses GalNAc conjugates with each one serinol-linked GalNAc moiety at both termini of the second strand and with 2'-F at positions 7-9 of the second strand reducing ALDH2 mRNA levels in vitro.
- Figure 31 discloses GalNAc siRNA conjugates with vinylphosphonate at the 5' end of the first strand and phosphodiester internucleotide linkages at the 5' end of the first strand effect reduction of ALDH2 target mRNA levels in vitro.
- Figure 32 discloses GalNAc siRNA conjugates with vinylphosphonate at the 5' end of the first strand and phosphodiester internucleotide linkages at the 5' end of the first strand effect improved reduction of ALDH2 target mRNA levels in vitro.
- Figure 33 shows that GalNAc siRNA conjugates with vinylphosphonate at the 5' end of the first strand and phosphodiester internucleotide linkages at the 5' end of the first strand are stable in acidic tritosome lysate.
- Figure 34 shows that GalNAc siRNA conjugates with vinylphosphonate at the 5' end of the first strand and phosphodiester internucleotide linkages at the 5' end of the first strand are stable in acidic tritosome lysate.
- Figure 35 discloses GalNAc siRNA conjugates with vinylphosphonate at the 5' end of the first strand and phosphodiester internucleotide linkages at the 5' end of the first strand effect reduction of ALDH2 target mRNA levels in vitro.
- Figure 36 shows improved duration of Aldh2 mRNA knockdown in mice following s.c. administration of 1 mg/kg ALDH2-hcm9 siRNA conjugate 16 with 5' & 3' serinol-GalNAc second strand conjugation as compared to conjugate 14 with 5' second strand
- Figure 37 depicts Conjugate 4.
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide.
- the serinol-GalNAc-linkers are conjugated via a phosphodiester bond to the 3' end and the 5' end of the sense strand.
- Figure 38 depicts Conjugate 5.
- the last three nucleotides at the 5' and 3' ends of the antisense strand are connected by a phosphorothioate linker between each nucleotide.
- the serinol-GalNAc-linkers are conjugated via a phosphorothioate bond to the 3' end and the 5' end of the sense strand.
- Figure 39 depicts Conjugate 6.
- the last three nucleotides at the 5' and 3' ends of the antisense strand are connected by a phosphorothioate linker between each nucleotide.
- the serinol-GalNAc-linkers are conjugated via a phosphodiester bond to the 3' end and the 5' end of the sense strand.
- Figure 40 depicts Conjugate 7.
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide.
- the serinol-GalNAc-linkers are conjugated via a phosphorothioate bond to the 3' end and the 5' end of the sense strand.
- the serinol-GalNAc-linkers are connected to each other via a phosphorothioate bond.
- Figure 41 depicts Conjugate 8.
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide.
- a GalNAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand and a GalNAc-C7-amino-modifier linker is conjugated at the 3' end of the sense strand.
- Figure 42 depicts Conjugate 9. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide. A GalNAc-GlyC3-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.
- Figure 43 depicts Conjugate 10. The last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide, and sense strands are connected by a phosphorothioate linker between each nucleotide. A GalNAc-piperidyl-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.
- Figure 44 depicts Conjugate 1 1 .
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide.
- a GalNAc-C3-amino-modifier linker is conjugated at the 5' and 3' ends of the sense strand.
- Figure 45 depicts Conjugate 12.
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide.
- a GalNAc-C6-amino-modifier linker is conjugated at the 5' end of the sense strand and a GalNAc-GlyC3-amino-modifier linker is conjugated at the 3' end of the sense strand.
- Figure 46 depicts Conjugates 15, 16, 18 and 19 which differ only by their RNA sequences.
- the last three nucleotides at the 5' and 3' ends of the antisense and sense strands are connected by a phosphorothioate linker between each nucleotide in each conjugate.
- the serinol-GalNAc-linkers are conjugated via a phosphorothioate bond to the 3' end and the 5' end of the sense strand.
- Figure 47 depicts Reference Conjugate 5 and Reference Conjugate 9 which differ only by their RNA sequences.
- the last three nucleotides at the 5' and 3' ends of the antisense strand and 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide in both conjugates.
- the trimeric GalNAc-linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.
- Figure 48 depicts Reference Conjugate 6 and Reference Conjugate 7 which differ only by their RNA sequences.
- the last three nucleotides at the 5' and 3' ends of the antisense strand and 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide in both conjugates.
- the trimeric GalNAc-linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand in both conjugates.
- Figure 49 depicts Reference Conjugate 8.
- the last three nucleotides at the 5' and 3' ends of the antisense strand and 3' end of the sense strand are connected by a phosphorothioate linker between each nucleotide.
- the trimeric GalNAc-linker is conjugated via a phosphorothioate bond to the 5' end of the sense strand.
- Figure 50 illustrates the in vitro determination of TTR knockdown.
- Figure 50A shows the in vitro determination of TTR knockdown by Conjugates 4, 5, 6 and 2 compared to "Luc" (Reference Conjugate 3) as well as the untreated control "UT”
- Figure 50B shows the in vitro determination of TTR knockdown by Conjugates 7 and 2, compared to "Luc” (Reference Conjugate 3) as well as the untreated control "UT”.
- Luc or Reference Conjugate 3 represents a non-targeting GalNAc siRNA and "untreated" (“UT”) represents untreated cells. Both RC3 and UT are negative controls. mRNA level were normalised against Ptenll.
- Figure 51 illustrates the in vitro determination of TTR knockdown.
- Figure 51 A shows the in vitro determination of TTR knockdown by Conjugates 8, 9, 10, 1 1 and 2 compared to "Luc" (Reference Conjugate 3) as well as the untreated control "UT”
- Figure 51 B shows the in vitro determination of TTR knockdown by Conjugates 12 and 2, compared to "Luc" (Reference Conjugate 3) as well as the untreated control "UT”.
- Luc or Reference Conjugate 3 represents a non-targeting GalNAc siRNA and "untreated" (“UT”) represents untreated cells. Both RC3 and UT are negative controls. mRNA levels were normalised against Ptenll.
- Figure 52 illustrates the in vitro determination of LP A mRNA knockdown by Conjugate 19 compared to controls.
- Ctr represents a non-targeting GalNAc siRNA and "untreated" ("UT") represents untreated cells. Both Ctr and UT are negative controls.
- mRNA level were normalised against ACTB.
- Figure 56 shows serum stability of Conjugates 4, 5, 6, 7 and 2, and untreated control (UT) at 37 °C over 3 days.
- Figure 57 shows serum stability of Conjugates 8, 9, 10, 1 1 , 12 and 2, and untreated control (UT) at 37 °C over 3 days.
- Figure 58 shows extended reduction of Aldh2 mRNA levels in mice by an ALDH2-t.arget.ing GalNAc conjugates with an optimised 2'-OMe/2'-F modification pattern in the second strand and with end-stabilising inverted RNA at the 3' end of the second strand.
- Figure 59 shows schematic representations of varous embodiments of nucleic acids conjugated with ligands via linkers.
- Example 1 The numbering referred to in each example is specific for said example.
- Example 1 The numbering referred to in each example is specific for said example.
- oligonucleotides as set out in the tables below.
- the method of synthesis was as follows, using one of the sequences of the invention as an example:
- Second strand (SEQ ID NO: 87, based on SEQ ID NO: 4)
- a further example is ALDH2-hcm-6
- Second strand (SEQ ID NO: 89, based on SEQ ID NO: 12)
- Second strand (SEQ ID NO: 91 , based on SEQ ID NO: 12)
- a further example is ALDH2-hcm-9
- ST23, ST41 and ST43 are as below.
- ST23-phos is a GalNac C4 phosphoramidite (structure components as below)
- STKS long trebler
- ST41 ST43-phos is as follows and as described in WO2017/174657:
- oligonucleotides were either obtained from a commercial oligonucleotide manufacturer (Biospring, Frankfurt, Germany) or synthesized on an AKTA oligopilot synthesizer (in house) using standard phosphoramidite chemistry.
- Commercially available solid support and 2O-Methyl RNA phosphoramidites, 2 ' Fluoro DNA phosphoramidites (all standard protection) and commercially available long trebler phosphoramidite (Glen research) were used. Synthesis was performed using 0.1 M solutions of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). All other reagents were commercially available standard reagents.
- Conjugation of the respective GalNac synthon (e.g., ST23, ST41 or ST43) was achieved by coupling of the respective phosphoramidite to the 5 ' end of the oligochain under standard phosphoramidite coupling conditions.
- Phosphorothioates were introduced using standard commercially available thiolation reagents (EDITH, Link technologies).
- the single strands were cleaved off the CPG by using methylamine (40% aqueous) and the resulting crude oligonucleotide was purified by Ion exchange chromatography (Resource Q, 6ml_, GE Healthcare) on a AKTA Pure HPLC System using a Sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.
- equimolar amounts of the respective single strands were dissolved in water and heated to 80°C for 5min. After gradual cooling to RT the resulting duplex was lyophilised.
- siRNAs The sequences of the resulting nucleic acids (siRNAs) are set out in Table 1 below.
- Table 1 nucleic acid sequences tested for inhibition of ALDH2 expression. Nucleic acids were synthesized by Biospring, Frankfurt, Germany.
- Example 3 Dose responses of ALDH2-siRNA molecules in human hepatocarinoma HepG2 cells were measured as follows.
- RNA samples were plated at a density of 150,000 cells per well in 6-well format. 24 hours post seeding, cells were transfected with indicated amounts of non-conjugated siRNAs using 1 ⁇ g/ml of AtuFECT as liposomal transfection reagent. 48 hours after transfection, RNA was isolated using the Spin Cell Mini Kit 250 (Stratec). ALDH2 mRNA levels were determined by qRT-PCR relative to the geometric mean of PTEN and ACTIN mRNA expression as housekeepers. Values were normalized to the amount of ALDH2 mRNA in untreated cells. Means and SD of normalized triplicate measurements are shown. I C50 values, 95% confidence intervals and coefficients of determination are given. Results are shown in Figure 2.
- Example 4 ALDH2 mRNA and protein levels and reduction of ALDH enzyme activity by ALDH2-hcm- 2 siRNA in human HepG2-cells were investigated as follows.
- Cells were seeded at 500,000 cells per well in 6-cm dishes. 24 hours post seeding, cells were transfected in triplicate with 1 nM of ALDH2-hcm-2 siRNA or an siRNA against firefly luciferase as a non-targeting control (Flue) using ⁇ g/ml of Atufect as liposomal transfection reagent. Three days post transfection, cells were lysed using InviTrap Spin Cell RNA Mini Kit (Stratec). ALDH2 mRNA levels were determined by qRT-PCR relative to actin expression as housekeeper. Mean and SD of normalized values are shown.
- Membranes were probed with antibodies against ALDH2 (Abeam #194587, 1 :1200) and alpha-ACTININ (NEB #3134, 1 :1000). Quantification was performed Using a Stella Imaging System (Raytest). Aldehyde-Dehydrogenase activity assay was performed with total protein (12.2 micro g) extract using the Mitochondrial Aldehyde Dehydrogenase Activity Assay Kit (Abeam) according to manufacturer's protocol. Results are shown in Figure 3.
- Mouse hepatocytes were plated at a density of 20.000 cells per well on collagen-coated 96-well plates (Life Technologies). 3 hours after plating, cells were rinsed once with PBS and L1-GalNAc-conjugated siRNAs were added at the indicated concentrations in triplicates. 24 hours post treatment, cells were lysed with InviTrap Spin Cell RNA Mini Kit (Stratec). Aldh2 mRNA levels were determined by qRT-PCR relative to Actin mRNA expression as housekeeper. Values were normalized to the amount of Aldh2 in untreated cells. Means and SD of normalized triplicate values are shown. Results are shown in Figure 4.
- Example 6 Dose-response for GalNAc- L1 -linker-coupled siRNAs against ALDH2 in primary human hepatocytes was measured as follows.
- Human hepatocytes (Thermo Fisher) were plated at a density of 30.000 cells per well on collagen-coated 96-well plates (Life Technologies). GalNAc-L1 linker conjugated siRNAs were added at the indicated concentrations in triplicates immediately after plating. 24 hours post treatment, cells were lysed with InviTrap Spin Cell RNA Mini Kit (Stratec). ALDH2 mRNA levels were determined by qRT-PCR relative to ACTIN mRNA expression as housekeeper. Values were normalized to the amount of ALDH2 mRNA in untreated cells. Means and SD of normalized triplicate values are shown. Results are shown in Figure 5.
- mice Male mice (C57BI/6J) were treated subcutaneously with 3 or 10 mg/kg of the indicated compounds (6 animals per group). At necropsy 10 days post treatment, liver was harvested. Tissue was homogenized in a Tissue Lyser II machine (Qiagen).
- B - Protein was extracted from 10 mg of liver tissue in RIPA buffer (Cell Signaling) from three animals treated with 10 mg/kg of the indicated compounds per group. Protein amount was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific), and 30 ⁇ g per sample were used for electrophoresis in Laemmli buffer. Membranes were probed with antibodies against ALDH2 (Abeam #194587, 1 :1200) and alpha-ACTININ (NEB #3134, 1 :1000) and developed with HRP secondary antbody. Visualizations of the membranes are shown in the lower panel. Quantification (upper left panel) was performed Using a Stella Imaging System (Raytest).
- Aldehyde-Dehydrogenase activity assay (upper right panel) was performed using liver protein extracts of three mice treated with the indicated siRNAs or PBS using the Mitochondrial Aldehyde Dehydrogenase Activity Assay Kit (Abeam) according to manufacturer's protocol. Results are shown in Figure 8B.
- mice Male mice (C57BI/6J) were treated once subcutaneously with 1 , 3 or 10 mg/kg of the indicated compounds (5 animals per group). At necropsy at 10, 20, 30, and 41 days post treatment, liver was harvested. Tissue was homogenized in a Tissue Lyser II machine (Qiagen). Total RNA was extracted from 10 mg of liver tissue using an InviTrap Spin
- ALD02-ALD05 (Table 3) contain inverted RNA in addition to the last nucleotide in the first strand and second strand.
- ALD06-ALD09 contain inverted RNA instead of the last nucleotide in first strand and second strand. All inverted RNA nucleotides substitute for terminally used phosphorothioates. In both designs, ivA and ivG confer higher stability to the tested sequence than ivU and ivC. "UT" indicates untreated samples.
- FBS indicates siRNA duplexes which were incubated at 37 °C at 5 ⁇ concentration with 50 % FBS for 3 days, before being phenol/chloroform-extracted and precipitated with Ethanol. Samples were analyzed on 20% TBE polyacrylamide gels in native gel electrophoresis. UV pictures of the ethidium bromide-stained gels are shown in Figure 1 1 .
- ALD01 contains phosphorothioates at all termini
- ALD02-ALD05 contain ivA (ALD01 ), ivU (ALD03), ivC (ALD04) and ivG (ALD05) at the 3'- end of the first strand and at the 3'-end of the second strand.
- Both inverted nucleotides are present in addition to the terminal nucleotide of the respective strands and substitute for terminal phosphorothioates.
- a non-related siRNA (PTEN) and a control siRNA targeting firefly luciferase (Luci) were included. All tested variants show comparable activity under the tested conditions.
- the experiment was conducted in the human hepatocarcinoma cell line Hep3B. Cells were seeded at a density of 150,000 cells per 6- well, transfected with 0.1 nM and 1 nM siRNA, respectively, using 1 ⁇ g/ml Atufect as a liposomal transfection reagent after 24 h. Cells were lysed after 48 h. Total RNA was extracted and ALDH2 and ACTIN mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD of three technical replicates normalized to the house keeping gene expression (ACTIN). Results are shown in Figure 12.
- ALD01 contains phosphorothioates at all termini
- ALD06-ALD09 contain ivA (ALD06), ivU (ALD07), ivC (ALD08) and ivG (ALD09) at the 3'- end of the first strand and at the 3'-end of the second strand.
- Both inverted nucleotides replace the terminal nucleotide of the respective strands and substitute for terminal phosphorothioates used in other derivatives for stabilization.
- a non-related siRNA (PTEN) and a control siRNA targeting firefly luciferase (Luci) were included. All tested variants show comparable activity under the tested conditions.
- the experiment was conducted in the human hepatocarcinoma cell line Hep3B. Cells were seeded at a density of 150,000 cells per 6-well, transfected with 0.1 nM and 1 nM siRNA, respectively,using and 1 ⁇ g/ml Atufect as a liposomal transfection reagent after 24 h. Cells were lysed after 48 h. Total RNA was extracted and ALDH2 and ACTIN mRNA levels were determined by Taqman qRT-PCR. Each bar represents mean ⁇ SD of three technical replicates normalized to the house keeping gene expression (ACTIN). Results are shown in Figure 13.
- STS22002L6 contains phosphorothioates at all non-conjugated ends, whereas STS22002V1-L6 and
- STS22002V2-L6 contain inverted RNA nucleotides at the second strand's 3'-end, where the respective inverted nucleotide is present instead of the last nucleotide.
- STS22002V3- L6 and STS22002V4-L6 contain inverted RNA nucleotides at the first strand's 3'-end, whereby the respective inverted nucleotide is present in addition to the last nucleotide.
- ivA was used in STS22002V1 -L6 and STS22002V3-L6, whereas ivG was used in
- STS22002V2-L6 and STS22002V4-L6 All inverted RNA nucleotides substitute for terminal phosphorothioates used in other derivatives for stabilization.
- STS22002V1 -L6 and STS22002V2-L6 are slightly more stable than the other variants tested here.
- "UT" indicates untreated samples.
- JUK04 represents a control compound not serum stabilized as a positive control.
- FBS indicates GalNAc-siRNA conjugates which were incubated at 37 °C at 5 ⁇ concentration with 50% FBS for 3 days before being phenol/chloroform- extracted and precipitated with Ethanol. Samples were analyzed on 20% TBE
- the dose response of inverted RNA nucleotides at terminal 3' positions on the efficacy of ALDH2 mRNA knockdown was analyzed using GalNac-L6-conjugated derivatives of the siRNA ALDH2-hcm2 (see Table 4) by ASGPR receptor complex-mediated uptake in mouse primary hepatocytes.
- STS22002L6 contains phosphorothioates at all non- conjugated ends
- STS22002V1-L6 and STS22002V2-L6 contain inverted RNA nucleotides at the second strand's 3'-end, where the respective inverted nucleotide is present instead of the last nucleotide.
- STS22002V3-L6 and STS22002V4-L6 contain inverted RNA nucleotides at the first strand's 3'-end, whereby the nucleotide is present in addition to the last nucleotide.
- ivA was used in STS22002V1-L6 and STS22002V3-L6, whereas ivG was used in STS22002V2-L6 and STS22002V4-L6. All inverted RNA nucleotides substitute for terminal phosphorothioates used in other derivatives for stabilization. All tested variants show comparable activity. The experiment was conducted in mouse primary hepatocytes.
- GalNAc-L6 derivatives of ALDH2-hcm6 and ALDH2-hcm9 siRNA conjugates targeting Aldh2 were used in an in vivo efficacy study.
- STS22002L6 contains phosphorothioates at all non-conjugated termini, whereas
- STS22002V3-L6 contains an ivA at the first strand's 3'-end in addition to the last nucleotide.
- PBS vehicle
- Seven days after treatment at necropsy liver tissue was harvested and total RNA was extracted.
- Aldh2 and ApoB mRNA levels were analyzed by Taqman qRT-PCR. Each bar represents mean normalized (ApoB) expression ⁇ SD of six animals. Individual values normalized to the house keeping gene expression (ApoB) are plotted, additionally. Results are shown in Figure 16.
- Example 17 Example 17
- V1 variants contain a different 2'-OMe/2'-F modification pattern in the second strand and an ivA nucleotide at the 3'-end of the second strand, substituting for the last nucleotide and for terminal phosphorothioates at this end.
- V2 variants additionally contain a different 2'-OMe/2'-F modification pattern in the first strand as specified in Table 5.
- PBS vehicle
- GalNAc-L6 conjugate derivatives 3 mg/kg and 1 mg/kg GalNAc-L6 conjugate derivatives, respectively.
- RNA was extracted.
- Aldh2 and ApoB mRNA levels were analyzed by Taqman qRT-PCR. Each bar represents mean normalized (ApoB) expression ⁇ SD of six animals. Individual values normalized to the house keeping gene expression (ApoB) are plotted, additionally.
- Example compounds were synthesised according to methods described below and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology. GalNAc conjugation was achieved by peptide bond formation of a GalNAc- carboxylic acid building block to the prior assembled and purified oligonucleotide having the necessary number of amino modified linker building blocks attached.
- Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 15 min. A Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac20/NMI/Lutidine/Acetonitrile, Oxidizer: 0.1 M in pyridine/H20). Phosphorothioates were introduced using standard commercially available thiolation reagent (EDITH, Link technologies). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All oligonucleotides were synthesized in DMT-off mode.
- BTT benzylthiotetrazole
- the single strands were cleaved off the CPG by 40% aq. methylamine treatment.
- the resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6mL, GE Healthcare) on a AKTA Pure HPLC System using a sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.
- GalNAc Synthon (9) Synthesis of the GalNAc synthon 9 was performed as described in Nair et al. J. Am. Chem. Soc, 2014, 136 (49), pp 16958-16961 , in 46% yield over two steps.
- 5 ⁇ x NH2 means refers to the position (5' end) and number (1 x NH2) of free serinol derived amino groups which are available for conjugation.
- 1x3'NH2 on A0264 means there is free amino group which can be reacted with GalNAc synthon 9 at the 3' end of the strand A0264.
- 3'5'1 xNH2 means there is one serinol-derived free amino group which can be reacted with GalNAc linker 9 at the 3' end and the 5' end of the strand.
- Conjugation of the GalNac synthon (9) was achieved by coupling to the serinol-amino function of the respective oligonucleotide strand 11 using a peptide coupling reagent. Therefore, the respective amino-modified precursor molecule 11 was dissolved in H2O (500 OD/mL) and DMSO (DMSO/H2O, 2/1 , v/v) was added, followed by DIPEA (2.5% of total volume). In a separate reaction vessel pre-activation of the GalN(Ac4)-C4-acid (9) was performed by reacting 2 eq. (per amino function in the amino-modified precursor oligonucleotide 11 ) of the carboxylic acid component with 2 eq.
- the resulting pellet was dissolved in 40% MeNH2 (1 mL per 500 OD) and after 15 min at RT diluted in H 2 0 (1 :10) and finally purified again by anion exchange and size exclusion chromatography and lyophilised to yield the final product 12.
- Double strand formation was performed according to the methods described above.
- the double strand purity is given in % double strand which is the percentage of the UV-area under the assigned product signal in the UV-trace of the IP-RP-HPLC analysis.
- f denotes 2 ' Fluoro 2 ' deoxyribonucleotide or 2'-fluoro ribonucleotide (the terms are interchangeable)
- m denotes 2 ⁇ Methyl ribonucleotide
- Ser(GN) is a GalNAc-C4 building block attached to serinol derived linker moiety:
- O— is the linkage between the oxygen atom and e.g. H, phosphordiester linkage or phosphorothioate linkage.
- Antisense strand - STS16001AL33 (SEQ ID NO: 136)
- Antisense strand - STS16001A (SEQ ID NO: 138) mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fU mG (ps) fU (ps) mU Sense strand - STS16001 BV1 L42 (SEQ ID NO: 139) Ser(GN) (ps) fA (ps) mA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mC fU mA fU (ps) mA (ps) fA (ps) Ser(GN)
- Antisense strand - STS16001AL33 (SEQ ID NO: 136) 5 ' mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fU mG (ps) fU (ps) mU (ps) Ser(GN) 3 '
- Antisense strand - STS16001AL33 (SEQ ID NO: 136) mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fU mG (ps) fU (ps) mU (ps) Ser(GN) Sense strand - STS16001V1 B (SEQ ID NO: 140) fA (ps) mA (ps) fC mA fG mU fG mU fU mC fU mC fU mA fU (ps) mA (ps) fA Reference Conjugate 3
- Antisense strand - STS 18001 A (A0130; SEQ ID NO: 141 ) mU (ps) fC (ps) mG fA mA fG mU fA mU fU mC fC mG fC mG fU mA (ps) fC (ps) mG Sense strand - STS18001 BL4 (A0131 ; SEQ ID NO: 142)
- Antisense strand - STS16001AL33 (SEQ ID NO: 136) mU (ps) fU (ps) mA fU mA fG mA fG mC fA mA fG mA fA mC fA mC fU mG (ps) fU (ps) mU
- Murine primary hepatocytes were seeded into collagen pre-coated 96 well plates (Thermo Fisher Scientific, #A1 142803) at a cell density of 30,000 cells per well and treated with siRNA-conjugates at concentrations ranging from 10nM to 0.0001 nM.
- 24h post treatment cells were lysed and RNA extracted with InviTrap® RNA Cell HTS 96 Kit / C24 x 96 preps (Stratec #7061300400) according to the manufactures protocol. Transcripts levels of TTR and housekeeping mRNA (Ptenll) were quantified by TaqMan analysis.
- Figure 27 also shows the target gene expression levels of Reference Conjugates 1 and 2 which act as comparator conjugates.
- the conjugates of the invention decrease the target gene expression compared to Reference Conjugates 1 and 2.
- the most effective conjugate at 0.01 nM appears to be Conjugate 2.
- C57BL/6 mice were treated s.c. with 1 mg/kg siRNA-conjugates at day 0.
- Serum samples were taken at day 7, 14, and 27 by orbital sinus bleeding and stored at -20°C until analysis.
- Serum TTR quantification was performed with a Mouse Prealbumin ELISA (ALPCO, 41- PALMS/lot 22, 2008003B) according to the manufacturers protocol (sample dilution 1 :8000 or 1 :800).
- the conjugates of the invention are particularly effective at reducing target gene expression compared to the negative control (PBS) and Reference Conjugates 1 , 2, and in particular to Reference Conjugate 4.
- Conjugates 2 and 3 are also more effective than Reference Conjugates 1 , 2 and 4.
- the most effective conjugate is Conjugate 2.
- the dosing level of Conjugate 3 would be about three times lower to achieve the same initial knock down and would also result in longer duration of knock down as compared to Reference Conjugate 4.
- Example compounds were synthesised according to methods described below and methods known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology. GalNAc conjugation was achieved by peptide bond formation of a GalNAc- carboxylic acid building block to the prior assembled and purified oligonucleotide having the necessary number of amino modified linker building blocks attached.
- Oligonucleotide synthesis, deprotection and purification followed standard procedures that are known in the art. All Oligonucleotides were synthesized on an AKTA oligopilot synthesizer using standard phosphoramidite chemistry. Commercially available solid support and 2O-Methyl RNA phosphoramidites, 2 ' Fluoro, 2 ' Deoxy RNA phosphoramidites (all standard protection, ChemGenes, LinkTech) and commercially available 3'-Amino Modifier TFA Amino C-6 lcaa CPG 500A (Chemgenes) were used. Per-acetylated galactose amine 8 is commercially available.
- Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile and benzylthiotetrazole (BTT) was used as activator (0.3M in acetonitrile). Coupling time was 15 min. A Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac20/NMI/Lutidine/Acetonitrile, Oxidizer: 0.1 M in pyridine/hbO). Phosphorothioates were introduced using standard commercially available thiolation reagent (EDITH, Link technologies). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene.
- a diethylamine (DEA) wash was performed. All oligonucleotides were synthesized in DMT-off mode. Attachment of the serinol-derived linker moiety was achieved by use of either base-loaded (S)-DMT-Serinol(TFA)-succinate-lcaa-CPG 10 or a (S)-DMT-Serinol(TFA) phosphoramidite 7 (synthesis was performed as described in literature Hoevelmann etal. Chem. Sci., 2016,7, 128-135).
- Tri-antennary GalNAc clusters ST23/C4XLT or ST23/C6XLT were introduced by successive coupling of the respective trebler amidite derivatives (C4XLT-phos or C6XLT- phos) followed by the GalNAc amidite (ST23-phos).
- the single strands were cleaved off the CPG by 40% aq. methylamine treatment.
- the resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6ml_, GE Healthcare) on a AKTA Pure HPLC System using a sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised.
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